U.S. patent application number 09/751100 was filed with the patent office on 2002-10-03 for human adenylate cyclase and use therefor.
Invention is credited to Antoni, Ferenc, Paterson, Janice M..
Application Number | 20020142436 09/751100 |
Document ID | / |
Family ID | 26306498 |
Filed Date | 2002-10-03 |
United States Patent
Application |
20020142436 |
Kind Code |
A1 |
Antoni, Ferenc ; et
al. |
October 3, 2002 |
Human adenylate cyclase and use therefor
Abstract
There is provided a novel adenylate cyclase enzyme, the
nucleotide sequence of which is set out in SEQ ID NO: 98. The
activity of the adenylate cyclase is uniquely regulated by
calcineurin, a protein phosphatase. The calcineurin binding site
has been identified at amino acids 503 to 610 of the novel
adenylate cyclase. Regulation of the novel adenylate cycalse may be
useful in treating certain disorders. Suitable regulators include
agents which bind to the 503-610 amino acid site and include
calcineurin, its activators, inhibitors and competitors and
antibodies specific to that site. Specific disorders include
neurological disorders such as Parkinsons' disease, cardiovascular
disorders such as angina pectoris and tumours, especially ovarian
tumours.
Inventors: |
Antoni, Ferenc; (Edinburgh,
GB) ; Paterson, Janice M.; (Edinburgh, GB) |
Correspondence
Address: |
Ratner & Prestia
Suite 301
One Westlakes, Berwyn
P.O. Box 980
Valley Forge
PA
19482-0980
US
|
Family ID: |
26306498 |
Appl. No.: |
09/751100 |
Filed: |
December 28, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09751100 |
Dec 28, 2000 |
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09398193 |
Sep 17, 1999 |
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6197581 |
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09398193 |
Sep 17, 1999 |
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08894173 |
Aug 13, 1997 |
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6090612 |
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09398193 |
Sep 17, 1999 |
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PCT/GB96/00312 |
Feb 14, 1996 |
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Current U.S.
Class: |
435/199 ;
435/320.1; 435/325; 435/366; 435/69.1; 536/23.2 |
Current CPC
Class: |
A61K 38/13 20130101;
C12Y 406/01001 20130101; A61P 9/00 20180101; A61K 49/0004 20130101;
A61P 1/00 20180101; A61K 45/06 20130101; A61K 38/16 20130101; A61P
43/00 20180101; C07K 16/40 20130101; A61K 38/13 20130101; A61K
38/16 20130101; A61P 11/00 20180101; A61P 15/00 20180101; C12N 9/88
20130101; A61K 48/00 20130101; A61P 35/00 20180101; A61P 25/00
20180101; A61P 13/02 20180101; A61K 2039/505 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101 |
Class at
Publication: |
435/199 ;
435/69.1; 435/325; 435/366; 435/320.1; 536/23.2 |
International
Class: |
C12N 009/22; C07H
021/04; C12N 005/08; C12P 021/02; C12N 005/06 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 14, 1995 |
GB |
9502806.4 |
Aug 11, 1995 |
GB |
9516528.8 |
Claims
1. A protein or polypeptide having adenylate cyclase activity, said
activity being regulatable by calcineurin.
2. A protein or polypeptide as claimed in claim 1 which is encoded
by a polynucleotide comprising a nucleotide sequence derived from
the sequence set out in SEQ ID NO: 1 or in SEQ ID NO: 98, or a part
thereof.
3. A protein or polypeptide as claimed in either one of claims 1
and 2 comprising the amino acid sequence as set out in SEQ ID NO: 2
or in SEQ ID NO: 99, a functional equivalent or part thereof.
4. A protein or polypeptide as claimed in any one of claims 1 to 3
which includes amino acid Nos 503-570 of SEQ ID NO: 2 or which
includes amino acid Nos 503-570 of SEQ ID NO: 99.
5. An isolated polynucleotide comprising a nucleotide sequence as
set out in SEQ ID NO: 98, or a part thereof.
6. A recombinant polynucleotide construct comprising a
polynucleotide as claimed in claim 5.
7. A vector comprising a polynucleotide as claimed in claim 5 or a
construct as claimed in claim 6.
8. A host cell transformed with a vector as claimed in claim 7.
9. A host cell as claimed in claim 8 which is derived from the
Human Embryonic Kidney Cell line 293.
10. The use of a regulator of a protein or polypeptide as claimed
in any one of claims 1 to 4 to influence cellular metalbolism by
affecting the adenylate cyclase activity or the amount of said
protein or polypeptide.
11. The use as claimed in claim 10 wherein said regulator is
calcineurin, its activators, inhibitors and competitors; antibodies
to amino acids 503-570; or .beta.-adrenergic agonists and
.beta.-adrenergic antagonists.
12. The use as claimed in either one of claims 10 and 11 wherein
said regulator is an antibody to amino acids 503-570 of SEQ ID NOS:
2 or 99.
13. A method of treating a human or non-human animal body, said
method comprising administrating an agent to affect the adenylate
cyclase activity of, or the amount of a protein or polypeptide as
claimed in any one of claims 1 to 4, said protein or polypeptide
being present within said body.
14. A method as claimed in claim 13 wherein said protein or
polypeptide is naturally present within said body.
15. A method as claimed in either one of claims 13 and 14 to affect
or combat neurological-based disorders; psychiatrically-based
disorders; endocrine-based disorders; cardiovascular-based
disorders; pregnancy-based disorders; respiratory-based disorders;
bone-based disorders; kidney-based disorders; gut-based disorders;
or tumours.
16. The use of a protein or polypeptide as claimed in any one of
claims 1 to 4 or a polynucleotide as claimed in claim 5 to design
or test for a therapeutic agent which influences cellular
metabolism.
Description
[0001] This Application is a continuation-in-part of U.S. Ser. No.
08/894,173, filed Aug. 13, 1997, currently pending.
[0002] The present invention relates to the control of cellular
metabolic process by human adenylate cyclase.
[0003] Cells of multi-cellular organisms may be metabolically
affected by external factors, which are usually chemical. Hormones
are a well-known example of such chemical factors. Generally the
external chemical factors interact with a specific receptor located
on the membrane of the targeted cell. The binding event of the
factor to its receptor may induce alterations in cellular
metabolism via a "secondary messenger" mediator.
[0004] One of the key "secondary messengers" is cyclic AMP (cAMP)
(see Sutherland, Science 177:401-407 (1972)). Cyclic AMP is
produced from ATP through the action of an enzyme, adenylate
cyclase. It is now known that adenylate cyclase activity may be
affected by a factor/receptor binding event transmitted through an
associated G protein.
[0005] Alteration of the intracellular concentration of cAMP
affects many cellular reactions. For example, an increase in cAMP
intracellular concentration stimulates the activity of protein
kinases (enzymes that transfer terminal phosphate groups from ATP
to specific sites on targeted proteins). The action of the protein
kinases changes the activity or function of its substrate.
[0006] For a general review of cAMP and secondary messenger systems
reference is made to "Molecular Cell Biology", Darnell et al, 1986,
Chapter-16, incorporated herein by reference.
[0007] Further investigations of cAMP as a secondary messenger
revealed that an alteration in cAMP intracellular concentration was
caused by the interaction of several different external factors
with their distinct receptors. Further, it was found that different
receptors were associated with their own particular G-protein
intermediary which was itself associated with adenylate cyclase.
More recent investigations have shown that there are in fact
different types (isoenzymes) of adenylate cyclase, which display
considerable regulatory diversity.
[0008] To date eight distinct isoenzymes of adenylate cyclase have
been identified and described in the literature. The complete cDNA
sequences are known for isoenzymes types 1 to 8. A review of the
current understanding and knowledge of the known adenylate cyclase
isoenzymes is set out in Pieroni et al, Current Opinion in
Neurobiology 3:345-351 (1993); Kerwin Jr in Annual Reports in
Medicinal Chemistry, Section VI, Chapter 29, Pages 287-295 (ed
Venuti), (1994) and Premont, Methods in Enzymology 238:116-127
(1994).
[0009] A summary of the regulation of the known isoenzymes of
adenylate cyclase is set out below in Table 1.
1TABLE 1 cAMP Isoenzyme Regulated by concentration 1 Ca.sup.2+/CaM
.Arrow-up bold. .beta. .gamma. dimer .dwnarw. 2 G.sub.X1 + PKC
.Arrow-up bold. .beta. .gamma. dimer .Arrow-up bold. PKC .Arrow-up
bold. 3 Ca.sup.2+/CaM .Arrow-up bold. 4 .beta. .gamma. dimer
.Arrow-up bold. 5 Ca.sup.2+ .dwnarw. 6 Ca.sup.2+ .dwnarw. CaM =
calmodulin PCK = protein Kinase C
[0010] It has now been found, for the first time, that the protein
phosphatase calcineurin regulates an adenylate cyclase
isoenzyme.
[0011] It has further now been found that the isoenzyme regulated
by calcineurin is a novel previously uncharacterised adenylate
cyclase isoenzyme. The novel isoenzyme of the present invention was
originally referred to as adenylate cyclase 10 (AC10), but a review
of nomenclature has now caused the novel adenylate cyclase to be
referred to as adenylate cyclase 9 (AC9). To avoid confusion with
the different isoenzyme known before the nomenclature revision as
"adenylate cyclase 9", the novel adenylate cyclase of the present
invention will herein simply be referred to as "AC".
[0012] The nucleotide sequence encoding for mouse AC has been
identified, cloned and sequenced (see Example 2). The nucleotide
sequence encoding for mouse AC is given in SEQ ID No 1. The
sequence is also accessible in the Genbank.TM. database under
accession No. MMU30602 and in the EMBL database under accession No.
Z50190.
[0013] A nucleotide sequence purporting to be that for human AC is
described in WO-A-99/01540. The nucleotide sequence presented in
WO-A-99/01540 indicates that the C-terminal portion of the protein
is truncated relative to that of the mouse. We have now conducted
additional work in this area and, surprisingly, have cloned and
sequenced a human AC construct which does not have the truncated
C-terminal portion reported in WO-A-99/01540. The sequence obtained
by us for human AC is set out in SEQ ID No 98.
[0014] The present invention therefore provides a polypeptide
encoded by the nucleotide sequence of SEQ ID No 98 or as set out in
SEQ ID No 99 (or functional equivalents or parts of those
sequences).
[0015] The term "functional equivalents" is used herein to refer to
any modified version of a nucleotide or polypeptide which retains
the basic function of its unmodified form. As an example, it is
well-known that certain alterations in amino acid or nucleic acid
sequences may not affect the polypeptide encoded by that molecule
or the function of the polypeptide. It is also possible for deleted
versions of a molecule to perform a particular function as well as
the original molecule. Even where an alteration does affect whether
and to what degree a particular function is performed, such altered
molecules are included within the term "functional equivalent"
provided that the function of the molecule is not so deleteriously
affected as to render the molecule useless for its intended
purpose.
[0016] Whilst we do not wish to be bound to theoretical
considerations, it is believed that calcineurin regulates AC by
removal of phosphate group(s) required for the active form of the
enzyme. Thus, the adenylate cyclase activity of AC is believed to
decrease in the presence of calcineurin.
[0017] In a further aspect, therefore, the present invention
provides the use of calcineurin in the regulation of adenylate
cyclase activity, in particular in the regulation of AC.
[0018] The activity of calcineurin is itself enhanced by the
presence of Ca.sup.2+ ions and further enhanced by the additional
presence of calmodulin.
[0019] In a further aspect, the present invention provides an
adenylate cyclase isoenzyme, the activity of which can be regulated
by calcineurin.
[0020] Desirably the calcineurin regulatable adenylate cyclase
isoenzyme is encoded by the nucleotide sequence of SEQ ID No 98,
functional equivalents or parts thereof.
[0021] In a yet further aspect, the present invention provides a
polynucleotide comprising a sequence derived from the sequence set
out in SEQ ID No 98 or a part thereof.
[0022] The phrase "derived from" includes identical and
complementary copies of the sequence of SEQ ID No 1, whether of RNA
or DNA and whether in single or double-stranded form. The phrase
"derived from" further includes sequences with alterations which
(due to the degeneracy of the genetic code) do not affect the amino
acid sequence of the polypeptide expressed, as well as sequences
modified by deletions, additions or replacements of nucleotide(s)
which cause no substantial deleterious affection to function
(including the function of the polypeptide expressed).
[0023] The polynucleotide of the present invention includes all
recombinant constructs comprising a nucleotide sequence of the
invention as defined above. Such recombinant constructs may be
designed to express only part of AC. The constructs may include
expression control sequence(s) which differ to the control
sequence(s) naturally adjoining the AC gene. Optionally, the
construct may include a non-AC protein encoding region. Thus the
recombinant construct includes constructs encoding for chimeric
proteins, which comprise at least part of AC or a functional
equivalent thereof.
[0024] In a particular embodiment, the present invention provides a
vector (such as a cloning or expression vector) which comprises a
recombinant construct as defined above. Vectors include
conventional cloning and expression plasmids for bacterial and
yeast host cells as well as virus vectors such as vaccinia, which
may be useful for expression in eukaryotic cell lines. Such a
vector may be used to transform a suitable host cell (either for
cloning or expression purposes) and the transformed host cell also
forms a further aspect of the present invention. If the vector
produced is comprised only in part of a nucleotide sequence derived
from SEQ ID No 1 it may be appropriate to select a host cell type
which is compatible with the vector. Mention may be made of
prokaryotic host cells such as E coli as well as eukaryotic host
cells, including yeasts, algae and fish, insect or mammalian cells
in culture. Insect cells may be especially useful where a
baculovirus expression system is used. Suitable host cells will be
known to those skilled in the art.
[0025] As a general reference to genetic engineering techniques,
mention may be made of Sambrook, Fritsch, Maniatis, in "Molecular
Cloning, a Laboratory Manual", Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y., 1989; and also to Old and Primrose "Principles
of Genetic Manipulation", 5th edition, 1994.
[0026] In particular cell lines derived from the human embryonic
kidney cell line 293 (HEK293) have been generated and stably
express AC. Briefly, the cells were transfected with pcDNA3
containing the full length AC DNA (clone JP 173--see Example 3) and
cells were selected on the basis of resistance to G418 antibiotic
(0.8 mg/ml) for 4 weeks. Individual clones of resistant cells were
expanded and tested for cAMP production in response to CRF and the
effects of immunosuppressants were also examined. At least three
cell lines exhibited cAMP production in response to CRF which was
enhanced by both cyclosporin A and FK506. In the case of FK506,
this effect is blocked by L-685,818. Furthermore, the accumulation
of cAMP in the presence of inhibitors of phosphodiesterase is about
10-fold higher than in wild type HEK293 cells. This latter finding
suggests that the transfected cyclase is constitutively active and
thus may be a mutant of the wild type enzyme.
[0027] In a yet further aspect, the present invention provides
regulators of AC. Such regulators may act directly on AC itself,
and may include (but are not limited to) calcineurin and antibodies
specific to AC, or may affect production of AC, including (for
example) antisense oligonucleotides (which prevent expression of
the AC gene by binding to a portion of DNA preventing transcription
and/or by binding to the mRNA preventing translation), and agents
which bind to receptors and thus affect the activity of AC, for
example .beta.-adrenergic agonists and .beta.-adrenergic
antagonists. As .beta.-adrenergic agonists mention may be made of
salbutamol, clenbuterol, fenoterol and the like, whereas suitable
.beta.-adrenergic antagonists include propranolol.
[0028] Antibodies (including monoclonal antibodies) specific to AC
may be produced using conventional immunological techniques.
[0029] In a yet further aspect, the present invention provides a
method of regulating cellular metabolism, wherein said method
comprises altering the activity or amount of AC. For example, the
activity or amount of AC may be influenced by the regulators
described above. Alternatively, for example, the amount of AC could
be controlled through genetic manipulation of the genome, by
providing agents that affect transcription and/or translation of
the AC gene.
[0030] The distribution of AC varies between different tissues or
cell types and it is postulated that the intracellular
concentration of AC may fluctuate as a result of expression control
of the AC gene in response to particular stimuli. Studies of AC
distribution in tissues suggest that it is prominently present in
skeletal muscle and heart (FIG. 17). Its likely function in these
tissues is the coordination between contraction and metabolic
demand. Intracellular calcium ions promote contraction, and cAMP
promotes glycogenolysis. Feedback of calcium ions on cAMP synthesis
would delimit the accumulation of calcium and ensure that the
contractions are no longer than what can be metabolically tolerated
by the cells. In this respect it is plausible that the levels of
cyclase will be regulated by the trophic state of the muscle such
as that seen in the training of sportsmen and animals such as
racing horses. Thus the measurement of AC levels by means of
specific probes would constitute a means of predicting optimal
regulation of contraction and metabolism of nutrients.
[0031] Thus for example AC may be present at abnormal levels in
certain disease states or conditions. Mention may be made in this
regard of neurological-based disorders (for example Parkinson's
disease, epilepsy), psychiatrically-based disorders (for example,
anxiety, major depression disorder, mania, schizophrenia,
obsessive-compulsive disorder, Tourette's Syndrome and related
tics) endocrine-based disorders (for example Cushing's Syndrome and
disease, Nelson's Syndrome, Cohn's Syndrome, glucocorticoid
resistance, Graves' disease (thyrotoxicosis) with or without
exophthalmia, hyper and hypothyroidism; hyperprolactinaemia and its
effects); hypertrophy of the prostate; cardiovascular-based
disorders (for example, angina pectoris, cardiac infarction,
hypertension (benign and malignant)), pregnancy-based disorders
(for example, recurrent spontaneous abortion, pre-eclampsia and
eclampsia) respiratory-based disorders (for example, asthma,
bronchitis, chronic bronchitis emphysema and cor pulmonale),
bone-based disorders (for example, hyperparathyroidism,
osteomalacia, Paget's disease, osteoporosis), promotion of bone
healing, kidney-based disorders (for example, acute and chronic
glomerular nephritis, Albright's Syndrome--or the McCune Albright's
Syndrome), gut-based disorders (for example, ulcerative colitis,
irritable bowel, Crohn's disease, Hirschssprung's disease), tumours
(benign and malignant), especially ovarian tumours and prostate
tumours, and in the control or promotion of fertility. AC has been
found to be of greater prevalence in the brain (in particular the
cortex, striatum and hippocampus regions of the brain), in the
ovaries and in the lungs.
[0032] Finally, substances developed to regulate the activity of
this cyclase may be useful as improvers of metabolic balance in
ischemic heart muscle and syndromes of skeletal muscle atrophy.
[0033] In a further aspect, therefore, the present invention
provides a method of treating such conditions by control of AC
activity. An example of such control could be the design of
compounds that mimic the steric conformation of amino acids in
positions 503 to 610, especially 503 to 570, of SEQ ID No 99 which
may function as a calcineurin binding-site.
[0034] The amino acid sequence of human AC has been analysed
(analogously to that of the mouse sequence in Example 3) and a
domain corresponding to the immunophilin protein FKBP12 (which
potently inhibits calcineurin) has been located in residues 594 to
611. The sequence similarity strongly suggests that this is the
calcineurin binding site.
[0035] Other adenylyl cyclases were also examined for potential
sequence similarities with the FKPB12 and 13 proteins as described
in Example 3. A summary of the findings is shown in FIG. 15.
Briefly, all of the known adenylyl cyclase sequences appear to show
significant similarity with FKBPs in the area that corresponds to
the junction of the C1.alpha. and C1.beta. domains (see Example 3
for nomenclature). As shown in FIG. 16 the alignments of individual
cyclases with FKBP12 are distinct i.e. different portions of the
C1.alpha. high homology region are part of the FKBP like sequence.
These alignments are possible because of modular repeats in the
cyclase amino acid sequence in this region. The guiding motif for
these alignments is the requirement of the presence of an "80s"
loop of FKBP12 in a homologous position in the cyclase "FKBP like"
sequence (Yang et al, (1993) Journal of the American Chemical
Society 115:819-820). It is of note for instance, that ACtype1, a
calcium activated adenylyl cyclase contains a significant portion
(sequence: FGPLI) of the 80s loop of FKBP12 which is essential for
the interaction with calcineurin (Yang et al, 1993 supra).
[0036] Taken together with the current knowledge on the regulation
of ACtype1 it appears that the C1.alpha.-C1.beta. junction domain
is an important regulatory site of adenylyl cyclase activity. It is
proposed that all adenylyl cyclases are regulated by tightly
associated calcineurin bound in this region. This association may
eventually prove to be dependent on calcium ions. It is also of
note that there are potential protein kinase A, CAM kinase II and
casein kinase II phosphorylation sites in this area in most
adenylyl cyclases, which could be substrates of calcineurin and/or
may influence the avidity of association of calcineurin with the
cyclases.
[0037] The FKBP12 like domain (residues 594-611) of mouse AC has
been expressed as a Glutathione-S-transferase (GST) fusion protein
in the expression vector pGEX-2T (Pharmacia). The cyclase portion
of this fusion protein can be phosphorylated by cAMP dependent
protein kinase and dephosphorylated in a calcium dependent manner
by calcineurin. These data support the notion that the region of
the cyclase described here is a clacineurin binding site and
contains phosphorylated amino acids that are substrates for
calcineurin.
[0038] Data recently published show a remarkable similarity between
a phosphorylation site for myotonic dystrophy protein kinase on the
.beta.-subunit of the L type skeletal muscle calcium channel
(Timchenko et al, (1995) Proceedings of the National Academy of
Sciences of the United States of America 92:5366-5370) and the
FKBP-like domain of AC as shown below (wherein .circle-solid. shows
amino acid identity and .vertline. shows functionally conservative
subsitutions):
2 IDDSRESSGPR AC.sub.594-604 (SEQ ID NO:4) .vertline.
.vertline..cndot..cndot. .cndot..cndot..vertline. .vertline.
LRQSRLSSS-K calcium channel .beta.-sub- (SEQ ID NO:3)
unit.sub.176-185
[0039] In addition, it has been reported that the calcium
.beta.-subunit is selectively dephosphorylated by calcineurin (Lai
et al. (1993) Journal of Neurochemistry 61:1333-1339). It is
therefore likely that Ser.sub.600 or Ser.sub.601 in AC may be
phosphorylated by myotonic dystrophy protein kinase and
dephosphorylated by calcineurin. Abnormalities in this process may
occur in myotonic dystrophy and contribute to the symptoms of this
hereditary disorder, especially with respect to the pathological
changes of brain, muscle, heart and anterior pituitary
function.
[0040] In a yet further aspect, the present invention also provides
a diagnostic assay, to determine the presence and/or the amount of
AC within a sample, said method comprising contacting an agent
specific to AC with said sample and determining the presence and/or
amount of complex formed. The agent may be, for example, an
anti-sense oligonucleotide which is complementary to the mRNA of
AC. Alternatively, the agent may be an antibody specific to AC, or
may be calcineurin.
[0041] In a preferred embodiment the diagnostic agent is
immobilised on a support, for example a membrane. Further, it is
also preferred that a labelling moiety is present in the assay, so
that measurement of the agent/AC complex is simplified.
[0042] The present invention will now be further described with
reference to the following examples and with references to the
accompanying figures in which:
[0043] FIG. 1
[0044] Effect of FK506, cyclosporin A (CsA) and
MeVal.sup.4-cyclosporin A (MeVal.sup.4CsA) (SDZ202-384) on cAMP
accumulation induced by 10 nmol/l CRF in AtT20 cells in the
presence of 0.5 mmol/l isobutylmethylxanthine. Basal cAMP
production was 0.6.+-.0.08 pmol/well. Data are means.+-.SEM,
expressed as percentage of the increment caused by 10 nmol/l CRF
which was 6.1 pmol/well.
[0045] FIG. 2
[0046] A: Effect of cyclosporin A on the time-course of basal and
CRF-induced cAMP formation. Data are from a representative of 2
experiments. Means.+-.SEM, n=3 for CRF treated groups (o=10 nmol/l
CRF+vehicle, .circle-solid.=CRF+1 .mu.mol/l cyclosporin A) and n=1
for basal (.DELTA.=vehicle, .tangle-solidup.=cyclosporin A), 0.5
mmol/l IBM, 0.1 mM rolipram and cyclosporin A were given as
pre-treatment for 30 minutes at 37.degree. C. before the
application of 3 nM CRF for 10 minutesat 24.degree. C. *P<0.05
compared with respective control group (one way ANOVA followed by
orthogonal contrasts).
[0047] B: Dependence of the effect of 1 .mu.mol/l FK506 on
CRF-induced cAMP formation in the presence of 0.5 mM IBMX. FK506
and IBMX are given as for FIG. 2A. Data are means.+-.SEM,
n=4-6/group. (.quadrature.=FK506 group, .DELTA.=control group) *
P<0.05 compared with respective vehicle treated group (one-way
ANOVA followed by orthogonal contrasts).
[0048] FIG. 3
[0049] A: The effect of L685,818 on the enhancement of CRF-induced
cAMP formation by FK506. Data are means.+-.SEM, the cAMP level in
the presence of 10 nmol/l CRF taken as 100% was 12
pmol/well*10minutes, basal levels were 1.6 pmol/well*10minutes.
[0050] B: The effect of L685,818 on the inhibition of calcineurin
activity by 1 .mu.mol/l FK506 in AtT20 cells. Calcineurin activity
was measured by the phosphocasein method, the activity measured in
the absence of FK506 was taken as 100%.
[0051] FIG. 4
[0052] Effects of various manipulations that reduce intracellular
free calcium concentration on CRF-induced cAMP accumulation in
AtT20 cells. IBMX 0.5 mmol/l present throughout. All data are
expressed as the percentage of CRF-induced cAMP-formation,
means.+-.SEM, n=4/group.
[0053] EGTA: cells exposed to 2mmol/l EGTA in calcium free medium
during the preincubation period; nimodipine: cells exposed to 1
.mu.mol/l nimodipine during preincubation period;
[0054] BAPTA-AM: cells incubated in 20.mu.mol/l
[1,2-bis-(o-Aninophenoxy)-- ethane-N,N,N',N'-tetraacetic acid
tetra-(acetoxymethyl)-ester] during the preincubation period.
[0055] FIG. 5
[0056] Effect of FK506 on the inhibition of CRF-induced cAMP
accumulation by extracellular calcium ions in AtT20 cells. Cells
were depleted of calcium by preincubation in 2 mmol/l EGTA, calcium
free medium, containing 5 .mu.mol/l A23187 and 5 .mu.mol/l
nimodipine, and graded amounts of CaCl.sub.2 were added with 10
nmol/l CRF. The values on the abscissa give the nominal free
extracellular calcium ion concentration. Data are means.+-.SEM,
n=6/group.
[0057] FIG. 6
[0058] Effect of pertussis toxin (1 .mu.g/ml for 18 hours) on the
modulation of CRF-induced cAMP formation by FK506 and somatostatin.
AtT20 cells were preincubated with FK506 or somatostatin for 30
minutes. IBMX (0.5 mmol/l) present throughout. Data are
means.+-.SEM, n=6/group.
[0059] FIG. 7
[0060] A: FK506 (0.5 .mu.mol/l) on basal and CRF-induced ACTH
secretion in AtT20 cells.
[0061] B: Antagonism of the effect of FK506 (0.5 .mu.mol/l ) on
CRF-induced ACTH release by L685,818, which had no effect on basal
ACTH secretion in this system even at 5 .mu.mol/l. n=4/group,
means.+-.SEM.
[0062] In panel A the values for basal and CRF-stimulated ACTH
release taken as 100% were 15.+-.1 and 22.+-.1fmol/well*30 minutes,
respectively.
[0063] FIG. 8
[0064] Comparison of the sequence (SEQ ID NO: 64) amplified from
AtT20 cell RNA using primer set B, with the corresponding sequences
with other adenylyl cyclases found in current databases (EMBL,
GenBank, SwissProt (SEQ ID NOS: 48 to 64)). The numbers relate to
the amino acid sequence of rabbit ACtype5 (ocmradcyv) (SEQ ID NO:
60). The sequence is annotated by .circle-solid. showing amino acid
identity of the novel sequence with at least one previously
reported AC, .largecircle. shows funtionally conservative
substitutions, --denotes non-conservative subsitutions.
[0065] Abbreviations and sequence identification numbers
[0066] hum7--Human ACtype7 (GenBank #D25538) (SEQ ID NO: 48);
[0067] mmul2919--mouse ACtype7 (SEQ ID NO: 49);
[0068] cya2_rat--rat ACtype2 (SEQ ID NO: 50);
[0069] cya4_rat--rat ACtype4 (SEQ ID NO: 51);
[0070] hsadencyr8--Human ACtype8 (SEQ ID NO: 52);
[0071] ratacviii--rat ACtype8 (SEQ ID NO: 53);
[0072] a46187--human ACtype5 (SEQ ID NO: 54);
[0073] cya6--mouse--mouse ACtype6 (SEQ ID NO: 55);
[0074] a49201--mouse ACtype5 (now renamed as mouse AC6) (SEQ ID NO:
56);
[0075] cya6_rat--rat ACtype6 (SEQ ID NO: 57);
[0076] cya6_canfa--dog ACtype6 (SEQ ID NO: 58);
[0077] s29717--rat ACtype5 (SEQ ID NO: 59);
[0078] ocmradcyv--rabbit ACtype5 (SEQ ID NO: 60);
[0079] cya5_canfa--dog ACtype5 (SEQ ID NO: 61);
[0080] cya1_bovin--bovine ACtype1 (SEQ ID NO: 62);
[0081] cya3_rat--rat ACtype3 (SEQ ID NO: 63);
[0082] AC--AtT20 derived new sequence, ie AC (SEQ ID NO: 64).
[0083] FIG. 9
[0084] Pileup analysis of adenylyl cyclase sequences found in the
EMBL and GenBank data bases.
[0085] FIG. 10
[0086] Hydrophobicity plot of AC according to Kyte et al (1982) J
Mol Biol 157:105. The numbers show predicted intramembrane
domains.
[0087] FIG. 11
[0088] Functional analysis of cloned AC in transfected host
cells
[0089] Levels of cAMP in transiently transfected HEK293 (a-c) and
COS7 (d) in the presence of phosphodiesterase inhibitors (1 mmol/l
isobutylmethylxanthine) and 0.1 mmol/l rolipram). Data shown are
means.+-.SEM, n=4/group. Representative data from two similar
series of experiments.
[0090] (a) HEK293 cells transfected with vector cDNA and pretreated
with 0.2% (v/v) ethanol (empty columns) or 2 .mu.mol/l FK506
(striped columns) before being challenged with 0.5 nmol/l CRF.
[0091] (b) as (a) except with cells transfected with AC (Data are
means.+-.SEM, n=4/group. *P<0.05 when compared with the
respective vehicle-treated group, 1-way ANOVA, followed by
Newman-Keuls test).
[0092] (c) The effect of FK506 on CRF-induced cAMP production was
analysed further in (c). The enhancement of the CRF-induced cAMP
response by FK506 was blocked by the FK506 antagonist drug
L-685,818 (100 .mu.mol/l) (Dumont et al, (1992) J. Exp. Med.
176:751-760), which had no effect on CRF-induced cAMP formation
when given alone. It did, however, similarly to FK506, increase the
unstimulated levels of cAMP to 3-fold above basal (not shown).
*P<0.05 when compared with the vehicle treated group, 1-way
ANOVA, followed by Newman-Keuls test.
[0093] (d) Application of cyclosporin A, another calcineurin
blocking immunosuppressant enhanced the cAMP response to 1 nmol/l
CRF in COS7 cells transfected with AC (*P<0.05, when compared
with the vehicle-treated group).
[0094] FIG. 12
[0095] An FKBP-like domain in AC
[0096] (A) Schematic drawing of the predicted structure of AC,
nomenclature as suggested by Gilman and coworkers (Taussig et al,
(1995) J. Biol. Chem. 270:1-4). N=N-terminal intracellular loop; M1
and M2 =membrane spanning segments; C1.alpha. and C2.alpha. (thick
line)=the highly conserved, putative catalytic cyclase domains;
C1.beta. and C2.beta.=the non-conserved, putative regulatory
domains of the intracellular loops.
[0097] (B) AC.sub.(503-610) (SEQ ID NO: 65) shows approximately 40%
overall sequence similarity with FK506 binding-protein 12 (FKBP12)
(SEQ ID NOs: 66 to 69). Residue numbers in the top row correspond
to AC, in the bottom row to mammalian FKBP12s. A dot indicates
sequence identity between AC and at least one of the FKBPs.
Vertical lines denote conserved substitutions which were defind as
1) C 2) STPAG 3) NDEQ 4) HRK 5) MILV 6) FYW (see Krupinski et al,
(1989) Science 244:1558-1564).
[0098] yst=Saccharomyces cerevisiae, hum=human, mus=mouse,
ncr=Neurospora crassa.
[0099] (C) Alignment of the C1.alpha. and C1.beta. junction region
of known mammalian adenylyl cyclase isotypes (SEQ ID NOs: 70 to
78). The underlined region in bovACtpl (SEQ ID NO: 70) corresponds
to 24 residues of the 28 amino acid residue putative calmodulin
binding site (495-522) (Vorherr et al, (1993) Biochemistry (USA)
32:6081-6088; and Wu et al, (1993) J. Biol. Chem. 286:23766-23768),
bov=bovine. Abbreviations as for FIG. 15.
[0100] FIG. 13
[0101] Northern analysis of AtT20-cell, HEK293-cell and mouse
striatal RNA
[0102] .sup.32P-labelled cDNA probes derived from the AC sequence
were used. Note approximately 9 kb band in AtT20 cells that
hybridizes with both probes. A similar size RNA species is also
intensively hybridizing in mouse striatal RNA and a weak, barely
discernible band is found in HEK293 cells. The relative
hybridisation intensities (average of the two lanes) with probe jp
164 were: AtT20 2.1; HEK293 0.3; mouse striatum 1.4.
[0103] FIG. 14
[0104] Localisation of AC mRNA in mouse brain
[0105] AC mRNA detected by a .sup.35S-CTP labelled antisense
ribonucleic acid probe derived from plasmid JP142 in (A) the
hippocampus (pyramidal cell layer of CA1-CA4 (a) and of the
subiculum (b), granule cells in dentate gyrus (c) and in various
parts of the cereral cortex (posterior cingulate cortex indicated
by (d)). (B) .sup.35S-CTP-sense RNA probe control.
[0106] Magnification: 25.times..
[0107] FIG. 15
[0108] An alignment of adenylyl cyclases with FKBPs (SEQ ID NOs: 79
to 86).
[0109] .circle-solid. indicates identity; .vertline. indicates
conserved substitution in at least one of the FKBPs. Conserved
substitutions are defined as
[0110] 1) C
[0111] 2) STPAG
[0112] 3) NDEQ
[0113] 4) HRK
[0114] 5) MILV
[0115] 6) FYW
[0116] Abbreviations and sequence identifiers
[0117] YeastFKBP12
[0118] Yeast FKPB12 (SEQ ID NO: 79)
[0119] humFKBP12
[0120] human FKBP12 (SEQ ID NO: 80)
[0121] musFKBP12
[0122] mouse FKBP12 (SEQ ID NO: 81)
[0123] NcrFKBP12
[0124] Neurospora crassa FKBP 12 (SEQ ID NO: 83)
[0125] humFKBP13
[0126] human FKBP13 (SEQ ID NO: 84)
[0127] fkb2.sub.--
[0128] hum human FKBP13 precursor (SEQ ID NO: 85)
[0129] fkb2.sub.--
[0130] yeast FKBP13 (SEQ ID NO: 86)
[0131] bovACtp1
[0132] bovine adenylyl cyclase type 1
[0133] ratACtp3
[0134] rat adenylyl cyclase type 3
[0135] ratACtp8
[0136] rat adenylyl cyclase type 8
[0137] rutabaga
[0138] drosophila calmodulin activated adenylyl cyclase
[0139] ratACtp5
[0140] rat adenylyl cyclase type 5
[0141] ACtp5sp1
[0142] dog adenylyl cyclase type 5 splice variant
[0143] ratACtp6
[0144] rat adenylyl cyclase type 6
[0145] musAC
[0146] mouse adenylyl cyclase type 9 (AC)
[0147] ratACtp2
[0148] rat adenylyl cyclase type 2
[0149] ratACtp4
[0150] rat adenylyl cyclase type 4
[0151] musACtp7
[0152] mouse adenylyl cyclase type 7
[0153] humACtp7
[0154] human adenylyl cyclase type 7
[0155] FIG. 16
[0156] Illustrates cyclases extracted from their alignments with
FKBPs.
[0157] Abbreviations and sequence identifiers
[0158] 1. bovine adenylyl cyclase type 1 (SEQ ID NO: 87)
[0159] 2. rat adenylyl cyclase type 3 (SEQ ID NO: 88)
[0160] 3. rat adenylyl cyclase type 8 (SEQ ID NO: 89)
[0161] 4. drosophila calmodulin activated adenylyl cyclase (SEQ ID
NO: 90)
[0162] 5. rat adenylyl cyclase type 5 (SEQ ID NO: 91)
[0163] 6. dog adenylyl cyclase type 5 splice variant (SEQ ID NO:
92)
[0164] 7. rat adenylyl cyclase type 6 (SEQ ID NO: 93)
[0165] 8. mouse adenylyl cyclase type 9 ie AC (SEQ ID NO: 94)
[0166] 9. rat adenylyl cyclase type 2 (SEQ ID NO: 95)
[0167] 10. rat adenylyl cyclase type 4 (SEQ ID NO: 96)
[0168] 11. mouse adenylyl cyclase type 7 (SEQ ID NO: 97)
[0169] FIG. 17
[0170] Illustrates the relative hybridisation intensity in
different tissues, showing the distribution of AC mRNA in mouse, as
measured by RNase protection assay.
[0171] FIG. 18
[0172] Construction of complete human AC9 cDNA
[0173] Scheme showing relationship of overlapping human AC9 cDNA
clones used in construction of the complete hAC9 cDNA (GenBank/EMBL
Accession Number AJ133123) by joining clones HiBBC33, pJP192,
pJP230 and 166657 at the Mlu I, Eco RV and Nco I sites indicated as
described in materials and methods. The relationship of these
clones to human AC9 cDNA clones isolated by other groups
(Genbank/EMBL Accession Numbers AF036927 and AB011092) identified
in subsequent searches of the GenBank/EMBL DNA sequence database is
also represented.
[0174] FIG. 19
[0175] Immunocytochemical detection of human AC9 protein Cells
cultured on coverslips were briefly fixed with 3% paraformaldehyde
and reacted with affinity purified chicken antibodies directed
against residues 1340-1353 of the mouse AC9 protein, with a
tyrosine added at the N-terminus. Subsequently, rabbit anti-chicken
IgG coupled to FITC was applied, and the cells were viewed under a
Leica TCS-NT confocal microscope system. Note (a) preferential
localisation of immunoreactivity in the vicinity of the plasma
membrane and (b) dividing cell with membrane localisation of AC9.
(c) Specificity control with antibody preincubated with 10 .mu.M of
peptide antigen. The bar represents 10 .mu.m.
[0176] FIG. 20
[0177] Detection of multiple human AC9 polypeptides by
immunoblotting
[0178] Protein extracts from membranes of HEK 293 cells, either
untransfected (a & b, lane 3) or stably transfected with human
AC9 cDNA (a & b, lanes 1,2,4&5) separated by SDS-PAGE and
electroblotted onto PVDF membrane were incubated with antisera
directed against mouse AC9 N-terminus (a) or C-terminus (b).
Positions of molecular weight markers (KDa) are indicated. A major
polypeptide of 175 KDa was detected in two independent stable human
AC9 transfectants (a & b, lanes 1 & 2). In addition, a
faster migrating polypeptide was also detected. Specificity was
confirmed by competition with peptide antigen (a & b, lanes 4
& 5). Increased background staining was apparent when
C-terminal peptide was included (b, lanes 4 & 5).
[0179] FIG. 21
[0180] Analysis of cAMP synthesis by HEK293 cells overexpressing
human AC9
[0181] a) Time-course of cAMP accumulation. Blockers of cyclic
nucleotide degrading phosphodiesterases (PDE)
(isobutylmethylxanthine 1 mM, and rolipram 0.1 mM) and various
amounts of Ca.sup.2+/EGTA were applied at time 0 to cells
previously depleted of Ca.sup.2+ in HBSS/EGTA medium containing no
added Ca.sup.2+, 1 .mu.M thapsigargin and 10 .mu.M ryanodine. The
final extracellular levels of Ca.sup.2+ were (.DELTA.) nominally 0,
(o) 2 mM, (n) 5 mM. Data are means.+-.S.E.M. n=4 representative of
2 similar experiments. * P<0.05 when compared with the zero
Ca.sup.2+ group, 1-way ANOVA followed by Dunnett's test. b) Effect
of FK506 on the inhibitory effect of Ca.sup.2+ in cells pretreated
with Ca.sup.2+ free medium containing 5 .mu.M 4-Br-A23187. Blockers
of PDE were added in Ca.sup.2+/EGTA to achive 0.25 and 0.5 mM
extracellular Ca.sup.2+. Data are expressed as multiples of the
cAMP content measured in the absence of PDE blockers,
means.+-.S.E.M. n=4 representative of 3 similar experiments. *
P<0.05 when compared with respective vehicle treated controls,
+P<0.05 when compared with respective 0 Ca.sup.2+ group. c)
Pharmacological analysis of the effect of various immunosuppressant
compounds on Ca.sup.2+, inhibition of cAMP production. FK506,
cyclosporin A (CsA), and rapamycin were used at 10 .mu.M, L865,818
was present at 50 .mu.M 20 min before the application of PDE
blockers and Ca.sup.2+/EGTA. Data are expressed as multiples of the
cAMP content measured in the absence of PDE blockers,
means.+-.S.E.M. n=4 representative of 2 similar experiments. *
P<0.05 when compared with respective vehicle treated
controls.
[0182] FIG. 22
[0183] Differential expression of human AC9 mRNA
[0184] a) A probe derived from 5' end of human AC9 cDNA (see
materials and methods) detected an 8.5 kb transcript in all tissues
represented on a northern blot of mRNAs from human heart (H), brain
(B), placenta (Pl), lung (Lg), liver (Lv), skeletal muscle (M),
kidney (K) and pancreas (Pn) as well as a 6.3 kb mRNA in heart and
skeletal muscle. The control probe (b) confirmed expression of a 2
kb .beta.-actin mRNA in all tissues as well as a 1.8 kb mRNA in
heart and skeletal muscle. c) The same northern blot re-probed with
a cDNA fragment derived from the extended 3' UTR of human AC9 clone
KIAA0520 (see materials and methods) shows exclusive detection of
an 8.5 kb mRNA. Positions of molecular weight markers (kb) are
indicated.
[0185] FIG. 23
[0186] Localisation of human AC9 mRNA in human forebrain
[0187] a) Autoradiogram showing protection of 314 bases of the
single-stranded human AC9 riboprobe derived from clone pJP195 in
samples of total RNA from post-mortem human cortex, hippocampus and
striatum as well as in the HEK 293 cell positive control (as
indicated by the arrow). No protection of probe by yeast RNA (Y)
was observed. Probe present prior to single-strand specific
nuclease digestion is shown in the first lane (pr). b) In situ
hybridisation using .sup.35S UTP-labelled anti-sense (i) and sense
(ii) riboprobes derived from clone pJP195 incubated with 15 .mu.m
sections from normal adult post-mortem human brain showing
detection of human AC9 mRNA by the anti-sense probe in hippocampal
neurones of the CA1-CA3 regions (upper arrow) and dentate gyrus
(lower arrow).
[0188] FIG. 24
[0189] Comparison of AC9 nucleotide and predicted amino acid
sequences
[0190] The amino acid sequence deduced from the human AC9 cDNA
nuleotide reported (AJ133123) is highly similar to that of the
mouse AC9 protein. The polypeptide deduced from the human AC9 cDNA
sequence AF036927 is divergent from mouse, predicting an
alternative C-terminus due to a frame shift resulting from a 2 bp
deletion.
3 Abbreviations: legend on dendrogram adenylyl cyclase isoform hum7
Human type 7 GenBank #D25538; mmu12919 mouse type 7; cya2_rat rat
type 2; cya4_rat rat type 4; cya5_canfa dog type 5; cya5_rabit
rabbit type 5; s29717 rat type 5; cya6_mouse mouse type 6;
cya1_bovin bovine type 1; ratacviii rat type 8; cya3_rat rat type
3; Ac mouse AC 9, ie AC.
EXAMPLE 1
CALCINEURIN FEEDBACK INHIBITION OF AGONIST EVOKED cAMP
FORMATION
INTRODUCTION
[0191] The major immunosuppressant compounds cyclosporin A and
FK506 are potent blockers of the Ca.sup.2+/calmodulin-regulated
protein phosphatase calcineurin (protein phosphatase 2B) in
leukocytes (Liu et al, (1991) Cell 66:807-815). This observation
has led to the discovery that calcineurin is an essential element
of the signal transduction pathway activated by the T-cell receptor
(Schreiber (1992) Cell 70:365-368 and Sigal et al, (1992) Ann. Rev.
Immunol. 10:519-560). Immunophilins, which are proteins that
mediate the effects of immunosuppressants on calcineurin activity
in lymphocytes (Schreiber (1992) Cell 70:365-368) have been
identified in the brain (Steiner et al, (1992) Nature 358:584-586)
providing the plausible molecular targets of the prominent
neurological side-effects of FK506 and cyclosporin A (Frank et al,
(1993) Transplantation Proceeding 25:1887-1888 and Reyes et al,
(1990) Transplantation 50:10434-1081).
[0192] While calcineurin is highly abundant in the brain (0.5-1% of
total protein) (Klee et al, (1988) Advances in Enzymology
61:149-200) its functions in excitable cells remain to be defined.
Calcineurin has been implicated in the control of voltage regulated
ion channel activity (Armstrong, (1989) Trends in Neurosciences
12:117-122), particularly with respect to L-type calcium channels
(Lai et al, (1993) J. Neurochem. 61:1333-1339). More recent studies
have shown that the synaptic vesicular protein dynamin, which is
thought to participate in synaptic vesicle recycling in nerve
endings, is a prominent substrate for calcineurin (Liu et al,
(1994) Science 265:970-973). Furthermore, blockage of calcineurin
by immunosuppressants enhanced glutamate release by synaptosomes
prepared from rat brain and this appeared to correlate with the
state of phosphorylation of dynamin (Nichols et al, (1994) J. Biol.
Chem. 269:23817-23823).
[0193] With respect to secretory function in other systems, it has
been reported (Antoni et al, (1993) Biochem. Biophys. Res. Commun.
194:226-233) that immunosuppressants block calcineurin activity in
pituitary corticotrope tumour (AtT20) cells and stimulate
Ca.sup.2+-dependent hormone release in correlation with their
calcineurin blocking activity. Cyclic AMP (cAMP) is a cardinal
signalling molecule in pituitary corticotrophs that causes an
increase of intracellular free Ca.sup.2+([Ca.sup.2+].sub.1) and
triggers the release of adrenocorticotrophic hormone (ACTH)
(Antoni, (1986) Endocr. Rev. 7:351-378). Because [Ca.sup.2+].sub.1
is known to inhibit cAMP formation in a variety of systems (Cooper
et al, (1993) Trends Pharm. Sci. 14:34-36) we have analyzed in
AtT20 cells the effects of FK506 and cyclosporin A on cAMP
production induced by the hypothalamic neuropeptide
corticotropin-releasing factor (CRF) and beta-adrenergic
stimulation.
EXPERIMENTAL PROCEDURES
[0194] AtT20 D16:16 mouse anterior pituitary tumour cells were
maintained in culture as previously described (Woods et al, (1992)
Endocrinology 131:2873-2880). For measurements of ACTH release,
cAMP production or calcineurin activity the cells were plated on
24-well tissue culture plates (5.times.10.sup.4 cells/well), used
4-6 days afterwards. ACTH (Woods et al, (1992) Endocrinology
131:2873-2880) and CAMP (Dufau et al, (1973) Endocrinology 92:6-11)
were measured by specific radioimmunoassays. Calcineurin protein
phosphatase activity was determined by the .sup.32P-labelled casein
assay (Tallant et al, (1984) Arch. Biochem. Biophys. 232:269-279)
or by using the .sup.32P-labelled RII peptide substrate (Blumenthal
et al, (1986) J. Biol. Chem. 261:8140-8145) adapted to measure
calcineurin phosphatase activity in AtT20 cell extracts as
previously described (Antoni et al, (1993) Biochem. Biophys. Res.
Commun. 194:226-233).
[0195] Experiments for cAMP were all carried out in Hanks balanced
salt solution containing 2 mM CaCl.sub.2 and 1 mM MgSO.sub.4
buffered with 25 mM HEPES pH 7.4 and supplemented with 0.25% (w/v)
of bovine serum albumin. The cells were preincubated in serum free
medium for 1 hour, after which blockers of phosphosdiesterase
(isobutylmethylxanthine 0.5 mM (IBMX) and/or rolipram 0.1 mM) along
with various other treatments were applied for 30 minutes at
37.degree. C. Subsequently the cells were cooled to 22.degree. C.
for 5 minutes in a water bath and agonists were added for 10
minutes. The reaction was stopped by the addition of 0.2 mol/l HCl
to achieve a final concentration of 0.1 mol/l (Brooker et al,
(1979) Adv. in Cyclic Nucl. Res. Vol. 10, G. Brooker, P. Greengard
and G. A. Robinson, Raven Press, New York, 2-34). In the absence of
phosphodiesterase blockers agonist-induced changes of total cAMP
content (cells+medium) were small (2-3 fold of basal) and no
increment of intracellular cAMP could be detected (Woods et al,
(1992) Endocrinology 131:2873-2880). In the presence of IBMX total
cAMP content increased linearly with time up to 10 minutes after
the addition of CRF and remained constant for up to 20 minutes. In
contrast, cellular cAMP content peaked between 2-5 minutes and
subsequently declined to basal levels even in the presence of the
phosphodiesterase blockers. Hence, after establishing that
immunosuppressant drugs had the same effect on peak cellular and
total cAMP content under these conditions, all experiments shown
here report total cAMP content.
[0196] In some experiments cells were preincubated for 30 minutes
in medium containing 2 mmol/l EGTA and no added Ca.sup.2+,
supplemented with 5 .mu.mol/l A23187 and 2.5 .mu.mol/l nifedipine
in order to deplete rapidly mobilized cellular stores of Ca.sup.2+.
This treatment also ensured that L-type Ca.sup.2+, channels, the
principal avenue of voltage-regulated Ca.sup.2+, influx in AtT20
cells (Luini et al, (1985) Proc. Natl. Acad. Sci. U.S.A.
82:8034-8038; Reisine et al, (1987) Mol. Pharmacol. 32:488-496 and
Antoni et al, (1992) J. Endocrinol. 133:R13-R16) were fully blocked
and Ca.sup.2+ subsequently added to the extracellular fluid would
enter largely through the pores made by the ionophore A23187. The
rationale for this pretreatment is that calcineurin reportedly
influences L-channel activity (Lai et al, (1993) J. Neurochem.
61:1333-1339; and Armstrong et al, (1987) Proc. Natl. Acad. Sci.
U.S.A. 84:2518-2522), whereas the treatment regimen used here would
make Ca.sup.2+ entry independent of this regulation.
[0197] Immunosuppressant analogues (FK506, courtesy of Fujisawa
Ltd, Osaka, Japan; cyclosporin A and SDZ 220-384
(MeVal.sup.4-cyclosporin A) (Fliri, H. (1993) Antibiotics and
antiviral compounds, Chemical synthesis and modification K. Krohn,
H. Kirst And H. Maasg, VCN Verlagsgesellschaft mbH, Weinheim,
229-240), courtesy of Sandoz Pharma, Basel, Switzerland, L685,818
(Dumont et al, (1992) J. Exp. Med. 176:751-760) courtesy of
Merck&Co, Rahway, NJ) were also applied during the
preincubation period. These compounds were made up in ethanol at
10.sup.-3 mol/l and diluted with the incubation medium to the
desired final concentrations. In some cases cells were preincubated
with L685,818 the structural analogue of FK506 that binds to
FKBP-12 and inhibits prolyl-isomerase activity but is devoid of
immunosuppressant activity (Dumont et al, (1992) J. Exp. Med.
176:751-760) for 10 minutes before the addition of FK506 for 30
minutes at 37.degree. C.
[0198] Incubations for ACTH secretion were carried out as
previously described (Woods et al, (1992) Endocrinology
131:2873-2880) except that the test incubation with CRF was at
22.degree. C.
Amplification and DNA Sequence Analysis of Adenylyl Cyclases cDNAs
in AtT20 Cells
[0199] Total RNA was prepared from approximately 10.sup.7 cells
using Trizol reagent (GIBCO, Paisley, UK) according to the
manufacturer's instructions. RT-PCR was carried out using an RNA
PCR kit (Perkin Elmer, Warrington, Cheshire, U.K.). Briefly, 0.8
.mu.g of total RNA was denatured at 95.degree. C. for 5 minutes
then annealed with 2.5 .mu.M random hexanucleotide primers for
first-strand cDNA synthesis which was carried out for 15 minutes at
42.degree. C. in a 20 .mu.l reaction mixture containing 10 mM
Tris.HCl, pH8.3, 50 mM KCl, 5 mM MgCl.sub.2, 1 mM each dNTP, 20 U
RNase inhibitor and 50 U MMLV reverse transcriptase. The reaction
was terminated at 99.degree. C. for 5 minutes then cooled to
4.degree. C. and stored on ice. PCR was performed using degenerate
oligonucleotides, either pair A or pair B corresponding to highly
conserved regions within the first (pair A: 5'
CTCATCGATGGIGAYTGYTAYTAYTG- 3' (SEQ ID NO: 5); 3'
GGCTCGAGCCAIACRTCRTAYTGCCA5' (SEQ ID NO: 6) expected product size
220 bp) and second (pair B:
[0200] 5'
GAAGCTTAARATIAARACIATIGGI.sup.T/.sub.A.sup.C/.sub.GIACITAYATGGC3- '
(SEQ ID NO: 7); 3' GGGATCCACRTTIACIGTRTTICCCCAIATRTCRTA5' (SEQ ID
NO: 8) expected product size 180 bp) cytoplasmic domains of
previously cloned mammalian adenylyl cyclases (Yoshimura et al,
(1992) Proc. Natl. Acad. Sci. U.S.A. 89:6716-6720; Krupinski et al,
(1992) J. Biol. Chem. 267:24859-24862; and Gao et al, (1991) Proc.
Natl. Acad. Sci. U.S.A. 88:10178-10182). For PCR the reverse
transcription reaction (20 .mu.l) was expanded to 100 .mu.l and
contained 10 mM Tris.HCl, pH 8.3, 50 mM KCl, 2 mM MgCl.sub.2, 200
.mu.M each dNTP, 35 pmol of each primer and 2.5 U Amplitaq DNA
polymerase. PCR reactions were overlaid with mineral oil (Sigma,
Poole, Dorset, U.K.) and denatured at 95.degree. C. for 3 minutes
followed by 5 cycles (60 seconds denaturation at 94.degree. C., 60
seconds annealing/extension at 45.degree. C.) then a further 35
cycles (60 seconds denaturation at 94.degree. C., 60 seconds
annealing/extension at 55.degree. C.) and finally 7 minutes
annealing/extension at 55.degree. C. An aliquot (5%) of each
reaction was analysed by agarose gel electrophoresis (3% FMC,
Flowgen Instruments Ltd, Sittingbourne, Kent, U.K.). Products
within the expected size range for each primer pair were excised
from the gel, purified using a Wizard.TM. PCR Prep kit (Promega,
Madison, Wis., U.S.A.) and ligated into the vector pGEM-T
(Promega). Clones containing an insert of the expected size were
identified and their DNA sequence determined by the
dideoxynucleotide method (Sequenase 2.0 kit, USB, Amersham
International, Aylesbury, U.K.).
Detection of mRNA Expression
[0201] Northern analysis was performed using standard procedures.
Briefly, 10 .mu.g of total RNA was separated by formaldehyde gel
electrophoresis and transferred by blotting onto positively charged
nylon membrane (Appligene,) then fixed by baking at 80.degree. C.
and prehybridised at 42.degree. C. for 2 hours in 50% deionised
formamide, 5.times.SSPE, 0.5.times.Denhardt's, 0.1% w/v SDS, 0.2
mg/ml denatured salmon sperm carrier DNA and 10% Dextran sulphate.
Random-primed labelled DNA probe (50 ng; >10.sup.9 cpm/.mu.g)
was then added and hybridisation continued overnight at 42.degree.
C. The membrane was washed twice for 20 minutes in 2.times.SSC/0.1%
SDS, followed by 20 minutes in 1.times.SSC/0.1% SDS at 42.degree.
C. and finally 20 minutes in 0.5.times.SSC/0.1% SDS at 50.degree.
C. before wrapping in cling-film an exposing to autoradiographic
film at -70.degree. C. Ribonuclease protection assays were
performed using an RPA II kit (Ambion, AMS Biotechnology, Witney,
Oxon, U.K.) according to the manufacturer's instructions. Briefly,
10 .mu.g of total RNA was hybridised overnight at 450.degree.C. to
10.sup.5 cpm of radiolabelled anti-sense riboprobe. Following
hybridisation reactions were digested with single-strand-specific
RNase and protected fragments resolved on a 6% denaturing
polyacrylamide gel which was fixed for 30 minutes in 15%
methanol/5% acetic acid, dried and exposed to autoradiographic film
at -70.degree. C.
RESULTS
Enhancement of CRF-stimulated cAMP Production by
Immunosuppressants
[0202] Immunosuppressant blockers of calcineurin activity, FK506,
cyclosporin A and MeVal.sup.4-cyclosporin A enhanced CRF-induced
cAMP production in a concentration dependent manner (FIG. 1). The
effect of cyclosporin A (FIG. 2) was statistically significant
(P<0.05 or less) at 2, 5 and 10 minutes after the addition of
CRF, similar data were also obtained with FK506. Enhancement of
cAMP formation by FK506 was apparent at lower concentrations
(0.1-10 nmol/l) of CRF, while the maximal response appeared
unchanged (FIG. 2B).
[0203] The effect of FK506 on CRF-induced cAMP production (FIG. 3)
could be antagonized by the FK506 analog L685,818, a potent
inhibitor FKBP-12 prolyl isomerase activity which has no
immunosuppressant activity and does not block calcineurin (Dumont
et al, (1992) J. Exp. Med. 176:751-760) and hence, is a specific
antagonist of the calcineurin inhibitory action of FK506.
Importantly, this compound also blocked the inhibitory effect of
FK506 on calcineurin-mediated dephosphorylation of phosphocasein
(FIG. 3) in AtT20 cells.
Receptor-evoked Synthesis of cAMP is Under Inhibitory Control by
Intracellular Ca.sup.2+ and Calcineurin
[0204] Lowering of intracellular free Ca.sup.2+ by a variety of
methods, such as depletion by EGTA and the calcium ionophore
A23187, loading of the cells with the intracellular calcium
chelator BAPTA-AM and blockage of calcium channels with the
dihydropyridine channel blocker nimodipine, all markedly increased
the cAMP response to CRF (FIG. 4). The effect of BAPTA-AM on
CRF-induced cAMP formation was statistically significant
(P<0.05) by 2 minutes after the addition of CRF and at all
subsequent time-points studied up to 20 minutes (not shown).
[0205] Addition of graded amounts of Ca.sup.2+, with CRF to cells
depleted of Ca.sup.2+ and pretreated with the ionophore A23187
produced a concentration-dependent inhibition of CRF-induced cAMP
production to levels seen in non-depleted cells incubated in medium
containing 2 mmol/l Ca.sup.2+. The effect of exogenous Ca.sup.2+
could be inhibited by FK506, which, in fact failed to alter cAMP
accumulation in the absence of Ca.sup.2+ (FIG. 5).
Site and Specificity of Immunosuppressant Action
[0206] The effect of FK506 on CRF-induced cAMP formation was also
evident after a 16 hours pretreatment of the cells with pertussis
toxin (1 .mu.g/ml) (FIG. 6) which strongly suppressed inhibitory
G-protein function as assessed by the attenuation of
somatostatin-mediated inhibition of cAMP formation. Pertussis toxin
treatment also had no effect on the suppression of CRF-induced cAMP
formation by extracelluar Ca.sup.2+ in Ca.sup.2+-depleted cells
(not shown).
[0207] FK506 had no significant effect on cAMP accumulation evoked
by 10 or 30 .mu.M forskolin, a drug that activates adenylyl cyclase
independently of Gs. Loading of the cells with BAPTA-AM caused a
small (15%), but statistically significant (P<0.05) enhancement
of forskolin-evoked cAMP accumulation (not shown).
[0208] Finally, in contrast to the effects of FK506 and cyclosporin
A, pretreatment with other blockers of protein phosphatases such as
calyculi A (1-30 nmol/l) okadaic acid (0.2-5 .mu.mol/l), caused a
concentration-dependent inhibition (up to 80%) of CRF-induced cAMP
accumulation (not shown and Koch et al, (1994) Cellular Signalling
6:467-473).
Enhancement of CRF Stimulated ACTH Release by FK506
[0209] Blockage of calcineurin activity by FK506 enhanced the
release of ACTH evoked by CRF (FIG. 7A), and this action was
prevented by L685,818 (FIG. 7B). Note, that the apparent EC.sub.50s
of FK506 to inhibit calcineurin activity in AtT20 cells (Antoni,
(1986) Endocr. Rev. 7:351-378), to stimulate ACTH release and to
augment cAMP accumulation induced by CRF are all approximately 10
nM.
.beta.-adrenergic stimulation is under similar regulation by
calcineurin
[0210] Isoproterenol induces cAMP accumulation through .beta..sub.2
adrenergic receptors in AtT20 cells (Heisler et al, (1983)
Biochemical And Biophysical Research Communications 111:112-119).
Isoproterenol induced cAMP formation was also enhanced by BAPTA-AM,
FK506 and Ca.sup.2+-depletion in AtT20 cells (Table 1).
Effect of Immunosuppressants on cAMP Accumulation in AtT20 Cells
Correlates with the Expression of a Novel Adenylyl Cyclase mRNA
[0211] In order to determine the profile of adenylyl cyclase
isoforms present in AtT20 cells two sets of degenerate
oligonucleotide primers were used to analyze AtT20 cell total RNA
for adenylyl cyclase related sequences by means of RT-PCR. Using
primer set B a PCR product of approximately 180 bp was obtained.
DNA sequence analysis revealed that approximately 8% of the
subcloned 180 bp cDNA fragments amplified proved to be identical to
Type 6 adenylyl cyclase. The majority (>90%), however, gave a
novel sequence exhibiting a high level of homology with the amino
acid sequences of known mammalian adenylyl cyclases present within
current databases but which was not identical to any previously
reported sequence (FIG. 8). Type 1 adenylyl cyclase was detected in
AtT20 cells using primer set A.
[0212] Northern blot analysis of total RNA using the novel adenylyl
cyclase 180 bp cDNA fragment as a probe indicated hybridisation to
an approximately 9 kb mRNA expressed in AtT20 cells. Expression of
this message was not detected in NCB20 or HEK293 cell RNA.
[0213] As a more sensitive alternative mRNA expression was also
assayed by ribonuclease protection of the message using a
radiolabelled anti-sense riboprobe transcribed from the novel
adenylyl cyclase 180 bp cDNA. The presence of a
ribonuclease-protected fragment migrating as a distinct,
approximately 155 bp band on a denaturing polyacrylamide gel
indicates that the novel adenylyl cyclase mRNA is highly abundant
in AtT20 cells whereas only very low levels are present in NCB20
cells and in HEK293 cells the message remains undetected.
[0214] Measurements of calcineurin activity in cell extracts from
all three cell lines revealed a similar sensitivity of calcineurin
protein phosphatase activity (substrate RII subunit peptide Fruman
et al, (1992) Proc. Natl. Acad. Sci. U.S.A. 89:3686-3690) towards
inhibition by FK506 and cyclosporin A. Furthermore, all three cell
lines responded to stimulation by CRF which was enhanced by
depletion of intracellular free calcium. FK506 and cyclosporin A
enhanced CRF-induced cAMP formation consistently in AtT20 cells; in
NCB20 cells only one out of 4 experiments gave a statistically
significant effect of cyclosporin A on CRF-induced cAMP
accumulation and no effects were observed in HEK293 cells.
DISCUSSION
[0215] These data show that receptor-stimulated cAMP formation may
be regulated by calcineurin, and that this regulation is associated
with the expression of a novel adenylyl cyclase mRNA.
[0216] All studies of cAMP formation reported here were carried out
in the presence of blocker(s) of phosphodiesterase (IBMX and/or
rolipram), and hence the effects observed relate to the synthesis
of cAMP rather than its degradation.
[0217] Evidence for the involvement of calcineurin in the control
of cAMP accumulation is provided by the use of immunosuppressant
analogs previously (Antoni et al, (1993) Biochem. Biophys. Res.
Commun. 194:226-233; and Fliri (1993) Antibiotics and antiviral
compounds, Chemical synthesis and modification K. Krohn, H. Kirst
And H. Maasg, VCN Verlagsgesellschaft mbH, Weinheim, 229-240) shown
to block calcineurin activity in AtT20 cells and T lymphocytes
cells with the same order of potency that they influenced cAMP
accumulation (present study). Furthermore, L685,818 an analogue of
FK506 (Dumont et al, (1992) J. Exp. Med. 176:751-760) that binds to
the prolyl-isomerase FKBP-12 in a manner similar to FK506, but does
not give rise to a drug protein complex that inhibits the activity
of calcineurin, reversed the effects of FK506 on calcineurin
activity, cAMP formation as well as ACTH release. Importantly, when
given alone L685,818 had no discernible effect on cAMP formation or
ACTH secretion further suggesting that the changes observed upon
treatment with FK506 are due to the inhibition of calcineurin.
Finally, neither FK506 nor cyclosporin A were effective in cells
depleted of Ca.sup.2+.
[0218] Taken together, these characteristics justify the conclusion
that the effects of immunosuppressants described here are
attributable to the inhibition of calcineurin.
[0219] The production of cAMP in AtT20 cells is under inhibitory
control by [Ca.sup.2+].sub.1. Stimulation with cAMP is known to
elicit a rise of [Ca.sup.2+].sub.1 in these cells which is largely
derived from the extracellular pool by influx through
dihydropyridine sensitive Ca.sup.2+-channels (Luini et al, (1985)
Proc. Natl. Acad. Sci. U.S.A. 82:8034-8038; Reisine et al, (1987)
Mol. Pharmacol. 32:488-496 and Antoni et al, (1992) J. Endocrinol.
133:R13-R16), as intracellular pools of Ca.sup.2+ are sparse
(Fiekers (1993) Abstracts of the 23rd Annual Meeting of the Society
for Neuroscience 1186 Abst 488.3). Thus the [Ca.sup.2+].sub.1
signal is a measure of electrical activity of the cell and in
addition to triggering hormone release provides feedback inhibition
to the chemical messenger system that generates it. In the case of
CRF-induced cAMP formation this feedback is largely mediated by
calcineurin.
[0220] Several possibilities have to be considered with respect to
the site of action of Ca.sup.2+/calcineurin in the signal
transduction cascade.
[0221] An action of calcineurin at the receptor level is
conceivable, however, the prevailing concept of G-protein coupled
receptors (Sibley et al, (1987) Cell 48:913-922) dictates that
receptor-down regulation or uncoupling is largely due to the action
of protein kinases while protein phosphatases reverse this process.
In contrast, the present data implicate calcineurin as an inhibitor
of receptor stimulated cAMP production.
[0222] Dephosphorylation of the coupling protein Gs is also a
possible site of regulation by calcineurin (Houslay (1994) GTPases
in Biology B. F. Dickey and L. Birnbaumer, Springer Verlag, Berlin,
Vol 108 Pt2,147-165). Once more, current evidence in the literature
associates protein phosphorylation with down-regulation of
G-protein function and implicates protein phosphatases in the
restoration of the cellular response (Pyne et al, (1992) Biochem.
Biophys. Res. Commun. 186:1081-1086; and Strassheim et al, (1994)
J. Biol. Chem. 269:14307-14313).
[0223] With respect to the effector adenylyl cyclase, these
proteins have lately emerged as dynamic sites of signal integration
(Taussig et al, (1995) J. Biol. Chem. 270:1-4). At least two types
of cyclase, Types 5 & 6 (Iyengar (1993) Advances in Second
Messenger and Phosphoprotein Research B. L. Brown and P. R. M.
Dobson, Raven Press Ltd, New York, 28:27-36), are inhibited by
Ca.sup.2+ but the mechanism of this effect has not been elucidated
(Yoshimura et al, (1992) Proc. Natl. Acad. Sci. U.S.A.
89:6716-6720). The inhibition of Type 5 and 6 cyclase by Ca.sup.2+
is most marked after stimulation by agonists such as isoproterenol
in chick heart cells (Yu et al, (1993) Mol. Pharmacol. 44:689-693),
or VIP in GH.sub.4C.sub.1 pituitary tumour cells (Boyajian et al,
(1990) Cell Calcium 11:299-307), but much less prominent after
activation with forskolin in GH.sub.4C.sub.1 cells (Boyajian et al,
(1990) Cell Calcium 11:299-307). Overall this is analogous to the
observations made here, which in the first instance suggest a
prominent action of calcineurin at or before the level of G-protein
effector coupling. However, multiple types of adenylyl cyclase
coexist in all cell types analyzed to date (Yoshimura et al, (1992)
Proc. Natl. Acad. Sci. U.S.A. 89:6716-6720; DeBernardini et al,
(1993) Biochem. J. 293:325-328 and Hellevuo et al, (1993) Biochem.
Biophys. Res. Commun. 192:311-318) and this applies to AtT20 cells
as RT-PCR analysis and sequencing of the amplified cDNAs clearly
showed coexpression of at least three types of adenylyl cyclase
mRNA (type 1 and 6 as well as a novel isotype). Forskolin appears
to activate adenylyl cyclase isotypes by different efficacies and
mechanisms (Iyengar (1993) Advances in Second Messenger and
Phosphoprotein Research B. L. Brown and P. R. M. Dobson, Raven
Press Ltd, New York, 28:27-36), hence the relative contribution of
individual cyclase isotypes to forskolin-induced cAMP may be
different from receptor activated cAMP formation, and as a result
forskolin-induced cAMP synthesis and receptor activated synthesis
may have different pharamcological profiles.
[0224] It is unlikely that type 1 cyclase is involved in the
effects of Ca.sup.2+ and calcineurin reported here as it is
invariably stimulated by Ca.sup.2+, whereas the predominant effect
on CRF and .beta.-adrenergic stimulation was an inhibition by
Ca.sup.2+. Type 6 adenylyl cyclase could be implicated because it
is strongly inhibited by Ca.sup.2+, however, although the Type 6
isozyme is abundant in NCB20 (Yoshimura et al, (1992) Proc. Natl.
Acad. Sci. U.S.A. 89:6716-6720) as well as HEK293 cells (Hellevuo
et al, (1993) Biochem. Biophys. Res. Commun. 192:311-318) the
stimulation of cAMP accumulation in these cells through
endogenously expressed receptors for CRF was not altered upon
blockage of calcineurin protein phosphatase activity. Importantly,
the novel adenylyl cyclase homologue mRNA was undetectable in
HEK293 cells and was found in very low amounts in NCB20 cells,
whereas it appears to be the predominant adenylyl cyclase isotype
mRNA in AtT20 cells. Work reported elsewhere (Paterson et al,
(1995) Biochem. Biophys. Res. Commun. 214:1000-1008) reports
isolation of a 4473 bp cDNA from AtT20 cells which contains the
complete coding sequence of this novel mouse adenylyl cyclase.
[0225] Taken together, these data suggest that the effects of
calcineurin inhibitors are associated with specific adenylyl
cyclase isotype previously not characterized.
[0226] The potential functional significance of calcineurin
negative feedback on cAMP formation in pituitary cells is
highlighted by previous findings, showing that adrenal
corticosteroid inhibition of CRF-induced ACTH release involves the
de novo synthesis of calmodulin, the calcium sensor regulatory
protein of calcineurin (Shipston et al, (1992) Biochem. Biophys.
Res. Commun. 189:1382-1388). Furthermore, the efficiency of
corticosteroid inhibition is reduced by approximately 10-fold by
immunosuppressant blockers of calcineurin (Shipston et al, (1994)
Ann. N. Y. Acad. Sci. 746:453-456; and Antoni et al, (1994) J.
Physiol-London 475:137-138).
[0227] In summary, calcineurin is a Ca.sup.2+-operated feedback
inhibitor of cAMP production activated by CRF or .beta.-adrenergic
receptors in pituitary cortictrope tumour cells. As
[Ca.sup.2+].sub.1 is largely derived through voltage-regulated
Ca.sup.2+, channels in AtT20 cells, these cells exemplify a case
where calcineurin functions as a link between the chemical and
electrical signalling systems of the cell. These findings conform
with previous reports (Armstrong (1989) Trends in Neurosciences
12:117-122; and Nichols et al, (1994) J. Biol. Chem.
269:23817-23823) suggesting that in excitable cells calcineurin is
a fundamental negative feedback regulator of cellular responses as
opposed to its role as a stimulatory element in non-excitable
systems (Kincaid et al, (1993) Adv. Prot. Phosphatases 7:543-583).
Furthermore, our findings extend the involvement of calcineurin
from calcium channels and synaptic vesicle recycling to the
adenylyl cyclase signalling pathway that is fundamental for
intracellular signalling throughout the CNS.
SUMMARY
[0228] The Ca.sup.2+- and calmodulin-activated protein phosphatase
calcineurin is highly abundant in the brain. While a physiological
role for calcineurin has been delineated in T lymphocyte
activation, little is known about its role in excitable cells. The
present study investigated the effects of immunosuppressant
blockers of calcineurin on agonist-induced cAMP formation and
hormone release by mouse pituitary tumour (AtT20) cells. Inhibition
of calcineurin with FK506 or cyclosporin A enhanced cAMP formation
and adrenocorticotropin secretion induced by corticotropin
releasing-factor (CRF). Further analysis of cAMP production
revealed that intracellular Ca.sup.2+ derived through
voltage-regulated calcium channels reduces cAMP formation induced
by corticotropin releasing-factor or .beta..sub.2-adrenergic
stimulation, and that this effect of Ca.sup.2+ is mediated by
calcineurin. Analysis of AtT20 cell RNA indicated the co-expression
of at least three species of adenylyl cyclase mRNA encoding types 1
and 6 as well as a novel isotype, which appeared to be the
predominant species. In two cell lines expressing very low or
undetectable levels of the novel cyclase mRNA (NCB20 and HEK293
respectively), CRF-induced cAMP formation was not altered by FK506
or cyclosporin A. In summary, these data identify calcineurin as a
Ca.sup.2+ sensor that mediates a negative feedback effect of
intracellular Ca.sup.2+ on receptor-stimulated cAMP production
thereby regulating the cellular response to cAMP generating
agonists. Furthermore, the effect of calcineurin on cAMP synthesis
appears to be associated with the expression of a novel adenylyl
cyclase isoform, which is highly abundant in AtT20 cells.
EXAMPLE 2
DNA CLONING AND TISSUE DISTRIBUTION OF AC
INTRODUCTION
[0229] Adenylyl cyclases convert ATP to cAMP, one of the earliest
recognized intracellular messenger molecules. The family of
previously known adenylyl cyclases consists of eight members
(Premont (1994) Meth. Enzymol. 238:116-127). Some of these enzymes
have been analyzed functionally, and appear to confer unique signal
processing capacities to cells (Taussig et al, (1995) J. Biol.
Chem. 270:1-4). In particular isotype specific regulation of
enzymatic activity by calmodulin, calcium (through as well as
independently of calmodulin), protein kinase C as well as G-protein
subunits has been demonstrated (for review see Taussig et al,
(1995) J. Biol. Chem. 270:1-4). In addition to functional diversity
adenylyl cyclase isotypes have distinct tissue distribution
profiles (Krupinski et al, (1992) J. Biol. Chem. 267:24859-24862)
and particularly marked regional differences of cyclase
distribution have been observed in the brain (Xia et al, (1994)
Mol. Brain Res. 22:236-244; and Glatt et al, (1993) Nature
361:536-538). Collectively, these observations indicate that the
particular adenylyl cyclase isotype profile of a cell is
fundamentally important with respect to cellular function.
[0230] Pharmacological analysis of the cAMP response to agonist
stimulation in mouse corticotroph tumor (AtT20) cells (Antoni et
al, (1994) Journal Of Physiology-London 475p:137-138) has shown
that calcium inhibition of cAMP accumulation in these cells is
mediated by the Ca.sup.2+/calmodulin activated protein phosphatase
calcineurin (protein phosphatase 2B ). This novel feature of the
cAMP response has led to the examination of the adenylyl cyclase
isotype profile of AtT20 cells (Antoni et al, (1995) EMBO Journal
submitted), which, amongst two known sequences (types 1 and 6),
revealed the presence of a novel isotype. The present study reports
the full cDNA sequence and tissue distribution of this novel
adenylyl cyclase which is the predominant species of the enzyme
expressed in AtT20 cells. The enzyme mRNA is also relatively
abundant in the brain as well as certain peripheral endocrine
organs, including the ovary and the adrenal gland.
MATERIALS AND METHODS
Isolation of a cDNA Containing the Complete Coding Sequence of a
Novel Mouse Adenylyl Cyclase Identified in AtT20 Cells
[0231] AtT20 cells were grown to sub-confluency in DMEM
supplemented with 10% foetal calf serum (Woods et al, (1992)
Endocrinology 131:2873-2880) and total RNA was isolated from
approximately 10.sup.7 cells using Trizol reagent (GIBCO, Paisley,
U.K.) according to the manufacturer's instructions. RT-PCR was
carried out using an RNA PCR kit (Perkin Elmer, Warrington,
Cheshire, U.K.) as previously described (Antoni et al, (1995) EMBO
Journal submitted), using degenerate oligonucleotides corresponding
to a highly conserved region within the second cytoplasmic domain
of previously cloned mammalian adenylyl cyclases (Krupinski et al,
(1992) J. Biol. Chem. 267:24859-24862; Yoshimura et al, (1992)
Proc. Natl. Acad. Sci. U.S.A. 89:6716-6720; and Gao et al, (1991)
Proc. Natl. Acad. Sci. U.S.A. 88:10178-10182) A 180 bp cDNA
fragment of a novel adenylyl cyclase amplified from AtT20 cells
(Antoni et al, (1995) EMBO Journal submitted) was used as a probe
to obtain a cDNA containing the complete coding sequence by
screening an oligo dT-primed cDNA library prepared from AtT20 cells
and constructed in the vector ZAP II (a generous gift of Dr. M J
Shipston, Edinburgh (see Shipston (1992) PhD Thesis, University of
Edinburgh)) together with 5' RACE PCR. Screening of approximately
5.times.10.sup.5 clones was carried out according to standard
procedures (Sambrook et al, (1989) Cold Spring Harbor Press, USA).
Plaque-purified positive clones were recovered as pBluescript
phagemids by excision using the helper phage ExAssist (Stratagene,
Cambridge, U.K.). The insert size of these clones ranged from 1-3
kb. DNA sequence was determined from several independent isolates
of each clone using Exo III/Mung Bean nuclease digestion and/or
clone-specific primers with the Sequenase 2.0 kit (USB, Amersham
International, Aylesbury, U.K.). Analysis of the DNA sequence data
generated revealed the presence of an open frame which lacked a
suitable initiator codon.
[0232] Two rounds of 5' RACE-PCR were performed to obtain the
remaining of the open reading frame using a 5' RACE System kit
(GIBCO, Paisley, U.K.) according to the manufacturer's instructions
and summarised in brief as follows. Based on the DNA sequence at
the 5' end of the largest cDNA clone isolated by library screening
(jp134) a set of three nested anti-sense oligonucleotides were
designed. The first of these (most proximal to the 3' end of the
cDNA isolated; primer 1: CGTCAATGACCTCAAAGCC (SEQ ID NO: 9)) was
used to prime synthesis of first strand cDNA by reverse
transcription of 0.8 .mu.g of total RNA isolated from AtT20 cells
in a 25 .mu.l reaction containing 20 mM Tris.HCl (pH 8.4), 50 mM
KCl, 3 mM MgCl.sub.2, 10 mM DTT, 100 nM primer 1, 400 .mu.M each
dNTP and 200 U Superscript II Reverse Transcriptase at 42.degree.
C. for 30 minutes. First strand cDNA was purified away from this
primer using the GlassMax purification system (GIBCO) and one-fifth
of the purified cDNA 3'-tailed with dCTP using 10 U terminal
deoxynucleotide transferase (TdT) in a 25 .mu.l reaction containing
20 mM Tris.HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl.sub.2, 200 .mu.M
dCTP at 37.degree. C. for 15 minutes. Following heat inactivation
of the TdT a second strand cDNA synthesis reaction was primed by
the anchor primer supplied with the 5' RACE system kit present at
200 nM and carried out as for first strand synthesis.
Double-stranded cDNA was subsequently purified using the GlassMax
system and one-fifth of this preparation used as a template in PCR
containing 20 mM Tris.HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl.sub.2,
200 mM each dNTP, 200 nM each of universal amplification primer
(UAP; supplied with the kit) and jp134-specific nested anti-sense
primer 2 (SEQ ID NO: 10) (GCCTCTGCACAGCTGCAGTGGGACTCC) and 0.03
U/.mu.l Amplitaq DNA polymerase (Perkin Elmer) (parameters for all
the PCR reactions can be found in Table II). A second round of PCR
using 0.05% of the primary PCR reaction (or 0.05% of size-selected
primary PCR products) as a template was performed as described
above except jp134-specific nested anti-sense primer 3 (SEQ ID NO:
11) (CCTGGCAGAACTGCTCGATGGCTTTTATCATGC) was substituted for primer
2. This first round of 5' RACE-PCR yielded a product of
approximately 1 kb which was purified from an agarose gel and
ligated into the plasmid vector pGEM-T (Promega, Madison,
Wis.,U.S.A.). Although DNA sequence analysis of this fragment (from
both strands of two independent clones) extended the open reading
frame observed in jp134 by 842 bp no suitable initiator codon was
yet apparent. Another set of three anti-sense primers was designed
on the basis of sequence data from the 5' end of the 1 kb fragment
obtained from the first round and used in a second round of 5'
RACE-PCR performed as described above except that 1) the first
strand cDNA was synthesized at 50.degree. C. for 30 minutes and 2)
substitution of primers 1, 2 and 3 with 4 (SEQ ID NO: 12)
(GGAGAAGCTTCCTACTTG), 5 (SEQ ID NO: 13)
(GTGGCCGTGAGAGTATGATTGGAGCTGTC) and 6 (SEQ ID NO: 14)
(GTCCAAACCTGAAACTGCGCACGCAG), respectively. This yielded a product
of approximately 650 bp which, when cloned into PGEM-T and several
independent isolates sequenced, completed the open reading frame
encoding the novel mouse adenylyl cyclase.
Detection of mRNA Expression
[0233] Northern analysis was performed using standard procedures.
Briefly, 20 .mu.g of total RNA was separated by formaldehyde gel
electrophoresis and transferred by blotting onto positively charged
nylon membrane (Appligene, Ullkirch, France) then fixed by baking
at 80.degree. C. and prehybridised at 42.degree. C. for 2 hours in
50% deionised formamide, 5.times.SSPE, 0.5.times.Denhardt's, 0.1%
w/v SDS, 0.2 mg/ml denatured salmon sperm carrier DNA and 10%
Dextran sulphate. Random-primed labelled DNA probe (50 ng;
>10.sup.9 cpm/.mu.g) was then added and hybridisation continued
overnight at 420.degree. C. The membrane was washed twice for 20
minutes in 2.times.SSC/0.1% SDS, followed by 20 minutes in
1.times.SSC/0.1% SDS at 42.degree. C. and finally 20 minutes in
0.5.times.SSC/0.1% SDS at 50.degree. C. before wrapping in plastic
an exposing to autoradiographic film at -70.degree. C. Ribonuclease
protection assays were performed using an RPA II kit (Ambion, AMS
Biotechnology, Witney, Oxon, U.K.) according to the manufacturer's
instructions. Briefly, 10 .mu.g of total RNA was hybridised
overnight at 45.degree. C. to 10.sup.5 cpm of radiolabelled
anti-sense riboprobe. Following hybridisation, reactions were
digested with single-strand-specific RNase and protected fragments
resolved on a 6% denaturing polyacrylamide gel which was fixed for
30 minutes in 15% methanol/5% acetic acid, dried and exposed to
autoradiographic film at -70.degree. C.
RESULTS AND DISCUSSION
Analysis of the cDNA and Amino Acid Sequence
[0234] The sequence of the full length cDNA isolated from AtT20
cells (sense strand on top) and the deduced primary structure of AC
are shown in SEQ ID No 1. A 4473 bp cDNA containing a 4062 bp open
reading frame which encodes a 1352 amino acid protein. A stretch of
10 amino acids near the 5' end of the cDNA strongly resembles the
Kozak consensus sequence for initiation of translation and contains
the presumed initiator codon of the novel adenylyl cyclase protein.
The presence of a translational stop codon upstream of this
methionine residue suggests that the complete open reading frame
encoding this protein has been cloned. The deduced amino acid
sequence between residues 1105 and 1192 appears to be identical
(except for a single asparagine to aspartic acid switch at 1191) to
a short sequence derived from mouse brain RNA reported in
preliminary form (Premont (1994) Meth. Enzymol. 238:116-127) and
designated as AC. Although this region of the enzyme contains at
least two highly preserved stretches of amino acid sequence common
to all cyclases, on the basis of the abundance of AC mRNA in the
brain (see below) it seems plausible that the full sequence
reported here corresponds to the same enzyme and will subsequently
referred to as AC.
[0235] While the key adenylyl cyclase consensus sequences thought
to be involved in catalytic activity (Taussig et al, (1995) J.
Biol. Chem. 270:1-4) are well conserved in AC, overall amino acid
sequence homology comparisons indicate that AC is not sufficiently
similar to currently known cyclases to be placed within one of the
previously suggested subfamilies as illustrated by FIG. 9.
Therefore AC represents a sixth adenylyl cyclase subfamily. It
cannot be excluded at present that perhaps other members of this
subfamily exist.
[0236] Hydrophobicity plots (Kyte et al, (1982) J. Mol. Biol.
157:105) return the previously reported adenylyl cyclase structure
(FIG. 10) a short N-terminal loop followed by two homologous
segments each consisting of a hydrophobic chain with 6
intermembrane helices and a large hydrophilic (putative
cytoplasmic) loop. The first cytoplasmic loop appears considerably
longer (by about 130 amino acids) in AC than previously reported
for other isotypes.
[0237] Analysis of putative phosphorylation sites (Pearson et al,
(1991) Meth. Enzymol. 200:61-81) indicates several strong consensus
protein kinase A and protein kinase C sites as well as two casein
kinase phosphorylation sites (Table 3). Elements of the
functionally relevant calmodulin binding site identified in ACtype1
(Wu et al, (1993) J. Biol. Chem. 286:23766-23768 and Vorherr et al,
(1993) Biochemistry (U.S.A.) 32:6081-6088) can be discerned on the
basis of conserved amino acid residues at positions 504-505 in the
first cytoplasmic domain. However, two important basic residues
Lys500 and Lys497 which are part of the calmodulin binding site in
ACtype1 are substituted by Gly in AC making calmodulin binding
unlikely in this region of the enzyme.
Distribution of mRNA for AC in rat tissues and mouse brain
[0238] Ribonuclease protection analysis of rat tissues showed
hybridizion of total RNA with probe JP114 gave a 125 bp double
stranded product that was most abundant in brain, with significant
hybridizing activity in the anterior pituitary gland, the ovary,
the adrenal gland, the lung and the kidney. Liver, pancreas,spleen,
thymus, heart gave no detectable signal, suggesting that the
distribution of the enzyme is highly tissue specific.
[0239] Gross dissection of the mouse brain suggests that AC mRNA is
most abundant in the cortex, striatum and the hippocampus, with
lower levels in the cerebellum and much lower but detectable
concentrations in the olfactory bulb, the diencephalon, the brain
stem and the pituitary gland. It is of note that the RNAse
resistant hybrid in mouse tissues and AtT20 cells is larger, about
155 bp, than in rat tissues indicating interspecies sequence
variation(s) in this region of the enzyme.
[0240] Northern mRNA analysis showed an approximately 9 kb size RNA
in AtT20 cells that hybridizes with a JP114 antisense .sup.32P-DNA
probe.
[0241] In summary, the present results show the existence of a
further member of the adenylyl cyclase family of proteins, which
has a restricted tissue distribution and a distinct regional
pattern in the brain. The functional properties of this cyclase
have yet to be explored in detail. Pharmacological analysis (Antoni
et al, (1994) Journal Of Physiology-London 475p:137-138 and Antoni
et al, (1995) EMBO Journal submitted) indicates that AC is
inhibited by Ca.sup.2+ through calcineurin and that this may be
relevant for corticosteroid inhibition of corticotropin secretion
in anterior pituitary cells (Shipston et al, (1994) Ann. N.Y. Acad.
Sci. 746:453-456). Furthermore, the abundance of AC mRNA in the
striatum and hippocampus, where calcineurin is particularly
abundant relative to other regions of the brain (Kuno et al, (1992)
J. Neurochem. 58:1643-1651) is of interest with respect to synaptic
function.
SUMMARY
[0242] The cDNA of a novel adenylyl cyclase isotype (AC) has been
cloned and sequenced from the mouse pituitary corticotrope tumour
cell line AtT20. Adenylyl cyclase cDNA sequences were amplified
from AtT20 cells using degenerate primers bracketing a highly
conserved motif near the carboxyl terminus region of the protein.
The majority of the sublcloned amplified cDNA products revealed a
novel sequence which was utilized to screen an AtT20 cell cDNA
library, from which a 3120 bp clone was isolated and sequenced. The
full length coding sequence was obtained upon two rounds of 5'
RACE-PCR, yielding a 4473 bp long cDNA containing an open reading
frame of 4062 bp which encodes a protein of 1352 amino acids. The
hydrophobicity profile of this protein resembles that of other
members of the adenylyl cyclase family in that two sets of six
hydrophobic, putatively membrane-spanning regions are predicted as
well as a large central cytoplasmic loop and long C-terminal
cytoplasmic tail. Amino acid sequence comparisons suggest that this
novel enzyme is quite distinct from other known mammalian adenylyl
cyclases and cannot be easily assigned to any of the previously
observed subfamilies. Tissue distribution of mRNA was examined by
RNAse protection assay and indicated the highest abundance of this
novel adenylyl cyclase to be in the brain followed by anterior
pituitary gland, ovary and adrenal gland, which appeared to express
approximately equal levels. Low level expression of this mRNA was
detected in lung and kidney while in the heart, liver and pancreas
none was apparent. Within the brain, relatively high levels of
novel adenylyl cyclase mRNA were detected in cortex, hippocampus,
striatum, cerebellum, with much less in diencephalon, olfactory
bulb and the brain stem.
EXAMPLE 3
FUNCTIONAL PROPERTIES OF AC
METHOD
[0243] The complete coding sequence of AC was subcloned into the
eukaryotic expression vector pcDNA3 (Invitrogen). The cDNA clone
for the CRF.sub.1 receptor cloned into the vector pcDNAI (Chang et
al, (1993) Neuron 11:1187-1195) was a gift of Drs. R. V. Pearse II
and M. G. Rosenfeld (University of California, San Diego). Both
expression plasmids (10 .mu.g) were transfected into
SV40-transformed monkey embryonic kidney (COS-7) cells grown in
DMEM and 10% fetal bovine serum to 70% confluency in 75 cm.sup.2
flasks following the double DEAE-dextran transfection protocol
(Ishikawa et al, (1992) Nucl. Acids Res. 20:4367), while only AC
cDNA was transfected into adenovirus-transformed human embryonic
kidney (HEK293) cells as these express endogenous receptors for CRF
(F. A. Antoni, unpublished data). In control transfections pcDNA3
vector DNA replaced the AC expression construct. Forty-eight hours
after the second transfection the cells were harvested in Hank's
Ca.sup.2+ and Mg.sup.2+ free balanced salt solution containing 0.1%
EDTA and centrifuged at 200 xg, for 10 minutes. Following
resuspension the cells were centrifuged again to remove serum
proteins and resuspended in 1 ml of HEPES (25 mmol/l, pH 7.4)
buffered Hank's balanced salt solution and a 50 .mu.l aliquot was
removed for the measurement of protein content. The cells were then
diluted further with 4 ml of HEPES buffered Hank's solution
containing 0.25% BSA, and preincubated for 1 hour at 37.degree. C.
under air. Subsequently the cells were pelleted again and
distributed (final concentration 3.times.10.sup.5 cells/ml) into
polypropylene vials and processed as AtT20 cells except that 1 mmol
IBMX and 0.1 mmol/l rolipram were used as inhibitors of
cAMP-degrading phosphodiesterases (see Example 1).
[0244] The functional properties of the cloned AC10 were
investigated in transiently transfected HEK293 and COS7 cells (FIG.
11). In HEK293 cells which have endogenous receptors for CRF,
unstimulated levels of cAMP and the cAMP response to CRF were
unchanged upon transfection with AC (FIG. 11a and b). However,
preincubation with FK506 increased unstimulated cAMP levels 3-fold
and significantly enhanced the cAMP response to 1 but not to 5
(FIG. 11b) or 25 nmol/l CRF (not shown). These effects were
specific to cells transfected with AC (FIG. 11a and b).
Surprisingly, in cells transfected with AC 100 .mu.mol/l L-685,818
increased unstimulated cAMP levels to the same extent as 2
.mu.mol/l FK506, while having no effect in sham-transfected or
pcDNA3-transfected cells (not shown). Importantly, L-685,818 had no
significant effect on CRF-induced cAMP production but blocked the
enhancement of the response to 1 nmol/l CRF by FK506 in cells
transfected with AC (FIG. 11c). Slightly different results were
obtained in COS7 cells. In this system unstimulated cAMP production
in cells transfected with AC cDNA (without or in combination with
the CRF receptor) was 1.9-fold higher than in controls receiving
vector DNA (unstimulated cAMP [pmol/mg protein] in COS7 cells
transfected with CRF receptor and pcDNA3: 20.9.+-.0.2; with CRF
receptor and AC cDNA: 38.2.+-.3.4; mean.+-.SEM, n=4/group
P<0.001, Student's t-test, representative of 4 separate
experiments) whereas the increment produced by CRF (1-25 nmol/l)
was not consistently altered (FIG. 11d and data not shown).
Preincubation with cyclosporin A significantly and selectively
enhanced the cAMP response to 1 nmol/l CRF in cells transfected
with AC (FIG. 11d).
[0245] These data confirm that the cDNA for AC encodes a cAMP
generating enzyme. At this point the cause of the differences in
unstimulated cAMP production between COS7 cells and HEK293 cells is
unknown, however, such discrepancies are not unprecedented in
eukaryotic expression systems (Premont (1994) Meth. Enzymol.
238:116-127; and Adie et al, (1994) Biochem. J. 303:803-808). With
respect to the paradoxical effect of L-685,818 on unstimulated cAMP
levels, it is likely that it has some calcineurin inhibiting
activity at a high concentration (100 .mu.mol/l) which may be
sufficient to enhance basal cAMP production. However, this small
inhibitory effect is not enough to cause a functionally relevant
reduction of activity when calcineurin is activated by the
cAMP-induced increase of intracellular free Ca.sup.2+ that is known
to occur upon a cAMP stimulation in both HEK293 and COS7 (Lin et
al, (1995) Mol. Pharmacol. 47:131-139; and Widman et al, (1994)
Mol. Pharmacol. 45:1029-1035 (1994)) cells. The consistent finding
of the transient transfection experiments is that cAMP production
in response to low CRF concentrations is enhanced by
immunosuppressant blockers of calcineurin and, in the case of
FK506, this is blocked by the antagonist L-685,818. In this respect
the findings with cloned and transiently transfected AC are in good
agreement with the findings in AtT20 cells and with pilot studies
in HEK293 cells stably transfected with AC (Antoni et al,
unpublished data).
SEQUENCE COMPARISON
METHOD:
[0246] Comparisons with other adenylyl cylases were made using the
GCG package available from the Human Genome Mapping Programme,
Cambridge U.K. Initial alignments with FKBP12 were made using
GeneJockeyII (Biosoft, Cambridge U.K.), and adjusted visually by
the aid of the programme.
[0247] Closer examination of the primary sequence of AC revealed
that residues 503 to 610 in the first cytoplasmic loop show
approximately 40% similarity with FKBP12 (FIG. 12a and b). The
first part of this FKBP12-like segment consists of the C-terminal
segment of the C1.alpha. domain which is highly conserved in all
adenylyl cyclases (residues 503-570 in FIG. 12c) (Taussig et al,
(1995) J. Biol. Chem. 270:1-4). The second part is the beginning of
the long non-conserved segment (C1.beta. domain). Importantly, some
of the sequence identities correspond to Asp37, Gly86, Phe87, Ile90
in FKBP12 (FIG. 12b) and are amino acid residues of key importance
for the high-affinity interaction of the FK506/FKBP12 complex with
calcineurin (Aldape et al, (1992) J. Biol. Chem. 267:16029-16032;
Yang et al, (1993) J. Am. Chem. Soc. 115:819-820; and Braun et al,
(1995) FASEB J. 9:63-72). The best overall sequence similarity is
observed with yeast FKBP12, which in fact has been shown to bind
calcineurin in the absence of FK506 (Cardenas et al, (1994) EMBO J.
13:5944-5957). Given the prominent actions of calcineurin blocker
immunosuppressants on cAMP formation in systems expressing AC it
seems reasonable to suggest that AC.sub.(503-610) may be a
physiologically relevant docking site for calcineurin.
[0248] Adenylyl cyclase type 1 (ACtp1) is also thought to interact
directly with a protein co-factor distinct from G-protein subunits
as this cyclase is markedly stimulated by Ca.sup.2+/calmodulin (for
review see Taussig et al, (1995) J. Biol. Chem. 270:1-4). Residues
495-522 of ACtp1 (495-518 shown underlined in FIG. 12c) bind
calmodulin with nanomolar affinity (Vorherr et al, (1993)
Biochemistry (USA) 32:6081-6088). Moreover, point mutations of
Phe.sub.503 and Lys.sub.504 virtually abolish the stimulatory
effect of Ca.sup.2+/calmodulin on this cyclase (Wu et al, (1993) J.
Biol. Chem. 286:23766-23768). Intriguingly, the position of
residues 495-522 along the cytoplasmic loop of AC corresponds to
the C-terminal part of the FKBP12-like sequence in AC (FIG. 12c).
It seems therefore that the junction between the C1.alpha. and
C1.beta. domains in adenylyl cyclase 1 and in AC is a site of
allosteric regulation by calcium binding proteins. In the case of
AC a strong candidate for such a regulatory protein is
calcineurin.
AC is Enriched in Nerve Cells
METHOD
[0249] Northern analysis of total RNA (20 .mu.g per lane) was
performed by transfer onto positively charged nylon membrane
(Appligene, Ullkirch, France) then hybridized to radio-labelled
probe in 50% formamide and washed according to standard procedures
(Sambrook et al, Molecular Cloning: a Laboratory Manual Ed. 2 (Cold
Spring Harbor Press, USA, 1989)). The cDNA probes used correspond
to amino acid residues 1-195 (JP164) and 1105-1165 (JP114).
[0250] Ribonuclease protection assays were performed using an RPA
II kit (Ambion, AMS Biotechnology, Witney, Oxon, U.K.) according to
the manufacturer's instructions using radio-labelled anti-sense
riboprobe derived from JP114 or JP142 (corresponding to amino acids
320-478). The AC probes showed no significant cross hybridisation
in Southern analysis with adenylyl cyclase isotype cDNAs 1, 2, 3, 5
or 6. A 250 bp probe hybridising with .beta.-actin was used as the
internal standard to correct for differences in RNA loading. Blots
and gels were exposed to X-ray film as well as to Molecular
Dynamics Phosphorimager casettes and quantified with the ImageQuant
software using the 28S RNA band and the .beta.-actin band as
standards for RNA loading in Northern and RNase protection
analysis, respectively. Division of the integrated volume of pixels
of the selected radiolabelled band with the integrated volume of
the internal standard band yields the relative hybridisation
intensity, which was used to compare the intensity of labelled RNA
bands within blots. The relative abundance of the protected RNA
species in a given tissue is expressed as the percentage of the
relative hybridisation intensity of the whole brain RNA band run in
the same assay.
[0251] Importantly, we have discovered that AC mRNA is present in
relatively high levels in the hypothalamic paraventricular nucleus
that contains the major contingent of CRF41 and vasopressin
producing neuroendocrine motoneurones and controls the secretion of
ACTH by the anterior pituitary gland. In addition, the levels of
mRNA are apparently controlled by adrenal corticosteroids, so that
lowering of adrenocortical steroid levels causes an increased
expression of AC mRNA. This reinforces the notion that AC is
expressed in areas of the brain important for the negative feedback
action of adrenal corticosteroids, in the
hypothalamic-pituitary-adrenocortical axis. This system is a
servomechanism where, when corticosteroid feedback is detected as
inadequate by the relevant as yet unidentified components of the
system, the levels of AC mRNA are increased.
RESULTS
[0252] Existence of a calcineurin-regulated adenylyl cyclase is of
great potential significance for brain function as calcineurin
constitutes about 1% of total brain protein (Klee et al, (1988)
Adv. Enzymol. 61:149-200). AC is in fact highly abundant in the
brain: Northern blot analysis shows the presence of a 9 kb mRNA in
mouse striatum and AtT20 cells (FIG. 13). RNAse protection anaylsis
of dissected regions of the mouse brain suggest the relative
abundance of AC mRNA to be
hippocampus.gtoreq.striatum.gtoreq.cortex.gtoreq.cerebellum.gtoreq.olfact-
ory bulb>brain stem>diencephalon.gtoreq.anterior pituitary
gland (range 3-fold, see FIG. 13 for methods). These findings
indicated a primarily neuronal localisation which was confirmed by
in situ hybridisation histochemistry, showing robust AC mRNA
bybridisation in hippocampal neurons (FIG. 14).
[0253] RNAse protection assay also shows the presence of AC10 mRNA,
amongst other tissues, in mouse kidney, thymus and spleen (relative
abundance 50, 10 and 8%, respectively if brain=100%, see FIG. 13
for methods) all of which are important targets of
immunosuppressants (Schreier et al, (1993) Transplant. Proc.
25:502-507; and Dumont et al, (1992) J. Exp. Med. 176:751-760).
[0254] Functional implications of calcineurin regulated adenylyl
cyclase
[0255] In summary, the present results show the existence of a new
member of the adenylyl cyclase family of proteins that is abundant
in the brain and is inhibited by calcineurin. The intricate control
of calcineurin activity by its EF-hand protein .beta.-subunit as
well as additional regulation by calmodulin (Schreier et al, (1993)
Transplant. Proc. 25:502-507) suggest a potentially fundamental
role for AC in situations where fine interplay between
intracellular Ca.sup.2+ and cAMP determines cellular function
(Cooper et al, (1995) Nature 374:421-424). Furthermore, the
abundance of AC mRNA in the striatum and hippocampus, where
calcineurin is particularly enriched relative to other regions of
the brain (Kuno et al, (1992) J. Neurochem. 58:1643-1651) is of
major interest with respect to synaptic function. Finally, it
remains to be explored whether or not the currently known Ca.sup.2+
regulated cyclases are also subject to control by calcineurin. A
recent study has in fact reported the facilitation of cAMP
formation by Ca.sup.2+/calcineurin suggestive of calcineurin
control of a Ca.sup.2+-stimulated adenylyl cyclase (Baukal et al,
(1994) J. Biol. Chem. 269:24546-24549).
EXAMPLE 4
HUMAN AC9
[0256] Isolation of a complete human AC9 cDNA clone
[0257] The assembly of a full length human AC9 cDNA clone is
represented in FIG. 18. The NCBI BLAST computer program
(http://www.ncbi.nlm.nih.gov/- BLAST/) was used to search the
GenBank/EMBL and dbEST (expressed sequence tag) DNA sequence
databases for human EST clones exhibiting similarity to mouse AC9
cDNA sequence Z50190. Two entries of interest were initially
identified under GenBank/EMBL Accession Numbers R88632 and T08314.
The EST cDNA clones, IMAGE 166657 (from adult human brain) and
HIBBC33 (from infant human brain), from which these sequences were
derived, were respectively obtained from the MRC Human Genome
Mapping Project Resource Centre (MRC HGMP-RC, Cambridge, UK) and
The Institute for Genome Research (TIGR, Gathersburg, Md., USA,
distributed by ATCC, Rockville, Md., USA). Due to the directed
nature of cDNA cloning (Adams et al, (1993) Nat. Genet. 4: 373-80)
the database sequence entries for these clones was presumed to
represent part of the sense strand of the cDNA. This holds true for
sequence R88632 (258 bp) which exhibits 77% sequence identity with
nucleotides 3855 to 4113 of mouse AC9 sequence Z50190. However,
sequence T08314 (228 bp) exhibited 82% identity with residues
682-909 on the anti-sense strand of mouse AC9 sequence Z50190
suggesting that this cDNA was cloned randomly rather than
directionally. This insert may have arisen due to fragmentation of
cDNA prior to vector ligation or as a result of mis-priming of cDNA
synthesis from a source other than the polyadenylated 3' end of
mRNA although a complete sequence representing the primer of cDNA
synthesis (Adams et al, (1993) supra) was not found. The inserts of
both EST clones were completely sequenced by the dideoxynucleotide
method using universal and sequence-specific primers using an ABI
Prism dye terminator DNA cycle sequencing kit (Perkin Elmer),
resolved on a 4.75% acrylamide denaturing gel and data collected on
an Applied Biosystems/Perkin Elmer 373 Stretch automated sequencer.
Sequences were analyzed independently using Gene Jockey II program
(Biosoft, Cambridge, U.K.) for the assessment of errors and visual
confirmation of base calling. IMAGE clone 166657 was found to
contain the 3' part of an open reading frame (ORF) encoding human
AC9 followed by an untranslated region (UTR) and polyadenylated
tail. Clone HIBBC33 contained 5' UTR sequence and the 5' part of
the ORF encoding human AC9. To complete cloning of the human AC9
cDNA, the gap between these EST cDNA clones containing the 5' and
3' parts of the ORF were isolated by polymerase chain reaction
(PCR) amplification using the High Fidelity Expand PCR system
(Boehringer Mannheim/Roche) with sequence-specific primers. A PCR
product of 2860 bp (1309-4169; pJP230) was amplified from a
denatured human skeletal muscle cDNA library (Clontech) using
primers 5'-GAC CTT TGG CTA CCA TTT CCG GGA TG-3' (SEQ ID NO: 100)
and 5'-CTT CGC TCA CCT GGA TGC GGC ACT C-3' (SEQ ID NO: 101)
respectively. The ends of this PCR product were blunt ended with T4
DNA polymerase (Boehringer Mannheim/Roche) prior to ligation into
the SmaI site of plasmid vector pUC9 and subsequently sequenced as
above. Additional overlapping clones of PCR products derived from
human AC9 cDNA (853-1196; pJP 192 and 853-1167 ; pJP195), amplified
as above or from a pool of denatured human cDNA libraries
(Clontech) using primers based on mouse AC9 cDNA (upstream primer
5'-GGA GCG CTG CTT TCC GCA G-3' (SEQ ID NO: 102) with downstream
primers 5'-CAT GTT CAC CAT CTC TTT CTT CTC C-3' (SEQ ID NO: 103)
and 5'-GTG GCC GTG AGA GTA TGA TTG GAG CTG TC-3' (SEQ ID NO: 104)
respectively), were propagated in the TA cloning vector pCRII
(Invitrogen) and sequenced as above. The complete human AC9 cDNA
(5515 bp, including 5' and 3' Not I cloning sites) was assembled
from the above clones using recombinant DNA techniques. Internal
PCR products pJP192 and pJP230 were joined at EcoRV site; 1735. The
resulting clone was joined to the 5'UTR, initiation codon and 5'
part of the ORF in clone HIBBC33 at MluI site; 1053. Finally the
insert from clone IMAGE 166657 containing the 3' ORF and UTR was
joined to the above clone at NcoI site; 4042 and the complete cDNA
cloned as a NotI fragment into pBluescript (Stratagene) and the
expression vector pcDNA3 (Invitrogen) for use in subsequent
experiments.
[0258] Functional expression of human AC9
[0259] The complete coding sequence of human AC9 was cloned into
the expression vector pcDNA3. This constuct and pcDNA3 (10 .mu.g)
were each linearised by PvuI digestion prior to electroporation
into HEK293 cells. Stably transfected clones were selected for
resistance to G418 (Gibco) at 0.6 .mu.g/ml in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% foetal calf serum. A
total of 12 clones were expanded and over-expression of human AC9
confirmed in 9 cell lines by immunoblotting using antisera raised
against a pentadecapeptide derived from the N-terminus of mouse AC9
(Paterson et al, (1995) Biochem. Biophys. Res. Commun. 214:1000-8).
All but one of these 9 clones showed the inhibitory effects of
Ca.sup.2+ on cAMP accumulation assayed in the presence of cyclic
nucleotide phosphdiesterase (PDE) blockers (Antoni et al, (1998) J.
Neurosci. 18:9650-61). Analysis of clone 7 is reported here.
[0260] HEK-293 cells display complex intracellular Ca2+ handling
mechanisms that may be controlled by cAMP and calcineurin (Lin et
al, (1995) Mol. Pharmacol. 47:131-9; Gromada et al, (1995) FEBS
Letts. 373:182-6; Querfurth et al, (1998) Biochem J. 334:79-86),
and thus complicate the interpretation of the data. Therefore,
intracellular stores of Ca.sup.2+ were depleted to reduce the
contribution of intracellular Ca.sup.2+ pools to the intracellular
free Ca.sup.2+ signal. All procedures were carried out at
37.degree. C., where required, immunosuppressant drugs FK506
(courtesy of Fujisawa GmbH, Munich, Germany) cyclosporin A
(courtesy of Novartis Ltd, U.K.) and L685,818 (Courtesy of Merck
& Co, Rahway, N.J.) rapamycin (courtesy of Dr Hans Fliri,
Sandoz Pharma, Basel, Switzerland) or ethanol vehicle (0.1% v/v)
were added to the medium at the onset of the Ca.sup.2+ depletion
period. Two different Ca.sup.2+ depletion protocols were used
(Antoni et al, (1998) supra). In the first, cells were incubated in
a balanced salt solution containing (mM) 133 NaCl, 5.4 KCl, 0.25
Na.sub.2HPO.sub.4, 0.44 KH.sub.2PO.sub.4, 1 MgSO4, 2 EGTA, 5.6
D-glucose, 25 Hepes pH7.40 and 0.1% (w/v) bovine serum albumin
(HBSS/EGTA) with 10 .mu.M ryanodine and 1 .mu.M thapsigargin (both
from LC Labs, Boston, Mass.). These conditions deplete rapidly
exchangeable Ca.sup.2+ pools and activate capacitative Ca.sup.2+
entry mechanisms (Cooper et al, (1994) Cellular Signalling
6:823-40). In the second protocol 5 .mu.M 4-Br-A23187 was used
instead of ryanodine and thapsigargin, and Ca/EGTA was added to
obtain similar initial levels of [Ca.sup.2+].sub.1 as in the
studies with ryanodine and thapsigargin. Under these conditions
intracellular Ca.sup.2+ pools are depleted and, in addition to
capacitative Ca.sup.2+ entry, Ca.sup.2+ influx may take place
through the pores formed by the ionophore. The cells were incubated
in Ca.sup.2+-depleting medium for 20 min. Subsequently HBSS/EGTA
containing various amounts of CaCl.sub.2 (Ca/EGTA) was added to
achieve extracellular concentrations of Ca.sup.2+ ranging between
0.125-2 mM. The pH of this solution was 7.65 in order to minimize
the change of pH due to the displacement of protons from EGTA--a pH
shift from 7.40 to 7.29 occurred upon achieving a free Ca.sup.2+
concentration of 2 mM. After Ca/EGTA was added, the cells were
incubated for 5 min after which the cyclic nucleotide PDE
inhibitors isobutylmethylxanthine (IBMX, Aldrich, Gillingham,
Dorset, U.K.) and rolipram (courtesy of Dr H Wachtel, Schering AG,
Berlin Germany), 1 mM and 0.1 mM respectively, were introduced.
Unless indicated otherwise, the cells were incubated for a further
10 min and the incubation was then terminated by the addition of
0.2 M HCl. The total cAMP content of the cells and the medium was
determined after freeze-thawing and acetylation by radioimmunoassay
(Antoni et al, (1995) J. Biol. Chem. 270:28055-61).
[0261] Analysis of human AC9 mRNA expression
[0262] Northern blotting
[0263] A multiple tissue northern blot (MTN; Clontech) of poly
A.sup.+ RNA from human heart, brain, placenta, lung, liver,
skeletal muscle, kidney and pancreas (2 .mu.g per lane) was probed
with a 528 bp SmaI-MluI fragment from the 5' end of the human AC9
cDNA and subsequently with a human .beta.-actin cDNA control probe
supplied with the blot (each labelled with 32P-dCTP by random
priming; Pharmacia "Ready-to-Go" system). Hybridization was carried
out in Express Hyb solution (Clontech) according to the
manufacturers instructions at 68.degree. C. for 1 hour, washed in
2.times.SSC/0.1 % SDS at room temperature followed by
0.1.times.SSC/0.1% SDS at 50.degree. C. then wrapped in plastic and
exposed to Fuji X-ray film for autoradiography. Following removal
of previous probes the filter was hybridised as above with a cDNA
fragment derived by PCR amplification from a human skeletal muscle
cDNA library (Clontech) corresponding to nucleotides 3816-4487 of
clone KIAA0520 (GenBank/EMBL Accession Number AB011092) (26).
[0264] Ribonuclease protection assays were performed using a
.sup.32P-CTP labelled riboprobe synthesised by in vitro
transcription (T7 RNA polymerase Riboprobe System, Promega) from a
HindIII-linearised plasmid (pJP195; FIG. 1) partial human AC9 cDNA
clone representing nucleotides 853-1167 according to the kit
manufacturer (Ambion). Probe fragments protected from nuclease
digestion following incubation with 5 .mu.g total RNA samples from
normal adult human brain obtained 4 h after death (JEB; Custodian,
MRC Brain Bank, Western General Hospital, Edinburgh) or HEK293
cells (control) were resolved on a 6% denaturing acrylamide gel (8
M Urea) and visualised by autoradiography.
[0265] In situ hybridisation was performed on 15 .mu.m frozen
sections from normal post mortem adult human brain obtained from
the MRC Alzheimer Disease Brain Bank, Western General Hospital,
Edinburgh--Professor Jeane E Bell, Custodian. Prepared sections
were courtesy of Dr Celia Yates and Professor George Fink.
.sup.35S-labelled riboprobes were prepared by in vitro
transcription from the human AC9 partial cDNA clone pJP195 (FIG.
18) using T7 (anti-sense) or SP6 (sense) RNA polymerase and used as
previously described for mouse AC9 (Paterson et al, (1995) Biochem.
Biophys. Res. Commun. 214:1000-8; Rosie et al, (1990) J.
Endocrinol. 124:285-9). Following hybridisation, brain sections
were exposed to .beta.-max autoradiogaphy film (Amersham-Pharmacia)
for 2 weeks. The autoradiogram was developed and photographed under
white light illumination.
[0266] Immunocytochemistry
[0267] HEK293 cells were plated on poly-D-lysine-coated coverslips
and cultured as described above. Two to four days after plating,
cells were washed in phosphate buffered saline (PBS) and fixed in
3% paraformaldehyde in PBS, pH 7.4, for 20 min at 4.degree. C.,
treated with chilled (-20.degree. C.) absolute methanol for 10 min
(4.degree. C.) and washed in 0.3 M glycine (3 times for 5 min) and
0.2% Triton X-100 (15 min) both in PBS at room temperature. Fixed
cells were incubated for 18-24 h at 4.degree. C. with affinity
purified chick antibodies (1:400-1:500) raised against the
C-terminus of mouse AC9 (Antoni et al, (1998) J. Neurosci.
18:9650-61). FITC-conjugated anti-chicken IgG (Jackson
ImmunoRes.Lab.) was applied at 1:400 dilution for 1 hr at RT. After
several washes in PBS specimens were mounted in CitiFluor (Sigma)
and examined using a Leica TCS-NT confocal microscope. Specificity
controls involved preincubation of the antibody with 10 .mu.M
antigen overnight at 4.degree. C., after which immunocytochemistry
was carried out as above.
[0268] Detection of human AC9 by immunoblotting
[0269] Membrane proteins were prepared from HEK293-derived cell
lines as described previously (Antoni et al, (1998) supra).
Approximately 10 .mu.g membrane protein per lane were separated by
SDS-PAGE and transferred by electroblotting on to PVDF membrane
(Bio-Rad) at 220 V in tank buffer (25 mMTris, 192 mM glycine,
0.036% SDS, 20% methanol) for 3 hrs at room temperature. PVDF
membranes were blocked at room temperature by shaking in PBS
containing 5% (w/v) skimmed milk powder and 0.1% Tween 20 for 2 hrs
at room temperature, then washed 5.times.5 mins in PBS/0.1% Tween
20 before shaking incubation in PBS/0.1% Tween 20 containing
primary antibody (rabbit anti-mouse AC9 N-terminus at 1:4,000
dilution or chicken anti-mouse AC9 C-terminus at 1:10,000 dilution
) for 1 hr at room temperature. PVDF membranes were then washed as
before and incubated in PBS/0.1% Tween 20 containing sheep
anti-rabbit IgG-Horseradish peroxidase (HRP) conjugate (kindly
provided by the Scottish Antibody Production Unit) at 1:8,000
dilution or goat anti-chicken IgY-HRP conjugate (Promega) at
1:10,000 dilution respectively) for 1 hour at room temperature then
finally washed as above, exposed to reagents for development of
chemiluminescence and visualised by exposure to Hyperfilm (ECL
system, Amersham-Pharmacia, Aylesbury, UK). To confirm specificity,
primary antisera were pre-incubated with mouse AC9-derived
pentadeca-peptide antigens at final concentrations 1 .mu.g/ml
(C-terminal) and 0.1 .mu.g/ml (N-terminal) for 20 & 45 mins
respectively prior to probing of the PVDF membranes.
[0270] Results
[0271] Cloning and Sequencing of Human AC9 cDNA
[0272] Searching of human EST databases for similarity to mouse AC9
identifed two cDNA clones: IMAGE clone 166657 and HIBBC33, which
were obtained as described in materials and methods.
Oligonucleotide primers based on the DNA sequence of the inserts of
these were used to construct a complete cDNA by PCR amplification
from human cDNA libraries as described in materials and methods and
outlined in FIG. 18. In the cDNA we describe (GenBank/EMBL
Accession Number AJ133123) a short open reading frame (ORF) of 72
bp exists near the 5' end (263-334). However, the initiation codon
of this ORF does not form part of a Kozak consensus sequence
suggesting that translation may not be initiated although this
remains to be confirmed. Downstream, a large ORF of 4059 bp is
apparent, from 539-4597. This encodes a putative protein of 1353
amino acids, identical in length to the mouse AC9 protein. The
deduced amino acid sequence of hAC9 reported here exhibits 92%
sequence identity with the mouse AC9 protein and 86.7% identity at
the nucleotide level. Structural features predicted from the amino
acid sequence using the computer program TopPred 2 (von Heijne
(1992) J. Mol. Biol. 225:487-494) are typical of
membrane-associated mammalian adenylyl cyclases cloned to date,
namely, two sets of six transmembrane regions (M1 and M2), each
followed by a large cytoplasmic region (C1 and C2 respectively).
These domains are conserved between the human and mouse AC9
proteins along with many motifs for potential post-translational
modifications. With N and C termini localised intracellularly, the
predicted topology of membrane-bound AC9 indicates conserved sites
which may be suitable for modification include those for N-linked
glycosylation, located extracellularly (residues 206, 955, 964), as
well as intracellular sites suitable for tyrosine phosphorylation
(residues 543, 1004, 1225) and serine/threonine phosphorylation by
cAMP-dependent protein kinase (residues 373, 374),
calmodulin-dependent protein kinase II (residues 1217), casein
kinase II (residues 433, 582, 589, 691, 766, 1200) and protein
kinase C (residues 27, 87, 112, 357, 365, 535, 620, 661, 762, 838,
1003, 1023, 1179, 1212, 1307).
[0273] Expression of Human AC9 Protein Detected by
Immunocytochemical Analysis
[0274] Immunocytochemical analysis using antisera raised against
the C-terminus of the mouse AC9 protein revealed fluorescent
labelling in HEK 293 cells stably transfected with cDNA encoding
human AC9 (FIG. 19a & b). No labelling was observed when the
antiserum was pre-incubated with peptide antigen (FIG. 19c)
indicating specific detection of human AC9 protein. A large
majority of human AC9 expressed in HEK 293 cells appeared to be
localised to the plasma membrane.
[0275] Detection of Multiple Human AC9 Polypeptides by
Immunoblotting
[0276] Immunoblotting of extracts from pcDNA3 and hAC9 stably
transfected HEK293 cells resolved by SDS-polyacrylamide gel
electrophoresis (PAGE) showed that hAC9 protein was expressed and
specifically recognised by polyclonal antisera raised against
peptides derived from the N- or the C-termini of mouse AC9 (FIG.
20). In each case, a broad immunoreactive band migrating between
160-180 kDa was detected, most probably attributable to
post-translational modification by phosphorylation and/or
N-glycosylation of the human AC9 protein in HEK 293. These
observations confirm the predicted conservation of the sequences of
the human and mouse AC9 at the N- and C-termini.
[0277] Ca.sup.2+/Calcineurin Inhibition of cAMP Production by Human
AC9
[0278] In the presence of phosphodiesterase inhibitors, the cAMP
content of stably transfected HEK293 cell lines expressing human
AC9 increased with time and this was reduced by the presence of
Ca.sup.2+ in the medium (FIG. 21a). No statistically significant
increase of cAMP was observed in wild type HEK293 cells or HEK293
cell lines transfected with pcDNA3 vector alone. Similar data were
obtained in cells treated in 5 .mu.M 4-Br-A23187 containing medium
(FIG. 21b) moreover, the inhibitory effect of Ca.sup.2+ was blocked
by FK506. L-685,818 and rapamycin, which both interact with
FK506-binding-protein-12, but do not inhibit calcineurin (29),
failed to block the inhibitory action of Ca.sup.2+ on cAMP
production (FIG. 21c). By contrast, cyclosporin A, which blocks
calcineurin through a complex formed with cyclophilin A (Schreiber
(1992) Cell 70:365-8) also abolished the effect of Ca.sup.2+. These
data indicate that the human AC9 expressed in HEK293 cells is able
to synthesize cAMP and its activity is inhibited by Ca.sup.2+ via
calcineurin.
[0279] Tissue distribution of two forms of Human AC9 mRNA
[0280] Analysis of hAC9 mRNA by Northern blotting (FIG. 22a) using
a radio-labelled probe derived from the 5' end of the hAC9 cDNA
(SmaI-MluI fragment; 528 bp) revealed hybridisation to a transcript
of approximately 8.5 kb in all tissues represented, including
brain, heart, skeletal muscle and pancreas. In addition, an
approximately 6.3 kb mRNA was detected which appeared to be
restricted to heart and skeletal muscle and gave a more intense
signal than the 8.5 kb species. A human cDNA sequence (GenBank/EMBL
Accession Number AB011092), described as part of a study reporting
the predicted coding sequences of 100 cDNA clones isolated from
human brain (Nagase et al, (1998) DNA Research 5:31-9), was
retrieved by database searching and found to be identical to
nucleotides 2900-5472 of the human AC9 cDNA sequence reported here
but continues with a 3' untranslated region extended by 2234 bp. A
cDNA fragment derived from this region used in hybridisation with
the same MTN Northern blot detected the 8.5 kb human AC9 mRNA
exclusively (FIG. 22c). These data are consistent with
tissue-specific differential use of alternative polyadenylation
signals on completion of mRNA transcription from the human AC9
gene. Subsequent searching of the GenBank/EMBL DNA sequence
database for comparison of human AC9 cDNA 3' UTR sequences with the
human AC9 gene locus (GenBank/EMBL Accession Number AC005736)
confirmed the positions of alternative polyadenylation sites.
[0281] Expression of AC9 mRNA in human brain
[0282] To address the potential role of AC9 within the human brain,
expression of AC9 mRNA was investigated by ribonuclease protection
assay of total RNA samples isolated from post-mortem hippocampus,
cortex and striatum (FIG. 23a). Protection of the anti-sense AC9
riboprobe was observed in each of these regions of human brain,
indicating expression of AC9 mRNA in areas important for learning
and memory functions. AC9 mRNA distribution in human forebrain was
also investigated by in situ hybridisation (FIG. 23b) where
enriched expression was detected specifically in neurones of the
dentate gyrus and hippocampal CA1-CA3 regions.
[0283] Discussion
[0284] We have isolated and sequenced a human cDNA encoding an
adenylyl cyclase which, distinct from a recently reported predicted
human AC9 polypeptide (Hacker et al, (1998) Genomics 50:97-104),
encodes a homologue of murine AC9. Cyclic AMP production by this
new human AC9 cDNA clone is inhibited by Ca.sup.2+ through the
protein phosphatase calcineurin. Analysis of the pattern of the
expression of the novel AC9 mRNA indicates differential tissue
specificity in the polyadenylation of transcripts. This has not
been observed in the mouse and may provide an additional level of
control in the regulation of AC9 expression in humans.
[0285] Confirmation of the Predicted Primary Sequence of the Human
AC9 Protein
[0286] The human AC9 cDNA we have cloned predicts a polypeptide of
the same length (1353 amino acid residues) as mouse AC9 and is 92%
identical with the mouse AC9 polypeptide sequence. The novel human
AC9 could be detected with anti-sera raised against the N- or the
C-terminal regions of the mouse AC9 polypeptide sequence,
confirming the predicted conservation of these regions between the
two proteins. The 3' end of the cDNA reported here is identical to
part of a 5'-truncated human AC9 cDNA clone KIAA0520 (Nagase et al,
(1998) DNA Research 5:31-9). These sequences differ from that of
another human AC9 cDNA clone reported recently by Hacker et al. who
predicted an alternative C-terminus for the encoded polypeptide
(Hacker et al, (1998) Genomics 50:97-104). By comparison of
sequences (FIG. 24), the prediction by Hacker et al. results from a
2 bp deletion causing a frame shift in the deduced amino acid
sequence. Our data, therefore, predict and confirm an amino acid
sequence for human AC9 which is distinct from that reported for a
previously isolated clone of the human enzyme but which is
conserved with mouse AC9.
[0287] Inhibition of human AC9 activity by
Ca.sup.2+/calcineurin
[0288] Stably transfected HEK293-derived cell lines which
over-express human AC9 showed a marked increase in cAMP production
in comparison to wild type or pcDNA3-transfected control cells. The
synthesis of cAMP was reduced by intracellular Ca.sup.2+
concentrations in the range of 100-150 nM which is similar to
previous data with HEK293 cells overexpressing mouse AC9 (Paterson
et al Biochem. Biophys. Res. Commun. 214:1000-8; Antoni et al,
(1998) J. Neurosci. 18:9650-61). The inhibitory effect of Ca.sup.2+
was blocked by FK506 or cyclosporin A, which are two
mechanistically distinct blockers of the Ca.sup.2+/calmodulin
activated protein phosphatase calcineurin (Sabatini et al, (1992)
Cell 70:365-8). In the presence of FK506 or cyclosporin A, cAMP
production in hAC9 expressing cells was enhanced. Furthermore,
application of L685,818 or rapamycin had no effect on cAMP
production. The results indicate that the activity of human AC9,
like mouse AC9 (Paterson et al, Biochem. Biophys. Res. Commun.
214:1000-8; Antoni et al, (1995) J. Biol. Chem. 270:28055-61;
Antoni et al, (1998) J. Neurosci. 18:9650-61), is under inhibitory
control by Ca.sup.2+ which is mediated by calcineurin. The lack of
Ca.sup.2+ inhibition of AC9 observed by others may be due to
analysis in insect cells (Premont et al, (1996) J. Biol. Chem.
271:13900-7) which may lack the necessary co-factors or may be
attributable to a feature of the cDNA and/or transfected cell clone
analysed (Hacker et al, (1998) Genomics 50:97-104). Immunological
detection of multiple human AC9 polypeptides may represent
differential post-translational modifications in HEK293 cells.
Correlation of these observations with multiple phosphorylation or
other post-translational modifications of human AC9 protein remains
to be addressed. Immunocytochemical analysis shown in FIG. 19
suggests that the great majority of human AC9 polypeptides
expressed in HEK 293 cells are localised to the plasma
membrane.
[0289] Differential Human AC9 mRNA Expression
[0290] In similarity with the mouse (Premont et al, (1996) J. Biol.
Chem. 271:13900-7) and in agreement with a recent study of another
human AC9 cDNA clone (Hacker et al, (1998) Genomics 50:97-104), a
broad pattern of expression was observed for an 8.5 kb AC9 mRNA in
human brain and peripheral tissues when assayed by Northern
blotting using a probe derived from the 5' end of the cDNA we have
isolated. In the present study, a 6.3 kb human AC9 mRNA also
detected by this probe appeared to be restricted to human heart and
skeletal muscle. Previously, however, a probe representing a
5'-truncated human AC9 cDNA detected a 6.3 kb transcript at high
levels in skeletal muscle, at low levels in kidney, liver, lung and
placenta but which was apparently absent from heart (Hacker et al,
(1998) supra). Although the probes used in these two studies
overlap by approximately 100 bp near the 5' end of the ORF, the
hybridisation conditions used may mean that these probes would be
capable of detecting differentially spliced human AC9 mRNA
transcripts if an intron/exon junction were located within this
region. Lack of detection of the 6.3 kb mRNA in heart may
potentially reflect mRNA splicing of upstream exons, detectable by
the 5' probe, to alternative downstream exons. Similarly, it is
possible that hybridisation to the 6.3 kb transcript by the
5'-truncated 4.65 kb partial cDNA probe reflects the presence of
alternative 5'-end sequences not detectable by the 5' probe used in
this study. However, comparison of our cDNA sequence with human AC9
genomic sequence available in the GenBank/EMBL database (Accession
Number AC005736) indicates no evidence of mRNA splicing in this
region. The .beta.-actin control in our Northern blotting analysis
suggests the presence of proportionally greater amounts of heart
and skeletal muscle mRNAs. Therefore, it is possible that the 6.3
kb AC9 mRNA was present in other tissues but not at a level
detectable under the conditions we have used and that expression of
this transcript is not necessarily restricted to heart and skeletal
muscle as our results suggest. A probe derived from the extended 3'
untranslated region of the 8.5 kb human AC9 transcript which is
present in the cDNA clone KIAA0520 exclusively detected the 8.5 kb
mRNA in northern blotting. This indicates the use of alternative
polyadenylation signals within the human AC9 gene to be the
mechanism accounting for the observation of two mRNAs.
Polyadenylation of many eukaryotic mRNAs is understood to
contribute to inhibition of mRNA degradation and/or potentiate mRNA
translation, reviewed in (Wickers et al, (1997) Curr. Opin. Genet.
Dev. 7:220-32). In addition, 5' and particularly 3' untranslated
regions (UTRs) have been found to be important for the regulation
of mRNA translation, stability and localisation, reviewed in
(Wickers et al, (1997) supra; Jackson et al, (1997) Curr. Opin.
Genet. Dev. 7:233-41), most significantly during development,
reviewed in (Curtis et al, (1995) Cell 81:171-8; Seydoux (1996)
Curr. Opin. Genet. Dev. 6:555-61; Macdonald et al, (1996) Curr.
Opin. Genet. Dev. 6:403-7). The spatial and/or temporal
distribution of trans-acting factors interacting with cis elements
within 3' UTRs and form a complex with the 5' UTR and associated
translation initiation factors has been proposed a molecular
mechanism for the control of gene expression by regulation of
translation efficiency (Wickers et al, (1997) supra). Although
sequences within the 3' UTRs of human and mouse AC9 mRNAs are
highly conserved, the 3' UTRs of human AC9 transcripts extend
beyond that of the mouse with the 8.5 kb mRNA possessing
considerable more 3' untranslated sequence than the 6.3 kb message.
Although the functional significance of 3' UTRs in AC9 mRNAs
remains to be addressed but may be subject to differential
post-transcriptional regulation providing an additional mechanism
of control at the level of mRNA stability and/or translational
efficiency facilitating differential regulation of AC9 expression
which may be relevant for the appropriate function of the enzyme in
humans.
[0291] In cardiac myocytes, adenylyl cyclase VI (AC6) expression
has been shown to be limiting for transmembrane .beta.-adrenergic
signal transduction. Furthermore, all currently known cardiac
adenylyl cyclases are under inhibitory control by Ca.sup.2+. Thus
precise control of cAMP levels mediated through regulation of AC
expression and control of AC activity appears essential for optimal
cardiac function (Gao et al, (1998) PNAS USA 95:1038-43).
Alterations to the levels of AC expression in heart, including AC9,
may therefore contribute to an impairment of cardiac function
related to reduction or loss of cAMP production. A role for
post-transcriptional regulation in the control of adenylyl cyclase
expression has recently been suggested from studies of ACVIII (AC8)
(Muglia et al, (1999) J. Neurosci. 19:2051-8) which have revealed
conservation of large 5' UTRs of AC8 mRNAs from mouse, rat and
human.
[0292] Detection of AC9 mRNA by ribonuclease protection assay
revealed expression in hippocampus, neocortex and striatum of
post-mortem human brain. In situ hybridisation analysis revealed
enriched expression of AC9 mRNA in hippocampus and indicated a
neuronal localisation in projection neurones of the CA1 -CA3
regions. The Ca.sup.2+/calmodulin activated protein phosphatase
calcineurin, which regulates AC9 activity, is also highly abundant
in these regions (Winder et al, (1998) Cell 92:25-37). These
findings are likely to be relevant to the function of AC9 in brain,
particularly in hippocampus, where precise control of Ca.sup.2+ and
cAMP levels is pivotal for synaptic plasticity and suggests
potential involvement in functions important for cognitive
behaviour (Mons et al, (1995) Trends in Neurosci. 18:536:42; Xia et
al, (1997) Curr. Opin. Neurobiol. 7:391-6; Antoni et al, (1998) J.
Neurosci. 18:9650-61; Winder et al, (1998) Cell 92:25-27; Mons et
al, (1998) Life Sciences 62:1647-52).
[0293] Isolation of a human AC9 has enabled our characterisation of
the enzyme, allowing comparison with that of the mouse.
Differential usage of polyadenylation signals within the gene
providing the potential for alternative mRNA post-transcriptional
regulation has been identified as a distinct feature of human AC9
likely to be important for precise control of expression. In
similarity with mouse AC9, cAMP production by human AC9 is under
inhibitory control by Ca.sup.2+/calcineurin. Post-translational
modifications of the human AC9 protein, including possible
phosphorylation, now require characterisation and their potential
functional consequences investigated. The physiological function of
AC9 and its potential involvement in human disease, particularly in
view of its broad expression pattern, are complex issues to
address. Although important differences have been described, many
features are common to the human and murine enzymes. The production
of mouse models may, therefore, be useful in the investigation of
specific functions of AC9 in vivo.
TABLE I
[0294] Effect of pretreatment with FK506 (1 .mu.mol/l) and BAPTA/AM
(20 .mu.mol/l) on cAMP accumulation induced by .beta.-adrenergic
stimulation in AtT20 cells. Data are means .+-.SEM, n=4/group. IBMX
0.5 mmol/l and rokpram 0.1 mmol/l present throughout. nd--not
determined, previous studies showed that there is no effect of
either preincubation protocol on basal cAMP levels.
4 Isoproterenol Pretreatment (nmol/l) None FK506 BAPTA/AM None 0.43
.+-. 04 nd nd 0.4 0.7 .+-. 0.04 0.94 .+-. 0.09 0.96 .+-. 0.06 2 2.0
.+-. 0.08 3.3 .+-. 0.7 3.6 .+-. 0.6 10 9.5 .+-. 0.33 15.3 .+-. 2.5
11.4 .+-. 0.9 100 19.5 .+-. .5 31.4 .+-. 1.9 35.5 .+-. 3.4 1000
32.1 .+-. 0.5 45.3 .+-. 9 46.9 .+-. 9
[0295]
5TABLE II PCR protocols for 5' RACE base cDNA cloning of AC from
AtT20 cell total RNA Reaction using primers 2 or 3 with UAP 1 cycle
95.degree. C. 5 min 80.degree. 5 min (Taq polymerase added) 5
cycles 94.degree. C. 45 sec 55.degree. C. 30 sec 72.degree. C. 90
sec 30 cycles 94.degree. C. 45 sec 57.degree. C. 30 sec 72.degree.
C. 90 sec 1 cycle 72.degree. C. 10 min Reaction using primer 5 with
UAP: 1 cycle 95.degree. C. 5 min 80.degree. C. 5 min (Taq
polymerase added) 35 cycles 94.degree. C. 45 sec 55.degree. C. 30
sec 72.degree. C. 90 sec 1 cycle 72.degree. C. 10 min Reaction
using primer 6 with UAP 1 cycle 95.degree. C. 5 min 80.degree. C. 5
min (Taq polymerase added) 35 cycles 94.degree. C. 60 sec
58.degree. C. 60 sec 72.degree. C. 90 sec 1 cycle 72.degree. C. 10
min
[0296]
6TABLE III Potential sites of phosphorylation by common S/T protein
kinases in AC Position A KINASE: Motif: RXS RAS 85 RES 597 RTS 763
RSS 919 Motif: RXXS RASS 85 (SEQ ID NO: 15) RVDS 201 (SEQ ID NO:
16) RSRS 304 (SEQ ID NO: 17) RESS 597 (SEQ ID NO: 18) RPAS 972 (SEQ
ID NO: 19) RVLS 1213 (SEQ ID NO: 20) C KINASE Motif: (S/T) X (K/R)
S preferred with hydrophobic AA on COOH side of S (23/37 of known
sites are like this) SIR 837 SMR 1022 SWR 1310 SVK 356 SVR 27 SYR
761 SYR 1002 SYR 1211 TAK 534 TLR 187 TVK 619 Motif (K/R) XX (S/T)
(S preferred with hydrophobic AA) KIKTI 1105 (SEQ ID NO: 21) KTATL
266 (SEQ ID NO: 22) KISTL 436 (SEQ ID NO: 23) KKSSI 370 (SEQ ID NO:
24) KEDSL 732 (SEQ ID NO: 25) KFDSM 94 (SEQ ID NO: 26) KIQSM 1019
(SEQ ID NO: 27) RPASL 972 (SEQ ID NO: 28) CASEIN KINASE II: Motif:
3 acidic aa-s after phosphorylation site are a strong indication
(*), highly S preferring SCAEA 581 (SEQ ID NO: 29) SGFEV 588* (SEQ
ID NO: 30) SLCEI 690 (SEQ ID NO: 31) SYQEE 765 (SEQ ID NO: 32)
SEFET 885* (SEQ ID NO: 33) SWREP 1310 (SEQ ID NO: 34) TTSET 261
(SEQ ID NO: 35) TKCEK 432 (SEQ ID NO: 36) TKCEK 432 TGVEC 1199 (SEQ
ID NO: 37) CAM KINASE II: Motif: XRXX (S/T) ERASS 84 (SEQ ID NO:
38) GRVDS 200 (SEQ ID NO: 39) VRSRS 303 (SEQ ID NO: 40) SRESS 596
(SEQ ID NO: 41) RRPAS 971 (SEQ ID NO: 42) YRVLS 1212 (SEQ ID NO:
43) KRHAT 358 (SEQ ID NO: 44) IREKT 719 (SEQ ID NO: 45) SRMDT 1194
(SEQ ID NO: 46) GRSPT 1270 (SEQ ID NO: 47)
TABLE IV
[0297] Inhibition by 1 .mu.mol/l FK506 of the effect of
extracellular calcium ions on 10 nmol/l CRF-induced cAMP formation
in calcium-depleted AtT20 cells in the presence of 0.5 mmol/l IBMX
(see Legend to FIG. 1 and Example 1 for methods). Data are
means.+-.SEM, pmol/well, n=6/group. 2-way ANOVA gave a significant
(P<0.05) interaction between extracellular calcium and FK506.
Unstimulated cAMP levels were 0.5.+-.0.06 pmol/l.
7 Extracellular free [Ca.sup.2+] [mmol/l] Vehicle FK506 0 12.1 .+-.
0.16 10.3 .+-. 0.10 0.5 10.4 .+-. 0.96 10.0 .+-. 0.11 1.0 9.7 .+-.
0.10 10.4 .+-. 0.10 1.5 6.2 .+-. 0.04 11.3 .+-. 0.13 2.0 5.2 .+-.
0.54 9.5 .+-. 0.11
[0298]
Sequence CWU 1
1
104 1 4473 DNA Mouse CDS (43)..(4101) Adenylate cyclase coding
region 1 tgcccctgac ctggtcggga aaggttccaa gagctcggca ac atg gct tcc
tca 54 Met Ala Ser Ser 1 ccc cac cag cag ctg ctg cat cac cat agc
acc gag gtg agc tgc gac 102 Pro His Gln Gln Leu Leu His His His Ser
Thr Glu Val Ser Cys Asp 5 10 15 20 tca agc gga gac agc aac agc gtg
agg gtc aag atc aac cct aag cag 150 Ser Ser Gly Asp Ser Asn Ser Val
Arg Val Lys Ile Asn Pro Lys Gln 25 30 35 ctg tcc tcc aac acc cac
ccg aag cac tgc aag tac agc atc tcc tcc 198 Leu Ser Ser Asn Thr His
Pro Lys His Cys Lys Tyr Ser Ile Ser Ser 40 45 50 agc tgt agc agc
tcg gga gac tca ggg ggc ctt ccc cgg agg gtt ggc 246 Ser Cys Ser Ser
Ser Gly Asp Ser Gly Gly Leu Pro Arg Arg Val Gly 55 60 65 ggc ggg
ggt cgc ctg cgc aga cag aag aag ctg ccc cag ctt ttt gag 294 Gly Gly
Gly Arg Leu Arg Arg Gln Lys Lys Leu Pro Gln Leu Phe Glu 70 75 80
agg gcc tcc agc cgg tgg tgg gac ccc aaa ttc gac tcc atg aac ctg 342
Arg Ala Ser Ser Arg Trp Trp Asp Pro Lys Phe Asp Ser Met Asn Leu 85
90 95 100 gag gag gcc tgc ctg gag cgc tgc ttt ccg cag acc cag cgc
cgc ttc 390 Glu Glu Ala Cys Leu Glu Arg Cys Phe Pro Gln Thr Gln Arg
Arg Phe 105 110 115 cgg tac gca ctc ttt tat gtg ggc ttc gcc tgc ctt
ctc tgg agc atc 438 Arg Tyr Ala Leu Phe Tyr Val Gly Phe Ala Cys Leu
Leu Trp Ser Ile 120 125 130 tat ttc gct gtc cac atg aaa tcc aaa gtg
att gtc atg gtg gtc cca 486 Tyr Phe Ala Val His Met Lys Ser Lys Val
Ile Val Met Val Val Pro 135 140 145 gct ctg tgc ttc ctg gtg gtg tgt
gtg ggc ttt ttc ctg ttt act ttc 534 Ala Leu Cys Phe Leu Val Val Cys
Val Gly Phe Phe Leu Phe Thr Phe 150 155 160 acc aag ctg tac gcc cgg
cat tat gcg tgg acc tcg ctg gct ctc acc 582 Thr Lys Leu Tyr Ala Arg
His Tyr Ala Trp Thr Ser Leu Ala Leu Thr 165 170 175 180 ctg ctg gtg
ttc gcc ctg acc ctg gct gcg cag ttt cag gtt tgg aca 630 Leu Leu Val
Phe Ala Leu Thr Leu Ala Ala Gln Phe Gln Val Trp Thr 185 190 195 cct
ctg tca gga cgt gtt gac agc tcc aat cat act ctc acg gcc act 678 Pro
Leu Ser Gly Arg Val Asp Ser Ser Asn His Thr Leu Thr Ala Thr 200 205
210 ccg gcg gac act tgc tta tct caa gta gga agc ttc tcc ata tgc atc
726 Pro Ala Asp Thr Cys Leu Ser Gln Val Gly Ser Phe Ser Ile Cys Ile
215 220 225 gaa gtg ctc ctt ttg ctc tac aca gtc atg cag tta cct ctg
tac ctg 774 Glu Val Leu Leu Leu Leu Tyr Thr Val Met Gln Leu Pro Leu
Tyr Leu 230 235 240 agc ttg ttt ttg ggg gtg gtc tat tct gtc ctt ttt
gag acc ttc ggc 822 Ser Leu Phe Leu Gly Val Val Tyr Ser Val Leu Phe
Glu Thr Phe Gly 245 250 255 260 tac cac ttc cga aac gaa gac tgc tac
cct tct ccg ggc cct ggg gcc 870 Tyr His Phe Arg Asn Glu Asp Cys Tyr
Pro Ser Pro Gly Pro Gly Ala 265 270 275 ctg cac tgg gag ctg ctg agc
aga gcc ctg ctt cac gtg tgc att cac 918 Leu His Trp Glu Leu Leu Ser
Arg Ala Leu Leu His Val Cys Ile His 280 285 290 gct atc ggg atc cat
ctg ttt gtc atg tct cag gtg agg tcc agg agc 966 Ala Ile Gly Ile His
Leu Phe Val Met Ser Gln Val Arg Ser Arg Ser 295 300 305 acc ttt ctc
aag gtg gga caa tcc att atg cac ggc aaa gat ctg gaa 1014 Thr Phe
Leu Lys Val Gly Gln Ser Ile Met His Gly Lys Asp Leu Glu 310 315 320
gta gag aaa gcc ctg aaa gag agg atg att cat tca gtg atg cca aga
1062 Val Glu Lys Ala Leu Lys Glu Arg Met Ile His Ser Val Met Pro
Arg 325 330 335 340 atc ata gcc gac gac tta atg aaa caa ggg gac gag
gag agt gag aat 1110 Ile Ile Ala Asp Asp Leu Met Lys Gln Gly Asp
Glu Glu Ser Glu Asn 345 350 355 tct gtc aag agg cat gcc acc tcc agt
ccc aag aac agg aag aag aag 1158 Ser Val Lys Arg His Ala Thr Ser
Ser Pro Lys Asn Arg Lys Lys Lys 360 365 370 tcc tcc ata cag aag gca
ccg ata gca ttc cgc ccc ttt aag atg cag 1206 Ser Ser Ile Gln Lys
Ala Pro Ile Ala Phe Arg Pro Phe Lys Met Gln 375 380 385 cag att gaa
gaa gtc agt att tta ttt gca gac att gtg ggt ttc acc 1254 Gln Ile
Glu Glu Val Ser Ile Leu Phe Ala Asp Ile Val Gly Phe Thr 390 395 400
aag atg agc gcc aac aaa tct gcg cat gcc ttg gta ggc cta ctc aat
1302 Lys Met Ser Ala Asn Lys Ser Ala His Ala Leu Val Gly Leu Leu
Asn 405 410 415 420 gac ctg ttc ggt cgc ttt gac cgc ctg tgt gag cag
acc aag tgt gag 1350 Asp Leu Phe Gly Arg Phe Asp Arg Leu Cys Glu
Gln Thr Lys Cys Glu 425 430 435 aag atc agc act ctg ggg gac tgt tat
tac tgt gtg gca ggg tgt ccg 1398 Lys Ile Ser Thr Leu Gly Asp Cys
Tyr Tyr Cys Val Ala Gly Cys Pro 440 445 450 gag ccc cgg gca gac cat
gcc tac tgc tgc att gaa atg ggc tta ggc 1446 Glu Pro Arg Ala Asp
His Ala Tyr Cys Cys Ile Glu Met Gly Leu Gly 455 460 465 atg ata aaa
gcc atc gag cag ttc tgc cag gag aag aaa gag atg gtg 1494 Met Ile
Lys Ala Ile Glu Gln Phe Cys Gln Glu Lys Lys Glu Met Val 470 475 480
aac atg cgt gtt ggg gtt cac acg ggg act gtc ctg tgt ggc atc ctg
1542 Asn Met Arg Val Gly Val His Thr Gly Thr Val Leu Cys Gly Ile
Leu 485 490 495 500 ggc atg agg agg ttt aaa ttt gat gtg tgg tcc aac
gat gtg aac ttg 1590 Gly Met Arg Arg Phe Lys Phe Asp Val Trp Ser
Asn Asp Val Asn Leu 505 510 515 gct aat ctc atg gag cag ctg gga gtg
gct ggc aaa gtt cac ata tct 1638 Ala Asn Leu Met Glu Gln Leu Gly
Val Ala Gly Lys Val His Ile Ser 520 525 530 gag gcc act gca aaa tac
tta gac gac agg tat gaa atg gaa gat ggg 1686 Glu Ala Thr Ala Lys
Tyr Leu Asp Asp Arg Tyr Glu Met Glu Asp Gly 535 540 545 aga gtt att
gag cgc ctt ggg cag agt gtg gtg gct gac cag ttg aaa 1734 Arg Val
Ile Glu Arg Leu Gly Gln Ser Val Val Ala Asp Gln Leu Lys 550 555 560
ggt ttg aag aca tac ctg ata tcg ggt cag aga gcc aag gag tcc cac
1782 Gly Leu Lys Thr Tyr Leu Ile Ser Gly Gln Arg Ala Lys Glu Ser
His 565 570 575 580 tgc agc tgt gca gag gcc ctg ctt tct ggc ttt gag
gtc att gac gac 1830 Cys Ser Cys Ala Glu Ala Leu Leu Ser Gly Phe
Glu Val Ile Asp Asp 585 590 595 tca cgg gag tcc tca ggc cct agg gga
cag ggg aca gca tcg cca ggg 1878 Ser Arg Glu Ser Ser Gly Pro Arg
Gly Gln Gly Thr Ala Ser Pro Gly 600 605 610 agt gtc agt gat ttg gcg
cag act gtc aaa acc ttt gat aac ctt aag 1926 Ser Val Ser Asp Leu
Ala Gln Thr Val Lys Thr Phe Asp Asn Leu Lys 615 620 625 act tgc cct
tct tgt gga atc aca ttt gct ccc aaa tct gaa gct ggt 1974 Thr Cys
Pro Ser Cys Gly Ile Thr Phe Ala Pro Lys Ser Glu Ala Gly 630 635 640
gca gaa gga gga act gtg caa aat ggc tgt caa gac gag cct aag acc
2022 Ala Glu Gly Gly Thr Val Gln Asn Gly Cys Gln Asp Glu Pro Lys
Thr 645 650 655 660 agc acc aag gct tct gga gga ccc aac tcc aaa acc
cag aat gga ctt 2070 Ser Thr Lys Ala Ser Gly Gly Pro Asn Ser Lys
Thr Gln Asn Gly Leu 665 670 675 ctg agc cct cct gca gag gag aag ctc
act aac agc cag acc tcc ctc 2118 Leu Ser Pro Pro Ala Glu Glu Lys
Leu Thr Asn Ser Gln Thr Ser Leu 680 685 690 tgt gag atc ctg caa gag
aag gga cgg tgg gca ggc gtg agc ttg gac 2166 Cys Glu Ile Leu Gln
Glu Lys Gly Arg Trp Ala Gly Val Ser Leu Asp 695 700 705 cag tca gcc
ctc ctc ccg ctc agg ttc aag aac atc cgt gag aaa act 2214 Gln Ser
Ala Leu Leu Pro Leu Arg Phe Lys Asn Ile Arg Glu Lys Thr 710 715 720
gat gcc cac ttt gtt gat gtc atc aaa gaa gac agc ctg atg aaa gat
2262 Asp Ala His Phe Val Asp Val Ile Lys Glu Asp Ser Leu Met Lys
Asp 725 730 735 740 tat ttc ttc aag ccg ccc atc aat cag ttc agc ctg
aac ttc ctg gac 2310 Tyr Phe Phe Lys Pro Pro Ile Asn Gln Phe Ser
Leu Asn Phe Leu Asp 745 750 755 cag gag ctg gag cga tca tat aga acc
agc tac cag gaa gag gtc ata 2358 Gln Glu Leu Glu Arg Ser Tyr Arg
Thr Ser Tyr Gln Glu Glu Val Ile 760 765 770 aag aat tct cct gtg aag
acg ttc gcc agt gcc acc ttc agc tcc ctt 2406 Lys Asn Ser Pro Val
Lys Thr Phe Ala Ser Ala Thr Phe Ser Ser Leu 775 780 785 ctg gat gtg
ttt ctg tca acc acc gtg ttc ctg att ctc tcc atc acc 2454 Leu Asp
Val Phe Leu Ser Thr Thr Val Phe Leu Ile Leu Ser Ile Thr 790 795 800
tgc ttc cta aag tat gga gcc acc gcc acc cct ccc cca ccg gct gcc
2502 Cys Phe Leu Lys Tyr Gly Ala Thr Ala Thr Pro Pro Pro Pro Ala
Ala 805 810 815 820 ctg gcc gtc ttt ggt gca gac ctg ctg ctg gag gtg
ctt tcc ctc ata 2550 Leu Ala Val Phe Gly Ala Asp Leu Leu Leu Glu
Val Leu Ser Leu Ile 825 830 835 gtg tcc atc aga atg gtg ttt ttc cta
gag gat gtc atg aca tgc aca 2598 Val Ser Ile Arg Met Val Phe Phe
Leu Glu Asp Val Met Thr Cys Thr 840 845 850 aag tgg ttg ctg gaa tgg
atc gct ggc tgg ctc cct cgc cac tgc att 2646 Lys Trp Leu Leu Glu
Trp Ile Ala Gly Trp Leu Pro Arg His Cys Ile 855 860 865 ggg gca atc
ttg gtg tct ctt cct gcc ctg gct gtc tat tca cac atc 2694 Gly Ala
Ile Leu Val Ser Leu Pro Ala Leu Ala Val Tyr Ser His Ile 870 875 880
acc tct gag ttt gag acc aac ata cat gtc acc atg ttc act ggc tct
2742 Thr Ser Glu Phe Glu Thr Asn Ile His Val Thr Met Phe Thr Gly
Ser 885 890 895 900 gcg gtg ctg gtg gcc gtt gtg cac tac tgt aac ttc
tgc cag ctc agc 2790 Ala Val Leu Val Ala Val Val His Tyr Cys Asn
Phe Cys Gln Leu Ser 905 910 915 tcc tgg atg agg tcc tcc ctt gcc acc
atc gtg ggg gct ggg ctg ctg 2838 Ser Trp Met Arg Ser Ser Leu Ala
Thr Ile Val Gly Ala Gly Leu Leu 920 925 930 ctt ctg ctc cac atc tcc
ctg tgt cag gac agt tcc att gtg atg tcc 2886 Leu Leu Leu His Ile
Ser Leu Cys Gln Asp Ser Ser Ile Val Met Ser 935 940 945 ccc ttg gac
tca gca cag aat ttc agt gcc cag agg aac cca tgc aac 2934 Pro Leu
Asp Ser Ala Gln Asn Phe Ser Ala Gln Arg Asn Pro Cys Asn 950 955 960
agc tca gtg ctg cag gac ggc agg agg ccg gcc agc ctc ata ggc aag
2982 Ser Ser Val Leu Gln Asp Gly Arg Arg Pro Ala Ser Leu Ile Gly
Lys 965 970 975 980 gag ctt atc ctc acc ttc ttc ctc ctg ctc ctc ttg
gtc tgg ttc ctg 3030 Glu Leu Ile Leu Thr Phe Phe Leu Leu Leu Leu
Leu Val Trp Phe Leu 985 990 995 aac cgg gag ttc gag gtc agc tac cgg
ctg cac tac cat ggg gat 3075 Asn Arg Glu Phe Glu Val Ser Tyr Arg
Leu His Tyr His Gly Asp 1000 1005 1010 gtg gag gcc gac cta cac cgc
acc aag atc cag agc atg aga gac 3120 Val Glu Ala Asp Leu His Arg
Thr Lys Ile Gln Ser Met Arg Asp 1015 1020 1025 cag gct gac tgg cta
ctg cgg aac atc atc ccc tac cat gtg gct 3165 Gln Ala Asp Trp Leu
Leu Arg Asn Ile Ile Pro Tyr His Val Ala 1030 1035 1040 gag cag ctc
aag gtc tct cag acc tac tcc aag aac cat gac agc 3210 Glu Gln Leu
Lys Val Ser Gln Thr Tyr Ser Lys Asn His Asp Ser 1045 1050 1055 ggg
gga gtc atc ttt gcc agc att gtc aac ttc agt gaa ttc tat 3255 Gly
Gly Val Ile Phe Ala Ser Ile Val Asn Phe Ser Glu Phe Tyr 1060 1065
1070 gag gag aac tat gag ggg ggc aag gag tgc tac cgt gtc ctc aac
3300 Glu Glu Asn Tyr Glu Gly Gly Lys Glu Cys Tyr Arg Val Leu Asn
1075 1080 1085 gag ctg atc ggt gac ttc gat gag ctc ttg agc aag ccg
gac tat 3345 Glu Leu Ile Gly Asp Phe Asp Glu Leu Leu Ser Lys Pro
Asp Tyr 1090 1095 1100 aat agc atc gag aag atc aag acc atc ggg gcc
aca tac atg gca 3390 Asn Ser Ile Glu Lys Ile Lys Thr Ile Gly Ala
Thr Tyr Met Ala 1105 1110 1115 gcc tca ggg ctg aac acg gcc cag tgt
cag gag ggt ggc cac cca 3435 Ala Ser Gly Leu Asn Thr Ala Gln Cys
Gln Glu Gly Gly His Pro 1120 1125 1130 cag gag cat ctg cgt atc ctc
ttc gag ttc gcc aag gag atg atg 3480 Gln Glu His Leu Arg Ile Leu
Phe Glu Phe Ala Lys Glu Met Met 1135 1140 1145 cgc gtg gtg gat gac
ttc aac aac aat atg tta tgg ttc aac ttc 3525 Arg Val Val Asp Asp
Phe Asn Asn Asn Met Leu Trp Phe Asn Phe 1150 1155 1160 aag ctc agg
gtc ggc ttt aac cac gga ccc ctc aca gca ggt gtc 3570 Lys Leu Arg
Val Gly Phe Asn His Gly Pro Leu Thr Ala Gly Val 1165 1170 1175 ata
ggt acc acc aag ctg ctg tat gac atc tgg ggg gac acc gtc 3615 Ile
Gly Thr Thr Lys Leu Leu Tyr Asp Ile Trp Gly Asp Thr Val 1180 1185
1190 aac atc gcc agc agg atg gac acc act ggt gtg gag tgc cgt atc
3660 Asn Ile Ala Ser Arg Met Asp Thr Thr Gly Val Glu Cys Arg Ile
1195 1200 1205 cag gtg agc gaa gag agc tac cgt gtg ctg agc aag atg
ggt tat 3705 Gln Val Ser Glu Glu Ser Tyr Arg Val Leu Ser Lys Met
Gly Tyr 1210 1215 1220 gac ttt gac tac cga ggg acc gtg aat gtc aag
ggg aaa ggg cag 3750 Asp Phe Asp Tyr Arg Gly Thr Val Asn Val Lys
Gly Lys Gly Gln 1225 1230 1235 atg aag acc tac ctt tac cca aag tgc
acg gac aat gga gtg gtt 3795 Met Lys Thr Tyr Leu Tyr Pro Lys Cys
Thr Asp Asn Gly Val Val 1240 1245 1250 ccc cag cac cag ctg tcc atc
tcc cca gac atc cga gtc cag gtg 3840 Pro Gln His Gln Leu Ser Ile
Ser Pro Asp Ile Arg Val Gln Val 1255 1260 1265 gac ggc agc att ggg
cgg tct ccc aca gat gag att gcc aac ttg 3885 Asp Gly Ser Ile Gly
Arg Ser Pro Thr Asp Glu Ile Ala Asn Leu 1270 1275 1280 gtg cct tcc
gtt cag tat tcg gac aag gct tcc ctg gga tct gat 3930 Val Pro Ser
Val Gln Tyr Ser Asp Lys Ala Ser Leu Gly Ser Asp 1285 1290 1295 gat
agc aca cag gct aag gaa gct cac ctg tcc tct aag agg tcc 3975 Asp
Ser Thr Gln Ala Lys Glu Ala His Leu Ser Ser Lys Arg Ser 1300 1305
1310 tgg aga gag cca gtc aaa gca gag gaa agg ttt cca ttt ggc aaa
4020 Trp Arg Glu Pro Val Lys Ala Glu Glu Arg Phe Pro Phe Gly Lys
1315 1320 1325 gcc ata gaa aag gac agc tgt gaa gac ata gga gta gaa
gag gcc 4065 Ala Ile Glu Lys Asp Ser Cys Glu Asp Ile Gly Val Glu
Glu Ala 1330 1335 1340 agt gaa ctc agc aag ctc aat gtc tca aag agt
gtg tgaggcagcg 4111 Ser Glu Leu Ser Lys Leu Asn Val Ser Lys Ser Val
1345 1350 ccgagagctg ccaaggtgct ctgcgtgtcc aaacacagta acatctgtgt
cgataggctg 4171 ttgtgctatc tagcacctca gtttctgtcc ccagatgtgg
tgtcacgtgg tcatttcagc 4231 ccgaatctct gtgtggagca cagttattca
gggttcattt ccacccattt cggttttcct 4291 ttacttgcgt tcctggaagc
cttttcctgg aagcctgccc ccagcccagc caggggatcc 4351 agtcagcagc
gtggagggat tcaagtgcct tcagggtctg gccttgcgtc tggggctgag 4411
gccactggtg gaatcatggc cctggggatt atttgacttc tttaagtttt tttttttttt
4471 tt 4473 2 1353 PRT Mouse 2 Met Ala Ser Ser Pro His Gln Gln Leu
Leu His His His Ser Thr Glu 1 5 10 15 Val Ser Cys Asp Ser Ser Gly
Asp Ser Asn Ser Val Arg Val Lys Ile 20 25 30 Asn Pro Lys Gln Leu
Ser Ser Asn Thr His Pro Lys His Cys Lys Tyr 35 40 45 Ser Ile Ser
Ser Ser Cys Ser Ser Ser Gly Asp Ser Gly Gly Leu Pro 50 55 60 Arg
Arg Val Gly Gly Gly Gly Arg Leu Arg Arg Gln Lys Lys Leu Pro 65 70
75 80 Gln Leu Phe Glu Arg Ala Ser Ser Arg Trp Trp Asp Pro Lys Phe
Asp 85 90 95 Ser Met Asn Leu Glu Glu Ala Cys Leu Glu Arg Cys Phe
Pro Gln Thr 100 105 110 Gln Arg Arg Phe Arg Tyr Ala Leu Phe Tyr Val
Gly Phe Ala Cys Leu 115
120 125 Leu Trp Ser Ile Tyr Phe Ala Val His Met Lys Ser Lys Val Ile
Val 130 135 140 Met Val Val Pro Ala Leu Cys Phe Leu Val Val Cys Val
Gly Phe Phe 145 150 155 160 Leu Phe Thr Phe Thr Lys Leu Tyr Ala Arg
His Tyr Ala Trp Thr Ser 165 170 175 Leu Ala Leu Thr Leu Leu Val Phe
Ala Leu Thr Leu Ala Ala Gln Phe 180 185 190 Gln Val Trp Thr Pro Leu
Ser Gly Arg Val Asp Ser Ser Asn His Thr 195 200 205 Leu Thr Ala Thr
Pro Ala Asp Thr Cys Leu Ser Gln Val Gly Ser Phe 210 215 220 Ser Ile
Cys Ile Glu Val Leu Leu Leu Leu Tyr Thr Val Met Gln Leu 225 230 235
240 Pro Leu Tyr Leu Ser Leu Phe Leu Gly Val Val Tyr Ser Val Leu Phe
245 250 255 Glu Thr Phe Gly Tyr His Phe Arg Asn Glu Asp Cys Tyr Pro
Ser Pro 260 265 270 Gly Pro Gly Ala Leu His Trp Glu Leu Leu Ser Arg
Ala Leu Leu His 275 280 285 Val Cys Ile His Ala Ile Gly Ile His Leu
Phe Val Met Ser Gln Val 290 295 300 Arg Ser Arg Ser Thr Phe Leu Lys
Val Gly Gln Ser Ile Met His Gly 305 310 315 320 Lys Asp Leu Glu Val
Glu Lys Ala Leu Lys Glu Arg Met Ile His Ser 325 330 335 Val Met Pro
Arg Ile Ile Ala Asp Asp Leu Met Lys Gln Gly Asp Glu 340 345 350 Glu
Ser Glu Asn Ser Val Lys Arg His Ala Thr Ser Ser Pro Lys Asn 355 360
365 Arg Lys Lys Lys Ser Ser Ile Gln Lys Ala Pro Ile Ala Phe Arg Pro
370 375 380 Phe Lys Met Gln Gln Ile Glu Glu Val Ser Ile Leu Phe Ala
Asp Ile 385 390 395 400 Val Gly Phe Thr Lys Met Ser Ala Asn Lys Ser
Ala His Ala Leu Val 405 410 415 Gly Leu Leu Asn Asp Leu Phe Gly Arg
Phe Asp Arg Leu Cys Glu Gln 420 425 430 Thr Lys Cys Glu Lys Ile Ser
Thr Leu Gly Asp Cys Tyr Tyr Cys Val 435 440 445 Ala Gly Cys Pro Glu
Pro Arg Ala Asp His Ala Tyr Cys Cys Ile Glu 450 455 460 Met Gly Leu
Gly Met Ile Lys Ala Ile Glu Gln Phe Cys Gln Glu Lys 465 470 475 480
Lys Glu Met Val Asn Met Arg Val Gly Val His Thr Gly Thr Val Leu 485
490 495 Cys Gly Ile Leu Gly Met Arg Arg Phe Lys Phe Asp Val Trp Ser
Asn 500 505 510 Asp Val Asn Leu Ala Asn Leu Met Glu Gln Leu Gly Val
Ala Gly Lys 515 520 525 Val His Ile Ser Glu Ala Thr Ala Lys Tyr Leu
Asp Asp Arg Tyr Glu 530 535 540 Met Glu Asp Gly Arg Val Ile Glu Arg
Leu Gly Gln Ser Val Val Ala 545 550 555 560 Asp Gln Leu Lys Gly Leu
Lys Thr Tyr Leu Ile Ser Gly Gln Arg Ala 565 570 575 Lys Glu Ser His
Cys Ser Cys Ala Glu Ala Leu Leu Ser Gly Phe Glu 580 585 590 Val Ile
Asp Asp Ser Arg Glu Ser Ser Gly Pro Arg Gly Gln Gly Thr 595 600 605
Ala Ser Pro Gly Ser Val Ser Asp Leu Ala Gln Thr Val Lys Thr Phe 610
615 620 Asp Asn Leu Lys Thr Cys Pro Ser Cys Gly Ile Thr Phe Ala Pro
Lys 625 630 635 640 Ser Glu Ala Gly Ala Glu Gly Gly Thr Val Gln Asn
Gly Cys Gln Asp 645 650 655 Glu Pro Lys Thr Ser Thr Lys Ala Ser Gly
Gly Pro Asn Ser Lys Thr 660 665 670 Gln Asn Gly Leu Leu Ser Pro Pro
Ala Glu Glu Lys Leu Thr Asn Ser 675 680 685 Gln Thr Ser Leu Cys Glu
Ile Leu Gln Glu Lys Gly Arg Trp Ala Gly 690 695 700 Val Ser Leu Asp
Gln Ser Ala Leu Leu Pro Leu Arg Phe Lys Asn Ile 705 710 715 720 Arg
Glu Lys Thr Asp Ala His Phe Val Asp Val Ile Lys Glu Asp Ser 725 730
735 Leu Met Lys Asp Tyr Phe Phe Lys Pro Pro Ile Asn Gln Phe Ser Leu
740 745 750 Asn Phe Leu Asp Gln Glu Leu Glu Arg Ser Tyr Arg Thr Ser
Tyr Gln 755 760 765 Glu Glu Val Ile Lys Asn Ser Pro Val Lys Thr Phe
Ala Ser Ala Thr 770 775 780 Phe Ser Ser Leu Leu Asp Val Phe Leu Ser
Thr Thr Val Phe Leu Ile 785 790 795 800 Leu Ser Ile Thr Cys Phe Leu
Lys Tyr Gly Ala Thr Ala Thr Pro Pro 805 810 815 Pro Pro Ala Ala Leu
Ala Val Phe Gly Ala Asp Leu Leu Leu Glu Val 820 825 830 Leu Ser Leu
Ile Val Ser Ile Arg Met Val Phe Phe Leu Glu Asp Val 835 840 845 Met
Thr Cys Thr Lys Trp Leu Leu Glu Trp Ile Ala Gly Trp Leu Pro 850 855
860 Arg His Cys Ile Gly Ala Ile Leu Val Ser Leu Pro Ala Leu Ala Val
865 870 875 880 Tyr Ser His Ile Thr Ser Glu Phe Glu Thr Asn Ile His
Val Thr Met 885 890 895 Phe Thr Gly Ser Ala Val Leu Val Ala Val Val
His Tyr Cys Asn Phe 900 905 910 Cys Gln Leu Ser Ser Trp Met Arg Ser
Ser Leu Ala Thr Ile Val Gly 915 920 925 Ala Gly Leu Leu Leu Leu Leu
His Ile Ser Leu Cys Gln Asp Ser Ser 930 935 940 Ile Val Met Ser Pro
Leu Asp Ser Ala Gln Asn Phe Ser Ala Gln Arg 945 950 955 960 Asn Pro
Cys Asn Ser Ser Val Leu Gln Asp Gly Arg Arg Pro Ala Ser 965 970 975
Leu Ile Gly Lys Glu Leu Ile Leu Thr Phe Phe Leu Leu Leu Leu Leu 980
985 990 Val Trp Phe Leu Asn Arg Glu Phe Glu Val Ser Tyr Arg Leu His
Tyr 995 1000 1005 His Gly Asp Val Glu Ala Asp Leu His Arg Thr Lys
Ile Gln Ser 1010 1015 1020 Met Arg Asp Gln Ala Asp Trp Leu Leu Arg
Asn Ile Ile Pro Tyr 1025 1030 1035 His Val Ala Glu Gln Leu Lys Val
Ser Gln Thr Tyr Ser Lys Asn 1040 1045 1050 His Asp Ser Gly Gly Val
Ile Phe Ala Ser Ile Val Asn Phe Ser 1055 1060 1065 Glu Phe Tyr Glu
Glu Asn Tyr Glu Gly Gly Lys Glu Cys Tyr Arg 1070 1075 1080 Val Leu
Asn Glu Leu Ile Gly Asp Phe Asp Glu Leu Leu Ser Lys 1085 1090 1095
Pro Asp Tyr Asn Ser Ile Glu Lys Ile Lys Thr Ile Gly Ala Thr 1100
1105 1110 Tyr Met Ala Ala Ser Gly Leu Asn Thr Ala Gln Cys Gln Glu
Gly 1115 1120 1125 Gly His Pro Gln Glu His Leu Arg Ile Leu Phe Glu
Phe Ala Lys 1130 1135 1140 Glu Met Met Arg Val Val Asp Asp Phe Asn
Asn Asn Met Leu Trp 1145 1150 1155 Phe Asn Phe Lys Leu Arg Val Gly
Phe Asn His Gly Pro Leu Thr 1160 1165 1170 Ala Gly Val Ile Gly Thr
Thr Lys Leu Leu Tyr Asp Ile Trp Gly 1175 1180 1185 Asp Thr Val Asn
Ile Ala Ser Arg Met Asp Thr Thr Gly Val Glu 1190 1195 1200 Cys Arg
Ile Gln Val Ser Glu Glu Ser Tyr Arg Val Leu Ser Lys 1205 1210 1215
Met Gly Tyr Asp Phe Asp Tyr Arg Gly Thr Val Asn Val Lys Gly 1220
1225 1230 Lys Gly Gln Met Lys Thr Tyr Leu Tyr Pro Lys Cys Thr Asp
Asn 1235 1240 1245 Gly Val Val Pro Gln His Gln Leu Ser Ile Ser Pro
Asp Ile Arg 1250 1255 1260 Val Gln Val Asp Gly Ser Ile Gly Arg Ser
Pro Thr Asp Glu Ile 1265 1270 1275 Ala Asn Leu Val Pro Ser Val Gln
Tyr Ser Asp Lys Ala Ser Leu 1280 1285 1290 Gly Ser Asp Asp Ser Thr
Gln Ala Lys Glu Ala His Leu Ser Ser 1295 1300 1305 Lys Arg Ser Trp
Arg Glu Pro Val Lys Ala Glu Glu Arg Phe Pro 1310 1315 1320 Phe Gly
Lys Ala Ile Glu Lys Asp Ser Cys Glu Asp Ile Gly Val 1325 1330 1335
Glu Glu Ala Ser Glu Leu Ser Lys Leu Asn Val Ser Lys Ser Val 1340
1345 1350 3 10 PRT Unidentified organism 3 Leu Arg Gln Ser Arg Leu
Ser Ser Ser Lys 1 5 10 4 11 PRT Mouse 4 Ile Asp Asp Ser Arg Glu Ser
Ser Gly Pro Arg 1 5 10 5 26 DNA Artificial Sequence PCR primer
corresponding to highly conserved first cytoplasmic domain of
mammalian adenyl cyclases. 5 ctcatcgatg gagaytgyta ytaytg 26 6 26
DNA Artificial Sequence PCR primer corresponding to highly
conserved first cytoplasmic domain of mammalian adenyl cyclases. 6
ggctcgagcc aaacrtcrta ytgcca 26 7 39 DNA Artificial Sequence PCR
primer corresponding to highly conserved second cytoplasmic domain
of mammallian adenyl cyclase. 7 gaagcttaar ataaaracaa taggawsaac
atayatggc 39 8 36 DNA Artificial Sequence PCR primer corresponding
to highly conserved second cytoplasmic domain of mammalian adenyl
cyclases 8 gggatccacr ttaacagtrt taccccaaat rtcrta 36 9 19 DNA
Artificial Sequence PCR primer to cDNA clone jp134 of AtT20 9
cgtcaatgac ctcaaagcc 19 10 27 DNA Artificial sequence PCR primer to
cDNA clone jp134 of AtT20 10 gcctctgcac agctgcagtg ggactcc 27 11 33
DNA Artificial sequence PCR primer to cDNA clone jp134 of AtT20 11
cctggcagaa ctgctcgatg gcttttatca tgc 33 12 18 DNA Artificial
sequence PCR primer to 1kb extension of cDNA clone jp134 of AtT20
12 ggagaagctt cctacttg 18 13 29 DNA Artificial sequence PCR primer
to 1kb extension of cDNA clone jp134 of AtT20. 13 gtggccgtga
gagtatgatt ggagctgtc 29 14 26 DNA Artificial sequence PCR primer to
1kb extension of cDNA clone jp134 of AtT20 14 gtccaaacct gaaactgcgc
acgcag 26 15 4 PRT Mouse 15 Arg Ala Ser Ser 1 16 4 PRT Mouse 16 Arg
Val Asp Ser 1 17 4 PRT Mouse 17 Arg Ser Arg Ser 1 18 4 PRT Mouse 18
Arg Glu Ser Ser 1 19 4 PRT Mouse 19 Arg Pro Ala Ser 1 20 4 PRT
Mouse 20 Arg Val Leu Ser 1 21 5 PRT Mouse 21 Lys Ile Lys Thr Ile 1
5 22 5 PRT Mouse 22 Lys Thr Ala Thr Leu 1 5 23 5 PRT Mouse 23 Lys
Ile Ser Thr Leu 1 5 24 5 PRT Mouse 24 Lys Lys Ser Ser Ile 1 5 25 5
PRT Mouse 25 Lys Glu Asp Ser Leu 1 5 26 5 PRT Mouse 26 Lys Phe Asp
Ser Met 1 5 27 5 PRT Mouse 27 Lys Ile Gln Ser Met 1 5 28 5 PRT
Mouse 28 Arg Pro Ala Ser Leu 1 5 29 5 PRT Mouse 29 Ser Cys Ala Glu
Ala 1 5 30 5 PRT Mouse 30 Ser Gly Phe Glu Val 1 5 31 5 PRT Mouse 31
Ser Leu Cys Glu Ile 1 5 32 5 PRT Mouse 32 Ser Tyr Gln Glu Glu 1 5
33 5 PRT Mouse 33 Ser Glu Phe Glu Thr 1 5 34 5 PRT Mouse 34 Ser Trp
Arg Glu Pro 1 5 35 5 PRT Mouse 35 Thr Thr Ser Glu Thr 1 5 36 5 PRT
Mouse 36 Thr Lys Cys Glu Lys 1 5 37 5 PRT Mouse 37 Thr Gly Val Glu
Cys 1 5 38 5 PRT Mouse 38 Glu Arg Ala Ser Ser 1 5 39 5 PRT Mouse 39
Gly Arg Val Asp Ser 1 5 40 5 PRT Mouse 40 Val Arg Ser Arg Ser 1 5
41 5 PRT Mouse 41 Ser Arg Glu Ser Ser 1 5 42 5 PRT Mouse 42 Arg Arg
Pro Ala Ser 1 5 43 5 PRT Mouse 43 Tyr Arg Val Leu Ser 1 5 44 5 PRT
Mouse 44 Lys Arg His Ala Thr 1 5 45 5 PRT Mouse 45 Ile Arg Glu Lys
Thr 1 5 46 5 PRT Mouse 46 Ser Arg Met Asp Thr 1 5 47 5 PRT Mouse 47
Gly Arg Ser Pro Thr 1 5 48 90 PRT Homo sapiens 48 Lys Ile Lys Thr
Ile Gly Ser Thr Tyr Met Ala Ala Ala Gly Leu Ser 1 5 10 15 Val Ala
Ser Gly His Glu Asn Gln Glu Leu Glu Arg Gln His Ala His 20 25 30
Ile Gly Val Met Val Glu Phe Ser Ile Ala Leu Met Ser Lys Leu Asp 35
40 45 Gly Ile Asn Arg His Ser Phe Asn Ser Phe Arg Leu Arg Val Gly
Ile 50 55 60 Asn His Gly Pro Val Ile Ala Gly Val Ile Gly Ala Arg
Lys Pro Gln 65 70 75 80 Tyr Asp Ile Trp Gly Asn Thr Val Asn Val 85
90 49 90 PRT Mouse 49 Lys Ile Lys Thr Ile Gly Ser Thr Tyr Met Ala
Ala Ala Gly Leu Ser 1 5 10 15 Ala Pro Ser Gly His Glu Asn Gln Asp
Leu Glu Arg Lys His Val His 20 25 30 Ile Gly Val Leu Val Glu Phe
Ser Met Ala Leu Met Ser Lys Leu Asp 35 40 45 Gly Ile Asn Arg His
Ser Phe Asn Ser Phe Arg Leu Arg Val Gly Ile 50 55 60 Asn His Gly
Pro Val Ile Ala Gly Val Ile Gly Ala Arg Lys Pro Gln 65 70 75 80 Tyr
Asp Ile Trp Gly Asn Thr Val Asn Val 85 90 50 91 PRT rat 50 Lys Ile
Lys Thr Ile Gly Ser Thr Tyr Met Ala Ala Thr Gly Leu Ser 1 5 10 15
Ala Ile Pro Ser Gln Glu His Ala Gln Glu Pro Glu Arg Gln Tyr Met 20
25 30 His Ile Gly Thr Met Val Glu Phe Ala Tyr Ala Leu Val Gly Lys
Leu 35 40 45 Asp Ala Ile Asn Lys His Ser Phe Asn Asp Phe Lys Leu
Arg Val Gly 50 55 60 Ile Asn His Gly Pro Val Ile Ala Gly Val Ile
Gly Ala Gln Lys Pro 65 70 75 80 Gln Tyr Asp Ile Trp Gly Asn Thr Val
Asn Val 85 90 51 91 PRT rat 51 Lys Ile Lys Thr Ile Gly Ser Thr Tyr
Met Ala Ala Thr Gly Leu Asn 1 5 10 15 Ala Thr Pro Gly Gln Asp Thr
Gln Gln Asp Ala Glu Arg Ser Cys Ser 20 25 30 His Leu Gly Thr Met
Val Glu Phe Ala Val Ala Leu Gly Ser Lys Leu 35 40 45 Gly Val Ile
Asn Lys His Ser Phe Asn Asn Phe Arg Leu Arg Val Gly 50 55 60 Leu
Asn His Gly Pro Val Val Ala Gly Val Ile Gly Ala Gln Lys Pro 65 70
75 80 Gln Tyr Asp Ile Trp Gly Asn Thr Val Asn Val 85 90 52 86 PRT
Homo sapiens 52 Lys Ile Lys Thr Ile Gly Ser Thr Tyr Met Ala Val Ser
Gly Leu Ser 1 5 10 15 Pro Glu Lys Gln Gln Cys Glu Asp Lys Trp Gly
His Leu Cys Ala Leu 20 25 30 Ala Asp Phe Ser Leu Ala Leu Thr Glu
Ser Ile Gln Glu Ile Asn Lys 35 40 45 His Ser Phe Asn Asn Phe Glu
Leu Arg Ile Gly Ile Ser His Gly Ser 50 55 60 Val Val Ala Gly Val
Ile Gly Ala Lys Lys Pro Gln Tyr Asp Ile Trp 65 70 75 80 Gly Lys Thr
Val Asn Leu 85 53 86 PRT rat 53 Lys Ile Lys Thr Ile Gly Ser Thr Tyr
Met Ala Val Ser Gly Leu Ser 1 5 10 15 Pro Glu Lys Gln Gln Cys Glu
Asp Lys Trp Gly His Leu Cys Ala Leu 20 25 30 Ala Asp Phe Ser Leu
Ala Leu Thr Glu Ser Ile Gln Glu Ile Asn Lys 35 40 45 His Ser Phe
Asn Asn Phe Glu Leu Arg Ile Gly Ile Ser His Gly Ser 50 55 60 Val
Val Ala Gly Val Ile Gly Ala Lys Lys Pro Gln Tyr Asp Ile Trp 65 70
75 80 Gly Lys Thr Val Asn Leu 85 54 85 PRT Homo sapiens 54 Lys Ile
Lys Thr Ile Gly Ser Thr Tyr Met Ala Ala Ser Gly Leu Asn 1 5 10 15
Ala Ser Thr Tyr Asp Gln Val Gly Arg Ser His Ile Thr Ala Leu Ala 20
25 30 Asp Tyr Ala Met Arg Leu Met Glu Gln Met Lys His Ile Asn Glu
His 35 40 45 Ser Phe Asn Asn Phe Gln Met Lys Ile Gly Leu Asn Met
Gly Pro Val 50 55 60 Val Ala Gly Val Ile Gly Ala Arg Lys Pro Gln
Tyr Asp Ile Trp Gly 65 70 75 80 Asn Thr Val Asn Val 85 55 85 PRT
Mouse 55 Lys Ile Lys Thr Ile Gly Ser Thr Tyr Met Ala Ala Ser Gly
Leu Asn 1 5 10 15 Ala Ser Thr Tyr Asp Gln Val Gly Arg Ser His Ile
Thr Ala Leu Ala 20 25 30 Asp Tyr Ala Met Arg Leu Met Glu Gln Met
Lys His Ile Asn Glu His 35 40 45 Ser Phe Asn Asn Phe Gln Met Lys
Ile Gly Leu Asn Met Gly Pro Val 50
55 60 Val Ala Gly Val Ile Gly Ala Arg Lys Pro Gln Tyr Asp Ile Trp
Gly 65 70 75 80 Asn Thr Val Asn Val 85 56 85 PRT Mouse 56 Lys Ile
Lys Thr Ile Gly Ser Thr Tyr Met Ala Ala Ser Gly Leu Asn 1 5 10 15
Ala Ser Thr Tyr Asp Gln Val Gly Arg Ser His Ile Thr Ala Leu Ala 20
25 30 Asp Tyr Ala Met Arg Leu Met Glu Gln Met Lys His Ile Asn Glu
His 35 40 45 Ser Phe Asn Asn Phe Gln Met Lys Ile Gly Leu Asn Met
Gly Pro Val 50 55 60 Val Ala Gly Val Ile Gly Ala Arg Lys Pro Gln
Tyr Asp Ile Trp Gly 65 70 75 80 Asn Thr Val Asn Val 85 57 85 PRT
Rat 57 Lys Ile Lys Thr Ile Gly Ser Thr Tyr Met Ala Ala Ser Gly Leu
Asn 1 5 10 15 Ala Ser Thr Tyr Asp Gln Val Gly Arg Ser His Ile Thr
Ala Leu Ala 20 25 30 Asp Tyr Ala Met Arg Leu Met Glu Gln Met Lys
His Ile Asn Glu His 35 40 45 Ser Phe Asn Asn Phe Gln Met Lys Ile
Gly Leu Asn Met Gly Pro Val 50 55 60 Val Ala Gly Val Ile Gly Ala
Arg Lys Pro Gln Tyr Asp Ile Trp Gly 65 70 75 80 Asn Thr Val Asn Val
85 58 85 PRT Dog 58 Lys Ile Lys Thr Ile Gly Ser Thr Tyr Met Ala Ala
Ser Gly Leu Asn 1 5 10 15 Ala Ser Thr Tyr Asp Gln Ala Gly Arg Ser
His Ile Thr Ala Leu Ala 20 25 30 Asp Tyr Ala Met Arg Leu Met Glu
Gln Met Lys His Ile Asn Glu His 35 40 45 Ser Phe Asn Asn Phe Gln
Met Lys Ile Gly Leu Asn Met Gly Pro Val 50 55 60 Val Ala Gly Val
Ile Gly Ala Arg Lys Pro Gln Tyr Asp Ile Trp Gly 65 70 75 80 Asn Thr
Val Asn Val 85 59 85 PRT Rat 59 Lys Ile Lys Thr Ile Gly Ser Thr Tyr
Met Ala Ala Ser Gly Leu Asn 1 5 10 15 Asp Ser Thr Tyr Asp Lys Ala
Gly Lys Thr His Ile Lys Ala Leu Ala 20 25 30 Asp Phe Ala Met Lys
Leu Met Asp Gln Met Lys Tyr Ile Asn Glu His 35 40 45 Ser Phe Asn
Asn Phe Gln Met Lys Ile Gly Leu Asn Ile Gly Pro Val 50 55 60 Val
Ala Gly Val Ile Gly Ala Arg Lys Pro Gln Tyr Asp Ile Trp Gly 65 70
75 80 Asn Thr Val Asn Val 85 60 85 PRT Rabbit 60 Lys Ile Lys Thr
Ile Gly Ser Thr Tyr Met Ala Ala Ser Gly Leu Asn 1 5 10 15 Asp Ser
Thr Tyr Asp Lys Val Gly Lys Thr His Ile Lys Ala Leu Ala 20 25 30
Asp Phe Ala Met Lys Leu Met Asp Gln Met Lys Tyr Ile Asn Glu His 35
40 45 Ser Phe Asn Asn Phe Gln Met Lys Ile Gly Leu Asn Ile Gly Pro
Val 50 55 60 Val Ala Gly Val Ile Gly Ala Arg Lys Pro Gln Tyr Asp
Ile Trp Gly 65 70 75 80 Asn Thr Val Asn Val 85 61 85 PRT Dog 61 Lys
Ile Lys Thr Ile Gly Ser Thr Tyr Met Ala Ala Ser Gly Leu Asn 1 5 10
15 Asp Ser Thr Tyr Asp Lys Val Gly Lys Thr His Ile Lys Ala Leu Ala
20 25 30 Asp Phe Ala Met Lys Leu Met Asp Gln Met Lys Tyr Ile Asn
Glu His 35 40 45 Ser Phe Asn Asn Phe Gln Met Lys Ile Gly Leu Asn
Ile Gly Pro Val 50 55 60 Val Ala Gly Val Ile Gly Ala Arg Lys Pro
Gln Tyr Asp Ile Trp Gly 65 70 75 80 Asn Thr Val Asn Val 85 62 88
PRT Bovine 62 Lys Ile Lys Thr Ile Gly Ser Thr Tyr Met Ala Ala Val
Gly Leu Ala 1 5 10 15 Pro Thr Ala Gly Thr Lys Ala Lys Lys Cys Ile
Ser Ser His Leu Ser 20 25 30 Thr Leu Ala Asp Phe Ala Ile Glu Met
Phe Asp Val Leu Asp Glu Ile 35 40 45 Asn Tyr Gln Ser Tyr Asn Asp
Phe Val Leu Arg Val Gly Ile Asn Val 50 55 60 Gly Pro Val Val Ala
Gly Val Ile Gly Ala Arg Arg Pro Gln Tyr Asp 65 70 75 80 Ile Trp Gly
Asn Thr Val Asn Val 85 63 98 PRT Rat 63 Lys Ile Lys Thr Ile Gly Ser
Thr Tyr Met Ala Ala Ser Gly Val Thr 1 5 10 15 Pro Asp Val Asn Thr
Asn Gly Phe Thr Ser Ser Ser Lys Glu Glu Lys 20 25 30 Ser Asp Lys
Glu Arg Trp Gln His Leu Ala Asp Leu Ala Asp Phe Ala 35 40 45 Leu
Ala Met Lys Asp Thr Leu Thr Asn Ile Asn Asn Gln Ser Phe Asn 50 55
60 Asn Phe Met Leu Arg Ile Gly Met Asn Lys Gly Gly Val Leu Ala Gly
65 70 75 80 Val Ile Gly Ala Arg Lys Pro His Tyr Asp Ile Trp Gly Asn
Thr Val 85 90 95 Asn Val 64 88 PRT Artificial sequence Primer based
on mouse adenyl cyclase 9 64 Lys Ile Lys Thr Ile Gly Ala Thr Tyr
Met Ala Ala Ser Gly Leu Asn 1 5 10 15 Thr Ala Gln Cys Gln Glu Gly
Gly His Pro Gln Glu His Leu Arg Ile 20 25 30 Leu Phe Glu Phe Ala
Lys Glu Met Met Arg Val Val Asp Asp Phe Asn 35 40 45 Asn Asn Met
Leu Trp Phe Asn Phe Lys Leu Arg Val Gly Phe Asn His 50 55 60 Gly
Pro Leu Thr Ala Gly Val Ile Gly Thr Thr Lys Leu Leu Tyr Asp 65 70
75 80 Ile Trp Gly Asp Thr Val Asn Ile 85 65 108 PRT Mouse 65 Arg
Phe Lys Phe Asp Val Trp Ser Asn Asp Val Asn Leu Ala Asn Leu 1 5 10
15 Met Glu Gln Leu Gly Val Ala Gly Lys Val His Ile Ser Glu Ala Thr
20 25 30 Ala Lys Tyr Leu Asp Asp Arg Tyr Glu Met Glu Asp Gly Arg
Val Ile 35 40 45 Glu Arg Leu Gly Gln Ser Val Val Ala Asp Gln Leu
Lys Gly Leu Lys 50 55 60 Thr Tyr Leu Ile Ser Gly Gln Arg Ala Lys
Glu Ser His Cys Ser Cys 65 70 75 80 Ala Glu Ala Leu Leu Ser Gly Phe
Glu Val Ile Asp Asp Ser Arg Glu 85 90 95 Ser Ser Gly Pro Arg Gly
Gln Gly Thr Ala Ser Pro 100 105 66 107 PRT Saccharomyces cerevisiae
66 Asn Val Lys Ile Asp Arg Ile Ser Pro Gly Asp Gly Ala Thr Phe Pro
1 5 10 15 Lys Thr Gly Asp Leu Val Thr Ile His Tyr Thr Gly Thr Leu
Glu Asn 20 25 30 Gly Gln Lys Phe Asp Ser Ser Val Asp Arg Gly Ser
Pro Phe Gln Cys 35 40 45 Asn Ile Gly Val Gly Gln Val Ile Lys Gly
Trp Asp Val Gly Ile Pro 50 55 60 Lys Leu Ser Val Gly Glu Lys Ala
Arg Leu Thr Ile Pro Gly Pro Tyr 65 70 75 80 Ala Tyr Gly Pro Arg Gly
Phe Pro Gly Leu Ile Pro Pro Asn Ser Thr 85 90 95 Leu Val Phe Asp
Val Glu Leu Leu Lys Val Asn 100 105 67 107 PRT Homo sapiens 67 Gly
Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro 1 5 10
15 Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp
20 25 30 Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe
Lys Phe 35 40 45 Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu
Glu Gly Val Ala 50 55 60 Gln Met Ser Val Gly Gln Arg Ala Lys Leu
Thr Ile Ser Pro Asp Tyr 65 70 75 80 Ala Tyr Gly Ala Thr Gly His Pro
Gly Ile Ile Pro Pro His Ala Thr 85 90 95 Leu Val Phe Asp Val Glu
Leu Leu Lys Leu Glu 100 105 68 107 PRT Mouse 68 Gly Val Gln Val Glu
Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro 1 5 10 15 Lys Arg Gly
Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp 20 25 30 Gly
Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe 35 40
45 Thr Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala
50 55 60 Gln Met Ser Val Gly Gln Arg Ala Lys Leu Ile Ile Ser Ser
Asp Tyr 65 70 75 80 Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro
Pro His Ala Thr 85 90 95 Leu Val Phe Asp Val Glu Leu Leu Lys Leu
Glu 100 105 69 107 PRT Neurospora crassa 69 Gly Leu Gln Ile Glu Val
Gln Gln Glu Gly Gln Gly Thr Arg Glu Thr 1 5 10 15 Arg Arg Gly Asp
Asn Val Asp Val His Tyr Lys Gly Val Leu Thr Ser 20 25 30 Gly Lys
Lys Phe Asp Ala Ser Tyr Asp Arg Gly Glu Pro Leu Asn Phe 35 40 45
Thr Val Gly Gln Gly Gln Val Ile Lys Gly Trp Asp Glu Gly Leu Leu 50
55 60 Gly Met Lys Ile Gly Glu Lys Arg Lys Leu Thr Ile Ala Pro His
Leu 65 70 75 80 Ala Tyr Gly Asn Arg Ala Val Gly Gly Ile Ile Pro Ala
Asn Ser Thr 85 90 95 Leu Ile Phe Glu Thr Glu Leu Val Gly Ile Lys
100 105 70 104 PRT Bovine 70 Lys Trp Gln Tyr Asp Val Trp Ser Asn
Asp Val Thr Leu Ala Asn Val 1 5 10 15 Met Glu Ala Ala Gly Leu Pro
Gly Lys Val His Ile Thr Lys Thr Thr 20 25 30 Leu Ala Cys Leu Asn
Gly Asp Tyr Glu Val Glu Pro Gly His Gly His 35 40 45 Glu Arg Asn
Ser Phe Leu Lys Thr His Asn Ile Glu Thr Phe Phe Ile 50 55 60 Val
Pro Ser His Arg Arg Lys Ile Phe Pro Gly Leu Ile Leu Ser Asp 65 70
75 80 Ile Lys Pro Ala Lys Arg Met Lys Phe Lys Thr Val Cys Tyr Leu
Leu 85 90 95 Val Gln Leu Met Tyr His Cys Arg 100 71 104 PRT Rat 71
Arg Trp Gln Tyr Asp Val Trp Ser Thr Asp Val Thr Val Ala Asn Lys 1 5
10 15 Met Glu Ala Gly Gly Ile Pro Gly Arg Val His Ile Ser Gln Ser
Thr 20 25 30 Met Asp Cys Leu Lys Gly Glu Phe Asp Val Glu Pro Gly
Asp Gly Gly 35 40 45 Ser Arg Cys Asp Tyr Leu Asp Glu Lys Gly Ile
Glu Thr Tyr Leu Ile 50 55 60 Ile Ala Ser Lys Pro Glu Val Lys Lys
Thr Ala Gln Asn Gly Leu Asn 65 70 75 80 Gly Ser Ala Leu Pro Asn Gly
Ala Pro Ala Ser Lys Pro Ser Ser Ser 85 90 95 Pro Ala Leu Ile Glu
Thr Lys Glu 100 72 104 PRT Rat 72 Lys Trp Gln Phe Asp Val Trp Ser
Trp Asp Val Asp Ile Ala Asn Lys 1 5 10 15 Leu Glu Ser Gly Gly Ile
Pro Gly Arg Ile His Ile Ser Lys Ala Thr 20 25 30 Leu Asp Cys Leu
Ser Gly Asp Tyr Asn Val Glu Glu Gly His Gly Lys 35 40 45 Glu Arg
Asn Glu Phe Leu Arg Lys His Asn Ile Glu Thr Tyr Leu Ile 50 55 60
Lys Gln Pro Glu Glu Ser Leu Leu Ser Leu Pro Glu Asp Ile Val Lys 65
70 75 80 Glu Ser Val Ser Cys Ser Asp Arg Arg Asn Ser Gly Ala Thr
Phe Thr 85 90 95 Glu Gly Ser Trp Ser Pro Glu Leu 100 73 104 PRT Rat
73 Lys Trp Gln Phe Asp Val Trp Ser Asn Asp Val Thr Leu Ala Asn His
1 5 10 15 Met Glu Ala Gly Gly Lys Ala Gly Arg Ile His Ile Thr Lys
Ala Thr 20 25 30 Leu Asn Tyr Leu Asn Gly Asp Tyr Glu Val Glu Pro
Gly Cys Gly Gly 35 40 45 Glu Arg Asn Ala Tyr Leu Lys Glu His Ser
Ile Glu Thr Phe Leu Ile 50 55 60 Leu Arg Cys Thr Gln Lys Arg Lys
Glu Glu Lys Ala Met Ile Ala Lys 65 70 75 80 Met Asn Arg Gln Arg Thr
Asn Ser Ile Gly His Asn Pro Pro His Trp 85 90 95 Gly Ala Glu Arg
Pro Phe Tyr Asn 100 74 104 PRT Rat 74 Lys Trp Gln Phe Asp Val Trp
Ser Asn Asp Val Thr Leu Ala Asn His 1 5 10 15 Met Glu Ala Gly Gly
Arg Ala Gly Arg Ile His Ile Thr Arg Ala Thr 20 25 30 Leu Gln Tyr
Leu Asn Gly Asp Tyr Glu Val Glu Pro Gly Arg Gly Gly 35 40 45 Glu
Arg Asn Gly Tyr Leu Lys Glu Gln Cys Ile Glu Thr Phe Leu Ile 50 55
60 Leu Gly Ala Ser Gln Lys Arg Lys Glu Glu Lys Ala Met Leu Val Lys
65 70 75 80 Leu Gln Arg Thr Arg Ala Asn Ser Met Glu Gly Leu Met Pro
His Trp 85 90 95 Gly Ala Glu Arg Pro Phe Tyr Asn 100 75 102 PRT Rat
75 Lys Trp Gln Tyr Asp Val Trp Ser His Asp Val Thr Leu Ala Asn His
1 5 10 15 Met Glu Ala Gly Gly Val Pro Gly Arg Val His Ile Ser Ser
Val Thr 20 25 30 Leu Glu His Leu Asn Gly Ala Tyr Lys Val Glu Glu
Gly Asp Gly Glu 35 40 45 Ile Arg Asp Pro Tyr Leu Lys Gln His Leu
Val Lys Thr Tyr Phe Val 50 55 60 Ile Asn Pro Lys Gly Glu Arg Arg
Ser Pro Gln His Leu Phe Arg Pro 65 70 75 80 Arg His Thr Leu Asp Gly
Ala Lys Met Arg Ala Ser Val Arg Met Thr 85 90 95 Arg Tyr Leu Ser
Trp Gly 100 76 103 PRT Rat 76 Lys Asn Gln Tyr Asp Val Asn Ser His
Asp Val Thr Leu Ala Asn His 1 5 10 15 Met Glu Ala Gly Gly Val Pro
Gly Arg Val His Ile Thr Gly Ala Thr 20 25 30 Leu Ala Leu Leu Ala
Gly Ala Tyr Ala Val Glu Arg Ala Asp Met Glu 35 40 45 His Arg Asp
Pro Tyr Leu Arg Glu Leu Gly Glu Pro Thr Tyr Leu Val 50 55 60 Ile
Asp Pro Trp Ala Glu Glu Glu Asp Glu Lys Gly Thr Glu Arg Gly 65 70
75 80 Leu Leu Ser Ser Leu Glu Gly His Thr Met Arg Pro Ser Leu Leu
Met 85 90 95 Thr Arg Tyr Leu Ser Trp Gly 100 77 103 PRT Mouse 77
Lys Trp Gln Tyr Asp Val Trp Ser His Asp Val Ser Leu Ala Asn Arg 1 5
10 15 Met Glu Ala Ala Gly Val Pro Gly Arg Val His Ile Thr Glu Ala
Thr 20 25 30 Leu Asn His Leu Asp Lys Ala Tyr Glu Val Glu Asp Gly
His Gly Glu 35 40 45 Gln Arg Asp Pro Tyr Leu Lys Glu Met Asn Ile
Arg Thr Tyr Leu Val 50 55 60 Ile Asp Pro Arg Ser Gln Gln Pro Pro
Pro Pro Ser His His Leu Ser 65 70 75 80 Lys Pro Lys Gly Asp Ala Thr
Leu Lys Met Arg Ala Ser Val Arg Val 85 90 95 Thr Arg Tyr Leu Ser
Trp Gly 100 78 103 PRT Homo sapiens 78 Lys Trp Gln Tyr Asp Val Trp
Ser His Asp Val Ser Leu Ala Asn Arg 1 5 10 15 Met Glu Ala Ala Gly
Val Pro Gly Arg Val His Ile Thr Glu Ala Thr 20 25 30 Leu Lys His
Leu Asp Lys Ala Tyr Glu Val Glu Asp Gly His Gly Gln 35 40 45 Gln
Arg Asp Pro Tyr Leu Lys Glu Met Asn Ile Arg Thr Tyr Leu Val 50 55
60 Ile Asp Pro Arg Ser Gln Gln Pro Pro Pro Pro Ser Gln His Leu Pro
65 70 75 80 Arg Pro Lys Gly Asp Ala Ala Leu Lys Met Arg Ala Ser Val
Arg Met 85 90 95 Thr Arg Tyr Leu Ser Trp Gly 100 79 107 PRT Yeast
79 Asn Val Lys Ile Asp Arg Ile Ser Pro Gly Asp Gly Ala Thr Phe Pro
1 5 10 15 Lys Thr Gly Asp Leu Val Thr Ile His Tyr Thr Gly Thr Leu
Glu Asn 20 25 30 Gly Gln Lys Phe Asp Ser Ser Val Asp Arg Gly Ser
Pro Phe Gln Cys 35 40 45 Asn Ile Gly Val Gly Gln Val Ile Lys Gly
Trp Asp Val Gly Ile Pro 50 55 60 Lys Leu Ser Val Gly Glu Lys Ala
Arg Leu Thr Ile Pro Gly Pro Tyr 65 70 75 80 Ala Tyr Gly Pro Arg Gly
Phe Pro Gly Leu Ile Pro Pro Asn Ser Thr 85 90 95 Leu Val Phe Asp
Val Glu Leu Leu Lys Val Asn 100 105 80 107 PRT Homo
sapiens 80 Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr
Phe Pro 1 5 10 15 Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly
Met Leu Glu Asp 20 25 30 Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg
Asn Lys Pro Phe Lys Phe 35 40 45 Met Leu Gly Lys Gln Glu Val Ile
Arg Gly Trp Glu Glu Gly Val Ala 50 55 60 Gln Met Ser Val Gly Gln
Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr 65 70 75 80 Ala Tyr Gly Ala
Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr 85 90 95 Leu Val
Phe Asp Val Glu Leu Leu Lys Leu Glu 100 105 81 107 PRT Mouse 81 Gly
Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro 1 5 10
15 Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp
20 25 30 Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe
Lys Phe 35 40 45 Thr Leu Gly Lys Gln Glu Val Ile Arg Gln Trp Glu
Glu Gly Val Ala 50 55 60 Gln Met Ser Val Gly Gln Arg Ala Lys Leu
Ile Ile Ser Ser Asp Tyr 65 70 75 80 Ala Tyr Gly Ala Thr Gly His Pro
Gly Ile Ile Pro Pro His Ala Thr 85 90 95 Leu Val Phe Asp Val Glu
Leu Leu Lys Leu Glu 100 105 82 107 PRT Bovine 82 Gly Val Glu Ile
Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro 1 5 10 15 Lys Lys
Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Gln Asn 20 25 30
Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe 35
40 45 Arg Ile Gly Lys Gln Glu Val Ile Lys Gly Phe Glu Glu Gly Ala
Ala 50 55 60 Gln Met Ser Leu Gly Gln Arg Ala Lys Leu Thr Cys Thr
Pro Asp Val 65 70 75 80 Ala Tyr Gly Ala Thr Gly His Pro Gly Val Ile
Pro Pro Asn Ala Thr 85 90 95 Leu Ile Phe Asp Val Glu Leu Leu Asn
Leu Glu 100 105 83 107 PRT Neurospora crassa 83 Gly Leu Gln Ile Glu
Val Gln Gln Glu Gly Gln Gly Thr Arg Glu Thr 1 5 10 15 Arg Arg Gly
Asp Asn Val Asp Val His Tyr Lys Gly Val Leu Thr Ser 20 25 30 Gly
Lys Lys Phe Asp Ala Ser Tyr Asp Arg Gly Glu Pro Leu Asn Phe 35 40
45 Thr Val Gly Gln Gly Gln Val Ile Lys Gly Trp Asp Glu Gly Leu Leu
50 55 60 Gly Met Lys Ile Gly Glu Lys Arg Lys Leu Thr Ile Ala Pro
His Leu 65 70 75 80 Ala Tyr Gly Asn Arg Ala Val Gly Gly Ile Ile Pro
Ala Asn Ser Thr 85 90 95 Leu Ile Phe Glu Thr Glu Leu Val Gly Ile
Lys 100 105 84 105 PRT Homo sapiens 84 Arg Val Asp His Cys Pro Ile
Lys Ser Arg Lys Gly Asp Val Leu His 1 5 10 15 Met His Tyr Thr Gly
Lys Leu Glu Asp Gly Thr Glu Phe Asp Ser Ser 20 25 30 Leu Pro Gln
Asn Gln Pro Phe Val Phe Ser Leu Gly Thr Gly Gln Val 35 40 45 Ile
Lys Gly Trp Asp Gln Gly Leu Leu Gly Met Tyr Glu Gly Glu Lys 50 55
60 Arg Lys Leu Val Ile Pro Ser Glu Leu Gly Tyr Gly Glu Arg Gly Ala
65 70 75 80 Pro Pro Lys Ile Pro Gly Gly Ala Thr Leu Val Phe Glu Val
Glu Leu 85 90 95 Leu Lys Ile Glu Arg Arg Thr Glu Leu 100 105 85 103
PRT Homo sapiens 85 Ser Val Leu Lys Lys Gly Asp Lys Thr Asn Phe Pro
Lys Lys Gly Asp 1 5 10 15 Val Val His Cys Trp Tyr Thr Gly Thr Leu
Gln Asp Gly Thr Val Phe 20 25 30 Asp Thr Asn Ile Gln Thr Ser Lys
Pro Leu Ser Phe Lys Val Gly Val 35 40 45 Gly Lys Val Ile Arg Gly
Trp Asp Glu Ala Leu Leu Thr Met Ser Lys 50 55 60 Gly Glu Lys Ala
Arg Leu Glu Ile Glu Pro Glu Trp Ala Tyr Gly Lys 65 70 75 80 Lys Gly
Gln Pro Ala Lys Ile Pro Pro Asn Ala Lys Leu Thr Phe Glu 85 90 95
Val Glu Leu Val Asp Ile Asp 100 86 104 PRT Yeast 86 Ile Lys Arg Ile
Pro Val Glu Asp Cys Leu Ile Lys Ala Met Pro Gly 1 5 10 15 Asp Lys
Val Lys Val His Tyr Thr Gly Ser Glu Glu Ser Gly Thr Val 20 25 30
Phe Asp Ser Ser Tyr Ser Arg Gly Ser Pro Ile Ala Phe Glu Leu Gly 35
40 45 Val Gly Arg Val Ile Lys Gly Trp Asp Gln Gly Val Ala Gly Met
Cys 50 55 60 Val Gly Glu Lys Arg Lys Leu Gln Ile Pro Ser Ser Leu
Ala Tyr Gly 65 70 75 80 Glu Arg Gly Val Pro Gly Val Ile Pro Pro Ser
Ala Asp Leu Val Phe 85 90 95 Asp Val Glu Leu Val Asp Val Lys 100 87
110 PRT Bovine 87 Thr Gly Arg Val Leu Cys Gly Val Leu Gly Leu Arg
Lys Trp Gln Tyr 1 5 10 15 Asp Val Trp Ser Asn Asp Val Thr Leu Ala
Asn Val Met Glu Ala Ala 20 25 30 Gly Leu Pro Gly Lys Val His Ile
Thr Lys Thr Thr Leu Ala Cys Leu 35 40 45 Asn Gly Asp Tyr Glu Val
Glu Pro Gly His Gly His Glu Arg Asn Ser 50 55 60 Phe Leu Lys Thr
His Asn Ile Glu Thr Phe Phe Ile Val Pro Ser His 65 70 75 80 Arg Arg
Lys Ile Phe Pro Gly Leu Ile Ile Ser Asp Ile Lys Pro Ala 85 90 95
Lys Arg Met Lys Phe Lys Thr Val Cys Tyr Leu Ile Val Gln 100 105 110
88 109 PRT Rat 88 Ala Asn Lys Met Glu Ala Gly Gly Ile Pro Gly Arg
Val His Ile Ser 1 5 10 15 Gln Ser Thr Met Asp Cys Leu Lys Gly Glu
Phe Asp Val Glu Pro Gly 20 25 30 Asp Gly Gly Ser Arg Cys Asp Tyr
Leu Asp Glu Lys Gly Ile Glu Thr 35 40 45 Tyr Leu Ile Ile Ala Ser
Lys Pro Glu Val Lys Lys Thr Ala Gln Asn 50 55 60 Gly Leu Asn Gly
Ser Ala Leu Pro Asn Gly Ala Pro Ala Ser Lys Pro 65 70 75 80 Ser Ser
Pro Ala Leu Ile Glu Thr Lys Glu Pro Asn Gly Ser Ala His 85 90 95
Ala Ser Gly Ser Thr Ser Glu Glu Ala Glu Glu Gln Glu 100 105 89 101
PRT Rat 89 Ala Asn Lys Leu Glu Ser Gly Gly Ile Pro Gly Arg Ile His
Ile Ser 1 5 10 15 Lys Ala Thr Leu Asp Cys Leu Ser Gly Asp Tyr Asn
Val Glu Glu Gly 20 25 30 His Gly Lys Glu Arg Asn Glu Phe Leu Arg
Lys His Asn Ile Glu Thr 35 40 45 Tyr Leu Ile Lys Gln Pro Glu Glu
Ser Leu Leu Ser Leu Pro Glu Asp 50 55 60 Ile Val Lys Glu Ser Val
Ser Cys Ser Asp Arg Arg Asn Ser Gly Ala 65 70 75 80 Thr Phe Thr Glu
Gly Ser Trp Ser Pro Glu Leu Pro Phe Asp Asn Ile 85 90 95 Val Gly
Lys Gln Asn 100 90 101 PRT Drosophila calmudolin 90 Ala Asn His Met
Glu Ser Gly Gly Glu Pro Gly Arg Val His Val Thr 1 5 10 15 Arg Ala
Thr Asp Ser Leu Ser Gly Glu Tyr Glu Val Glu Ala Gly His 20 25 30
Gly Asp Glu Arg Ser Ser Tyr Leu Arg Asp His Gly Val Asp Thr Phe 35
40 45 Phe Ile Val Pro Pro Pro His Arg Arg Lys Pro Leu Met Leu Asn
Thr 50 55 60 Leu Gly Val Arg Ser Ala Ile Gly Ser Arg Arg Lys Leu
Ser Phe Arg 65 70 75 80 Asn Val Ser Asn Val Val Met Gln Leu Leu His
Thr Ile Lys Phe Ser 85 90 95 Glu Pro Val Pro Phe 100 91 109 PRT Rat
91 Lys Trp Gln Phe Asp Val Trp Ser Asn Asp Val Thr Leu Ala Asn His
1 5 10 15 Met Glu Ala Gly Gly Lys Ala Gly Arg Ile His Ile Thr Lys
Ala Thr 20 25 30 Leu Asn Tyr Leu Asn Gly Asp Tyr Glu Val Glu Pro
Gly Cys Gly Gly 35 40 45 Glu Arg Asn Ala Tyr Leu Lys Glu His Ser
Ile Glu Thr Phe Leu Ile 50 55 60 Leu Arg Cys Thr Gln Lys Arg Lys
Glu Glu Lys Ala Met Ile Ala Lys 65 70 75 80 Met Asn Arg Gln Arg Thr
Asn Ser Ile Gly His Asn Pro Pro His Trp 85 90 95 Gly Ala Glu Arg
Pro Phe Tyr Asn His Leu Gly Gly Asn 100 105 92 119 PRT Dog 92 Gly
Met Asp Met Ile Glu Ala Ile Ser Leu Val Arg Glu Val Thr Gly 1 5 10
15 Val Asn Val Asn Met Arg Val Gly Ile His Ser Gly Arg Val His Cys
20 25 30 Gly Val Leu Gly Leu Arg Lys Trp Gln Phe Asp Val Trp Ser
Asn Asp 35 40 45 Val Thr Leu Ala Asn His Met Glu Ala Gly Gly Lys
Ala Gly Arg Ile 50 55 60 His Ile Thr Lys Ala Thr Leu Ser Tyr Leu
Asn Gly Asp Tyr Glu Val 65 70 75 80 Glu Pro Gly Cys Gly Gly Glu Arg
Asn Ala Tyr Leu Lys Glu His Ser 85 90 95 Ile Glu Thr Phe Leu Ile
Leu Arg Cys Thr Gln Lys Arg Val Arg Gly 100 105 110 Gly Gly Gly Pro
Arg Pro Gly 115 93 113 PRT Rat 93 Lys Trp Gln Phe Asp Val Trp Ser
Asn Asp Val Thr Leu Ala Asn His 1 5 10 15 Met Glu Ala Ala Arg Ala
Gly Arg Ile His Ile Thr Arg Ala Thr Leu 20 25 30 Gln Tyr Leu Asn
Gly Asp Tyr Glu Val Glu Pro Gly Arg Gly Gly Glu 35 40 45 Arg Asn
Ala Tyr Leu Lys Glu Gln His Ile Glu Thr Phe Leu Ile Leu 50 55 60
Gly Ala Ser Gln Lys Arg Lys Glu Glu Lys Ala Met Leu Ala Lys Leu 65
70 75 80 Gln Arg Thr Arg Ala Asn Ser Met Glu Gly Leu Met Pro Arg
Trp Val 85 90 95 Pro Asp Arg Ala Phe Ser Arg Thr Lys Asp Ser Lys
Ala Phe Arg Gln 100 105 110 Met 94 106 PRT Mouse 94 Arg Phe Lys Phe
Asp Val Trp Ser Asn Asp Val Asn Leu Ala Asn Leu 1 5 10 15 Met Glu
Gln Leu Gly Val Ala Gly Lys Val His Ile Ser Glu Ala Thr 20 25 30
Ala Lys Tyr Leu Asp Asp Arg Tyr Glu Met Glu Asp Gly Arg Val Ile 35
40 45 Glu Arg Leu Gly Gln Ser Val Val Ala Asp Gln Leu Lys Gly Leu
Lys 50 55 60 Thr Tyr Leu Ile Ser Gly Gln Arg Ala Lys Glu Ser His
Cys Ser Cys 65 70 75 80 Ala Glu Ala Leu Leu Gly Phe Glu Val Ile Asp
Asp Ser Arg Glu Ser 85 90 95 Ser Gly Pro Arg Gly Gln Gly Thr Ala
Ser 100 105 95 109 PRT Rat 95 Lys Trp Gln Tyr Asp Val Trp Ser His
Asp Val Thr Leu Ala Asn His 1 5 10 15 Met Glu Ala Gly Gly Val Pro
Gly Arg Val His Ile Ser Ser Val Thr 20 25 30 Leu Glu His Leu Asn
Gly Ala Tyr Lys Val Glu Glu Gly Asp Gly Glu 35 40 45 Ile Arg Asp
Pro Tyr Leu Lys Gln His Leu Val Lys Thr Tyr Phe Val 50 55 60 Ile
Asn Pro Lys Gly Glu Arg Arg Ser Pro Gln His Leu Phe Arg Pro 65 70
75 80 Arg His Thr Leu Asp Gly Ala Lys Met Arg Ala Ser Val Arg Met
Thr 85 90 95 Arg Tyr Leu Glu Ser Trp Gly Ala Ala Lys Pro Phe Ala
100 105 96 109 PRT Rat 96 Lys Trp Gln Tyr Asp Val Trp Ser His Asp
Val Thr Leu Ala Asn His 1 5 10 15 Met Glu Ala Gly Gly Val Pro Gly
Arg Val His Ile Thr Gly Ala Thr 20 25 30 Leu Ala Leu Leu Ala Gly
Ala Tyr Ala Val Glu Arg Ala Asp Met Glu 35 40 45 His Arg Asp Pro
Tyr Leu Arg Glu Leu Gly Glu Pro Thr Tyr Leu Val 50 55 60 Ile Asp
Pro Trp Ala Glu Glu Glu Asp Glu Lys Gly Thr Glu Arg Gly 65 70 75 80
Leu Leu Ser Ser Leu Glu Gly His Thr Met Arg Pro Ser Leu Leu Met 85
90 95 Thr Arg Tyr Leu Glu Ser Trp Gly Ala Ala Lys Pro Phe 100 105
97 114 PRT Rat 97 Lys Trp Gln Tyr Asp Val Trp Ser His Asp Val Ser
Leu Ala Asn Arg 1 5 10 15 Met Glu Ala Ala Gly Val Pro Gly Arg Val
His Ile Thr Glu Ala Thr 20 25 30 Leu Asn His Leu Asp Lys Ala Tyr
Glu Val Glu Asp Gly His Gly Glu 35 40 45 Gln Arg Asp Pro Tyr Leu
Lys Glu Met Asn Ile Arg Thr Tyr Leu Val 50 55 60 Ile Asp Pro Arg
Ser Gln Gln Pro Pro Pro Pro Ser His His Leu Ser 65 70 75 80 Lys Pro
Lys Gly Asp Lys Ala Thr Leu Arg Met Arg Ala Ser Val Arg 85 90 95
Val Thr Arg Tyr Leu Glu Ser Trp Gly Ala Ala Arg Pro Phe Ala His 100
105 110 Leu Asn 98 5515 DNA Homo sapiens CDS (539)..(4600) 98
gcggccgcgg cgggagcggc ggcgcactcg gggggaagca ggccccacta gacgggccgc
60 gccgacgggc tccgcgcact cgcagcccgg ggcgccccca cccccagcgc
ccgccgccgc 120 gggcagggcc ggcccggagc gcggggggcg gccggggagc
gcgggccggg ggcgcccgcc 180 gaagctcgct gctccgggtc ggggtcgggg
ccggggccgg ccgcgcgccg cctttgacgc 240 atcggagcgc ggctcctgca
ggatggaggg ctccgcgccg ccagcggagt tgtttgtgcg 300 caggcggctc
gcggggctgg gagcgctcaa ggtctgaact tccctccgga gccgcagctg 360
gaggaggcga gcgcgcgagg aggagaagcc gcgcggcgcg gaggccaccc tcggggcgag
420 aggcgcggaa ggcgagcgag caaagcggtc ccggagccac ggcggccacg
cggcggggac 480 ccccgggcgt tctaggtact ggtgaccccg gccggggcag
gccccgggac tcgacaac 538 atg gct tcc ccg ccc cac cag cag ctg ctg cat
cac cac agc acc gag 586 Met Ala Ser Pro Pro His Gln Gln Leu Leu His
His His Ser Thr Glu 1 5 10 15 gtg agc tgc gac tcc agc ggg gac agc
aac agc gtg cgc gtc aag atc 634 Val Ser Cys Asp Ser Ser Gly Asp Ser
Asn Ser Val Arg Val Lys Ile 20 25 30 aac ccc aag cag ctg tcc tcc
aac agc cac ccc aag cac tgc aaa tac 682 Asn Pro Lys Gln Leu Ser Ser
Asn Ser His Pro Lys His Cys Lys Tyr 35 40 45 agc atc tcc tct agc
tgc agc agc tct ggg gac tcc ggg ggc gtc ccc 730 Ser Ile Ser Ser Ser
Cys Ser Ser Ser Gly Asp Ser Gly Gly Val Pro 50 55 60 cgg cga gtg
ggc ggc gga ggc cgg ctg cgc agg cag aag aag ctg ccc 778 Arg Arg Val
Gly Gly Gly Gly Arg Leu Arg Arg Gln Lys Lys Leu Pro 65 70 75 80 cag
ctg ttc gag agg gcc tcc agc cgc tgg tgg gac ccc aag ttc gac 826 Gln
Leu Phe Glu Arg Ala Ser Ser Arg Trp Trp Asp Pro Lys Phe Asp 85 90
95 tcg gtg aac ctg gag gag gcc tgc ctg gag cgc tgc ttc ccg cag acc
874 Ser Val Asn Leu Glu Glu Ala Cys Leu Glu Arg Cys Phe Pro Gln Thr
100 105 110 cag cgc cgg ttc cgg tat gcg ctc ttc tac atc ggc ttc gcc
tgc ctt 922 Gln Arg Arg Phe Arg Tyr Ala Leu Phe Tyr Ile Gly Phe Ala
Cys Leu 115 120 125 ctg tgg agc atc tat ttt gcg gtc cac atg aga tcc
aga ctg atc gtc 970 Leu Trp Ser Ile Tyr Phe Ala Val His Met Arg Ser
Arg Leu Ile Val 130 135 140 atg gtc gcc ccc gcg ctg tgc ttc ctc ctg
gtg tgt gtg ggc ttc ttt 1018 Met Val Ala Pro Ala Leu Cys Phe Leu
Leu Val Cys Val Gly Phe Phe 145 150 155 160 ctg ttt acc ttc acc aag
ctg tac gcc cgg cat tac gcg tgg acc tcg 1066 Leu Phe Thr Phe Thr
Lys Leu Tyr Ala Arg His Tyr Ala Trp Thr Ser 165 170 175 ctg gct ctc
acc ctg ctg gtg ttc gcc ctg acc ctg gct gcg cag ttc 1114 Leu Ala
Leu Thr Leu Leu Val Phe Ala Leu Thr Leu Ala Ala Gln Phe 180 185 190
cag gtc ttg acg cct gtc tca gga cgc ggc gac agc tcc aac ctt acg
1162 Gln Val Leu Thr Pro Val Ser Gly Arg Gly Asp Ser Ser Asn Leu
Thr 195 200 205 gcc aca gcc cgg ccc aca gat act tgc tta tct caa gtg
ggg agc ttc 1210 Ala Thr Ala Arg Pro Thr Asp Thr Cys Leu Ser Gln
Val Gly Ser Phe 210 215 220 tcc atg
tgc atc gaa gtg ctc ttt ttg ctc tat acc gtc atg cac tta 1258 Ser
Met Cys Ile Glu Val Leu Phe Leu Leu Tyr Thr Val Met His Leu 225 230
235 240 cct ttg tac ctg agt ttg tgt ctg ggg gtg gcc tac tct gtc ctt
ttc 1306 Pro Leu Tyr Leu Ser Leu Cys Leu Gly Val Ala Tyr Ser Val
Leu Phe 245 250 255 gag acc ttt ggc tac cat ttc cgg gat gaa gcc tgc
ttc ccc tcg ccc 1354 Glu Thr Phe Gly Tyr His Phe Arg Asp Glu Ala
Cys Phe Pro Ser Pro 260 265 270 gga gcc ggg gcc ctg cac tgg gag ctg
ctg agc agg ggg ctg ctc cac 1402 Gly Ala Gly Ala Leu His Trp Glu
Leu Leu Ser Arg Gly Leu Leu His 275 280 285 ggc tgc atc cac gcc atc
ggg gtc cac ctg ttc gtc atg tcc cag gtg 1450 Gly Cys Ile His Ala
Ile Gly Val His Leu Phe Val Met Ser Gln Val 290 295 300 agg tcc agg
agc acc ttc ctc aag gtg ggg caa tcc att atg cac ggg 1498 Arg Ser
Arg Ser Thr Phe Leu Lys Val Gly Gln Ser Ile Met His Gly 305 310 315
320 aag gac ctg gaa gtg gaa aaa gcc ctc aaa gag agg atg att cat tcc
1546 Lys Asp Leu Glu Val Glu Lys Ala Leu Lys Glu Arg Met Ile His
Ser 325 330 335 gtg atg cca aga atc ata gcc gat gac tta atg aag cag
gga gat gag 1594 Val Met Pro Arg Ile Ile Ala Asp Asp Leu Met Lys
Gln Gly Asp Glu 340 345 350 gag agt gag aat tct gtc aag aga cat gcc
acc tcg agc ccc aag aac 1642 Glu Ser Glu Asn Ser Val Lys Arg His
Ala Thr Ser Ser Pro Lys Asn 355 360 365 agg aag aaa aag tct tcc atc
caa aaa gct cct ata gcc ttc cgc cct 1690 Arg Lys Lys Lys Ser Ser
Ile Gln Lys Ala Pro Ile Ala Phe Arg Pro 370 375 380 ttt aag atg cag
cag atc gaa gaa gtc agt att tta ttt gca gat atc 1738 Phe Lys Met
Gln Gln Ile Glu Glu Val Ser Ile Leu Phe Ala Asp Ile 385 390 395 400
gtg ggc ttc acc aag atg agt gcc aac aag tct gcc cac gcc ctg gtg
1786 Val Gly Phe Thr Lys Met Ser Ala Asn Lys Ser Ala His Ala Leu
Val 405 410 415 ggt ctc ctg aac gat ctg ttc ggt cgc ttc gac cgc ctg
tgt gag gag 1834 Gly Leu Leu Asn Asp Leu Phe Gly Arg Phe Asp Arg
Leu Cys Glu Glu 420 425 430 acc aag tgt gag aaa atc agc acc ctg gga
gac tgt tac tac tgc gtg 1882 Thr Lys Cys Glu Lys Ile Ser Thr Leu
Gly Asp Cys Tyr Tyr Cys Val 435 440 445 gcg ggc tgt ccc gag ccc cgg
gcc gac cat gcc tac tgc tgc atc gag 1930 Ala Gly Cys Pro Glu Pro
Arg Ala Asp His Ala Tyr Cys Cys Ile Glu 450 455 460 atg ggc ctg ggc
atg atc aag gcc atc gag cag ttc tgc cag gag aag 1978 Met Gly Leu
Gly Met Ile Lys Ala Ile Glu Gln Phe Cys Gln Glu Lys 465 470 475 480
aag gag atg gtg aac atg aga gtc ggg gtg cac acg cgc acc gtc ctt
2026 Lys Glu Met Val Asn Met Arg Val Gly Val His Thr Arg Thr Val
Leu 485 490 495 tgc ggc atc ctg ggc atg agg agg ttt aaa ttt gac gtg
tgg tcc aac 2074 Cys Gly Ile Leu Gly Met Arg Arg Phe Lys Phe Asp
Val Trp Ser Asn 500 505 510 gat gtg aac ctg gcc aat ctc atg gag cag
ctg gga gtg gcc ggc aaa 2122 Asp Val Asn Leu Ala Asn Leu Met Glu
Gln Leu Gly Val Ala Gly Lys 515 520 525 gtt cac att tct gag gcc acc
gca aaa tac tta gat gac cgg tac gaa 2170 Val His Ile Ser Glu Ala
Thr Ala Lys Tyr Leu Asp Asp Arg Tyr Glu 530 535 540 atg gaa gat ggg
aaa gtt att gaa cgg ctg ggc cag agc gtg gtt gct 2218 Met Glu Asp
Gly Lys Val Ile Glu Arg Leu Gly Gln Ser Val Val Ala 545 550 555 560
gac cag ttg aaa ggt ttg aag aca tac ctg ata tcg ggt cag aga gcc
2266 Asp Gln Leu Lys Gly Leu Lys Thr Tyr Leu Ile Ser Gly Gln Arg
Ala 565 570 575 aag gag tct cgc tgc agc tgt gca gag gcc ttg ctt tct
ggc ttt gag 2314 Lys Glu Ser Arg Cys Ser Cys Ala Glu Ala Leu Leu
Ser Gly Phe Glu 580 585 590 gtc att gac ggc tca cag gtg tcc tca ggc
cct agg gga cag ggg aca 2362 Val Ile Asp Gly Ser Gln Val Ser Ser
Gly Pro Arg Gly Gln Gly Thr 595 600 605 gcg tca tca ggg aat gtc agt
gac ttg gcg cag act gtc aaa acc ttt 2410 Ala Ser Ser Gly Asn Val
Ser Asp Leu Ala Gln Thr Val Lys Thr Phe 610 615 620 gat aac ctt aag
acc tgc cct tcg tgc gga atc aca ttt gct ccc aaa 2458 Asp Asn Leu
Lys Thr Cys Pro Ser Cys Gly Ile Thr Phe Ala Pro Lys 625 630 635 640
tct gaa gcc ggc gcc gag gga gga gca cct caa aac ggc tgc caa gac
2506 Ser Glu Ala Gly Ala Glu Gly Gly Ala Pro Gln Asn Gly Cys Gln
Asp 645 650 655 gag cat aaa aac agc acc aag gct tct gga gga cct aat
ccc aaa act 2554 Glu His Lys Asn Ser Thr Lys Ala Ser Gly Gly Pro
Asn Pro Lys Thr 660 665 670 cag aac ggg ctc ctc agc cct ccc caa gag
gag aag ctc acc aac agt 2602 Gln Asn Gly Leu Leu Ser Pro Pro Gln
Glu Glu Lys Leu Thr Asn Ser 675 680 685 cag act tct ctg tgt gag atc
ttg cag gag aag gga agg tgg gca ggg 2650 Gln Thr Ser Leu Cys Glu
Ile Leu Gln Glu Lys Gly Arg Trp Ala Gly 690 695 700 gtg agc ctg gac
cag tcg gct ctc ctt ccg ctg agg ttc aag aac atc 2698 Val Ser Leu
Asp Gln Ser Ala Leu Leu Pro Leu Arg Phe Lys Asn Ile 705 710 715 720
cgg gag aaa acg gac gcc cac ttt gtg gac gtt atc aaa gaa gac agc
2746 Arg Glu Lys Thr Asp Ala His Phe Val Asp Val Ile Lys Glu Asp
Ser 725 730 735 ctg atg aaa gat tac ttt ttt aag ccg ccc att aat cag
ttc agc ctg 2794 Leu Met Lys Asp Tyr Phe Phe Lys Pro Pro Ile Asn
Gln Phe Ser Leu 740 745 750 aac ttc ctg gat cag gag ctg gag cga tcc
tac agg acc agc tat cag 2842 Asn Phe Leu Asp Gln Glu Leu Glu Arg
Ser Tyr Arg Thr Ser Tyr Gln 755 760 765 gaa gag gtc ata aag aac tcc
ccc gtg aag acg ttt gct agt ccc acc 2890 Glu Glu Val Ile Lys Asn
Ser Pro Val Lys Thr Phe Ala Ser Pro Thr 770 775 780 ttc agc tcc ctc
ctg gat gtg ttt ctg tcg acc aca gtg ttt ctg acg 2938 Phe Ser Ser
Leu Leu Asp Val Phe Leu Ser Thr Thr Val Phe Leu Thr 785 790 795 800
ctg tcc acc acc tgc ttc ctg aag tac gag gcg gcc acc gtg cct ccc
2986 Leu Ser Thr Thr Cys Phe Leu Lys Tyr Glu Ala Ala Thr Val Pro
Pro 805 810 815 ccg ccc gcc gcc ctg gcg gtc ttc agt gca gcc ctg ctg
ctg gag gtg 3034 Pro Pro Ala Ala Leu Ala Val Phe Ser Ala Ala Leu
Leu Leu Glu Val 820 825 830 ctg tcc ctc gcg gtg tcc atc agg atg gtg
ttc ttc ctg gag gac gtc 3082 Leu Ser Leu Ala Val Ser Ile Arg Met
Val Phe Phe Leu Glu Asp Val 835 840 845 atg gcc tgc acc aag cgc ctg
ctg gag tgg atc gcc ggc tgg cta cca 3130 Met Ala Cys Thr Lys Arg
Leu Leu Glu Trp Ile Ala Gly Trp Leu Pro 850 855 860 cgt cac tgc atc
ggg gcc atc ctg gtg tcg ctt ccc gca ctg gcc gtc 3178 Arg His Cys
Ile Gly Ala Ile Leu Val Ser Leu Pro Ala Leu Ala Val 865 870 875 880
tac tcc cat gca acc tcc gaa tat gag acc aac ata cac ttc cca gtg
3226 Tyr Ser His Ala Thr Ser Glu Tyr Glu Thr Asn Ile His Phe Pro
Val 885 890 895 ttc aca ggc tcg gcc gcg ctg att gcc gtc gtg cac tac
tgt aac ttc 3274 Phe Thr Gly Ser Ala Ala Leu Ile Ala Val Val His
Tyr Cys Asn Phe 900 905 910 tgc cag ctc agc tcc tgg atg agg tcc tcc
ctc gcc acc gtc gtg ggg 3322 Cys Gln Leu Ser Ser Trp Met Arg Ser
Ser Leu Ala Thr Val Val Gly 915 920 925 gcc ggg ccg ctg ctc ctg ctc
tac gtc tcc ctg tgc cca gac agt tct 3370 Ala Gly Pro Leu Leu Leu
Leu Tyr Val Ser Leu Cys Pro Asp Ser Ser 930 935 940 gta tta act tcg
ccc ctt gac gca gta cag aat ttc agt tcc gag agg 3418 Val Leu Thr
Ser Pro Leu Asp Ala Val Gln Asn Phe Ser Ser Glu Arg 945 950 955 960
aac ccg tgc aat agt tcg gtg ccg cgt gac ctc cgg cgg ccc gcc agc
3466 Asn Pro Cys Asn Ser Ser Val Pro Arg Asp Leu Arg Arg Pro Ala
Ser 965 970 975 ctc atc ggc cag gag gtg gtt ctc gtc ttc ttt ctc ctg
ctc ttg ttg 3514 Leu Ile Gly Gln Glu Val Val Leu Val Phe Phe Leu
Leu Leu Leu Leu 980 985 990 gtc tgg ttc ctg aat cgc gaa ttt gaa gtc
agc tac cgc ctc cac tac 3562 Val Trp Phe Leu Asn Arg Glu Phe Glu
Val Ser Tyr Arg Leu His Tyr 995 1000 1005 cac gga gac gtg gaa gcg
gat ctt cac cgc acc aag atc cag agc 3607 His Gly Asp Val Glu Ala
Asp Leu His Arg Thr Lys Ile Gln Ser 1010 1015 1020 atg cgg gac cag
gca gac tgg ctg ctg agg aac atc atc ccc tac 3652 Met Arg Asp Gln
Ala Asp Trp Leu Leu Arg Asn Ile Ile Pro Tyr 1025 1030 1035 cac gtg
gct gag cag ctg aag gtg tcc cag acc tac tcc aag aac 3697 His Val
Ala Glu Gln Leu Lys Val Ser Gln Thr Tyr Ser Lys Asn 1040 1045 1050
cac gac agc gga ggg gtg atc ttc gcc agc atc gtc aac ttc agc 3742
His Asp Ser Gly Gly Val Ile Phe Ala Ser Ile Val Asn Phe Ser 1055
1060 1065 gag ttc tac gag gag aac tac gag ggc ggc aag gag tgc tac
cgg 3787 Glu Phe Tyr Glu Glu Asn Tyr Glu Gly Gly Lys Glu Cys Tyr
Arg 1070 1075 1080 gtc ctc aac gag ctc atc ggg gac ttt gac gag ctc
cta agc aag 3832 Val Leu Asn Glu Leu Ile Gly Asp Phe Asp Glu Leu
Leu Ser Lys 1085 1090 1095 ccg gac tac agc agc atc gag aag atc aag
acc atc gga gcc acg 3877 Pro Asp Tyr Ser Ser Ile Glu Lys Ile Lys
Thr Ile Gly Ala Thr 1100 1105 1110 tac atg gcg gcg tca ggg ctg aac
acc gcg cag gcc cag gac ggc 3922 Tyr Met Ala Ala Ser Gly Leu Asn
Thr Ala Gln Ala Gln Asp Gly 1115 1120 1125 agc cac ccg cag gag cac
ctg cag atc ctg ttc gag ttc gcc aag 3967 Ser His Pro Gln Glu His
Leu Gln Ile Leu Phe Glu Phe Ala Lys 1130 1135 1140 gag atg atg cgc
gtg gtg gac gac ttc aac agc aac atg ctg tgg 4012 Glu Met Met Arg
Val Val Asp Asp Phe Asn Ser Asn Met Leu Trp 1145 1150 1155 ttc aac
ttc aag ctc cgc gtc ggc ttc aac cat ggg ccc ctc acg 4057 Phe Asn
Phe Lys Leu Arg Val Gly Phe Asn His Gly Pro Leu Thr 1160 1165 1170
gcc ggg gtc atc ggc acc acc aag ctg ctg tac gac atc tgg gga 4102
Ala Gly Val Ile Gly Thr Thr Lys Leu Leu Tyr Asp Ile Trp Gly 1175
1180 1185 gac acc gtc aac atc gcc agc agg atg gac acc acc ggc gtg
gag 4147 Asp Thr Val Asn Ile Ala Ser Arg Met Asp Thr Thr Gly Val
Glu 1190 1195 1200 tgc cgc atc cag gtg agc gaa gag agc tac cgc gtc
ttg agc aag 4192 Cys Arg Ile Gln Val Ser Glu Glu Ser Tyr Arg Val
Leu Ser Lys 1205 1210 1215 atg ggc tat gac ttc gac tac aga ggg acc
gtg aat gtc aag ggg 4237 Met Gly Tyr Asp Phe Asp Tyr Arg Gly Thr
Val Asn Val Lys Gly 1220 1225 1230 aaa ggc cag atg aag acc tac ctg
tac cca aag tgc acg gat cac 4282 Lys Gly Gln Met Lys Thr Tyr Leu
Tyr Pro Lys Cys Thr Asp His 1235 1240 1245 agg gtc atc cca cag cac
cag ctg tcc atc tcc cca gac atc cgc 4327 Arg Val Ile Pro Gln His
Gln Leu Ser Ile Ser Pro Asp Ile Arg 1250 1255 1260 gtc cag gtg gat
ggc agc atc gga cgg tct ccc aca gac gag att 4372 Val Gln Val Asp
Gly Ser Ile Gly Arg Ser Pro Thr Asp Glu Ile 1265 1270 1275 gcc aac
ctg gtg cct tct gtc cag tat gtg gac aag aca tct ctg 4417 Ala Asn
Leu Val Pro Ser Val Gln Tyr Val Asp Lys Thr Ser Leu 1280 1285 1290
ggt tct gac agc agc acg cag gcc aag gat gcc cac ctg tcc cgc 4462
Gly Ser Asp Ser Ser Thr Gln Ala Lys Asp Ala His Leu Ser Arg 1295
1300 1305 aag aga ccg tgg aag gag ccc gtc aaa gcc gaa gaa agg ggt
cga 4507 Lys Arg Pro Trp Lys Glu Pro Val Lys Ala Glu Glu Arg Gly
Arg 1310 1315 1320 ttt ggc aaa gcc ata gag aaa gac gac tgt gac gaa
aca gga ata 4552 Phe Gly Lys Ala Ile Glu Lys Asp Asp Cys Asp Glu
Thr Gly Ile 1325 1330 1335 gaa gaa gcc aac gaa ctc acc aag ctc aac
gtt tca aag agt gtg 4597 Glu Glu Ala Asn Glu Leu Thr Lys Leu Asn
Val Ser Lys Ser Val 1340 1345 1350 tga ggcggcgccc acccgctgcc
cgaggtgctc tgtttgtcga aacacagtaa 4650 tatttgtatt tggctgttgt
gctttccaag cgccacagtt gccctccccg gacgtggtgt 4710 tatgtggtca
tttcagccct aacttctgtg tggatcacag ttattcaggg ttcattttca 4770
tccattcttc cctttcgctc ccttccctgg aaaccccgct gcctctgggt catccgttca
4830 gcacgtggtg gagaacaagt gccttcaggg ctggcctcgg cctcgagtct
cgggacagag 4890 gccgccagtg gagatcatgg ctttgggtat tatttgactt
ttagaacaaa agctgtggtt 4950 aagatctcat ttttattgct ttttcccacg
tcccacgaga cactattttc ggttctctgg 5010 ctaataccct gtttttgagt
ttattttgtt tctgtctatg tcacagtgtt cctctacgac 5070 ccgacctctc
tatgtaagca cacatgcgca cacacacttg cattcatgaa tctgatataa 5130
agtgccagta atccgccaag agggggtgcg aagggggcat gtcacgacag ctccgccacc
5190 ccccattgcc cacccgcact ttcccgagca ccgcgccccg tgggctgtgg
gtgagccgcg 5250 ctccctgcac tgagcgggtt taggggctcg cccacatgca
tgcaggccaa gacagcaaat 5310 gccagccggg cacgacgccc gtgtgcccag
gcctcggggg tctcagagcc gcctctcacc 5370 cccgaccctc cacccagggg
tcttcccgtc gggagtggag gcgttggtcc tggaagctga 5430 ctcatcggag
agggaaatac caaataaaca tccgaggttg caaaaaaaaa aaaaaaaaaa 5490
aaaaaaaaaa aaaaagcgcg gccgc 5515 99 1353 PRT Homo sapiens 99 Met
Ala Ser Pro Pro His Gln Gln Leu Leu His His His Ser Thr Glu 1 5 10
15 Val Ser Cys Asp Ser Ser Gly Asp Ser Asn Ser Val Arg Val Lys Ile
20 25 30 Asn Pro Lys Gln Leu Ser Ser Asn Ser His Pro Lys His Cys
Lys Tyr 35 40 45 Ser Ile Ser Ser Ser Cys Ser Ser Ser Gly Asp Ser
Gly Gly Val Pro 50 55 60 Arg Arg Val Gly Gly Gly Gly Arg Leu Arg
Arg Gln Lys Lys Leu Pro 65 70 75 80 Gln Leu Phe Glu Arg Ala Ser Ser
Arg Trp Trp Asp Pro Lys Phe Asp 85 90 95 Ser Val Asn Leu Glu Glu
Ala Cys Leu Glu Arg Cys Phe Pro Gln Thr 100 105 110 Gln Arg Arg Phe
Arg Tyr Ala Leu Phe Tyr Ile Gly Phe Ala Cys Leu 115 120 125 Leu Trp
Ser Ile Tyr Phe Ala Val His Met Arg Ser Arg Leu Ile Val 130 135 140
Met Val Ala Pro Ala Leu Cys Phe Leu Leu Val Cys Val Gly Phe Phe 145
150 155 160 Leu Phe Thr Phe Thr Lys Leu Tyr Ala Arg His Tyr Ala Trp
Thr Ser 165 170 175 Leu Ala Leu Thr Leu Leu Val Phe Ala Leu Thr Leu
Ala Ala Gln Phe 180 185 190 Gln Val Leu Thr Pro Val Ser Gly Arg Gly
Asp Ser Ser Asn Leu Thr 195 200 205 Ala Thr Ala Arg Pro Thr Asp Thr
Cys Leu Ser Gln Val Gly Ser Phe 210 215 220 Ser Met Cys Ile Glu Val
Leu Phe Leu Leu Tyr Thr Val Met His Leu 225 230 235 240 Pro Leu Tyr
Leu Ser Leu Cys Leu Gly Val Ala Tyr Ser Val Leu Phe 245 250 255 Glu
Thr Phe Gly Tyr His Phe Arg Asp Glu Ala Cys Phe Pro Ser Pro 260 265
270 Gly Ala Gly Ala Leu His Trp Glu Leu Leu Ser Arg Gly Leu Leu His
275 280 285 Gly Cys Ile His Ala Ile Gly Val His Leu Phe Val Met Ser
Gln Val 290 295 300 Arg Ser Arg Ser Thr Phe Leu Lys Val Gly Gln Ser
Ile Met His Gly 305 310 315 320 Lys Asp Leu Glu Val Glu Lys Ala Leu
Lys Glu Arg Met Ile His Ser 325 330 335 Val Met Pro Arg Ile Ile Ala
Asp Asp Leu Met Lys Gln Gly Asp Glu 340 345 350 Glu Ser Glu Asn Ser
Val Lys Arg His Ala Thr Ser Ser Pro Lys Asn 355 360 365 Arg Lys Lys
Lys Ser Ser Ile Gln Lys Ala Pro Ile Ala Phe Arg Pro 370 375 380 Phe
Lys Met Gln Gln Ile Glu Glu Val Ser Ile Leu Phe Ala Asp Ile 385 390
395 400 Val Gly Phe Thr Lys Met Ser Ala Asn Lys Ser Ala His Ala Leu
Val 405 410 415 Gly Leu Leu Asn Asp Leu Phe Gly Arg Phe Asp Arg Leu
Cys Glu Glu 420 425 430 Thr Lys Cys Glu Lys Ile Ser Thr Leu Gly Asp
Cys Tyr Tyr Cys Val 435 440 445 Ala Gly Cys Pro Glu Pro Arg
Ala Asp His Ala Tyr Cys Cys Ile Glu 450 455 460 Met Gly Leu Gly Met
Ile Lys Ala Ile Glu Gln Phe Cys Gln Glu Lys 465 470 475 480 Lys Glu
Met Val Asn Met Arg Val Gly Val His Thr Arg Thr Val Leu 485 490 495
Cys Gly Ile Leu Gly Met Arg Arg Phe Lys Phe Asp Val Trp Ser Asn 500
505 510 Asp Val Asn Leu Ala Asn Leu Met Glu Gln Leu Gly Val Ala Gly
Lys 515 520 525 Val His Ile Ser Glu Ala Thr Ala Lys Tyr Leu Asp Asp
Arg Tyr Glu 530 535 540 Met Glu Asp Gly Lys Val Ile Glu Arg Leu Gly
Gln Ser Val Val Ala 545 550 555 560 Asp Gln Leu Lys Gly Leu Lys Thr
Tyr Leu Ile Ser Gly Gln Arg Ala 565 570 575 Lys Glu Ser Arg Cys Ser
Cys Ala Glu Ala Leu Leu Ser Gly Phe Glu 580 585 590 Val Ile Asp Gly
Ser Gln Val Ser Ser Gly Pro Arg Gly Gln Gly Thr 595 600 605 Ala Ser
Ser Gly Asn Val Ser Asp Leu Ala Gln Thr Val Lys Thr Phe 610 615 620
Asp Asn Leu Lys Thr Cys Pro Ser Cys Gly Ile Thr Phe Ala Pro Lys 625
630 635 640 Ser Glu Ala Gly Ala Glu Gly Gly Ala Pro Gln Asn Gly Cys
Gln Asp 645 650 655 Glu His Lys Asn Ser Thr Lys Ala Ser Gly Gly Pro
Asn Pro Lys Thr 660 665 670 Gln Asn Gly Leu Leu Ser Pro Pro Gln Glu
Glu Lys Leu Thr Asn Ser 675 680 685 Gln Thr Ser Leu Cys Glu Ile Leu
Gln Glu Lys Gly Arg Trp Ala Gly 690 695 700 Val Ser Leu Asp Gln Ser
Ala Leu Leu Pro Leu Arg Phe Lys Asn Ile 705 710 715 720 Arg Glu Lys
Thr Asp Ala His Phe Val Asp Val Ile Lys Glu Asp Ser 725 730 735 Leu
Met Lys Asp Tyr Phe Phe Lys Pro Pro Ile Asn Gln Phe Ser Leu 740 745
750 Asn Phe Leu Asp Gln Glu Leu Glu Arg Ser Tyr Arg Thr Ser Tyr Gln
755 760 765 Glu Glu Val Ile Lys Asn Ser Pro Val Lys Thr Phe Ala Ser
Pro Thr 770 775 780 Phe Ser Ser Leu Leu Asp Val Phe Leu Ser Thr Thr
Val Phe Leu Thr 785 790 795 800 Leu Ser Thr Thr Cys Phe Leu Lys Tyr
Glu Ala Ala Thr Val Pro Pro 805 810 815 Pro Pro Ala Ala Leu Ala Val
Phe Ser Ala Ala Leu Leu Leu Glu Val 820 825 830 Leu Ser Leu Ala Val
Ser Ile Arg Met Val Phe Phe Leu Glu Asp Val 835 840 845 Met Ala Cys
Thr Lys Arg Leu Leu Glu Trp Ile Ala Gly Trp Leu Pro 850 855 860 Arg
His Cys Ile Gly Ala Ile Leu Val Ser Leu Pro Ala Leu Ala Val 865 870
875 880 Tyr Ser His Ala Thr Ser Glu Tyr Glu Thr Asn Ile His Phe Pro
Val 885 890 895 Phe Thr Gly Ser Ala Ala Leu Ile Ala Val Val His Tyr
Cys Asn Phe 900 905 910 Cys Gln Leu Ser Ser Trp Met Arg Ser Ser Leu
Ala Thr Val Val Gly 915 920 925 Ala Gly Pro Leu Leu Leu Leu Tyr Val
Ser Leu Cys Pro Asp Ser Ser 930 935 940 Val Leu Thr Ser Pro Leu Asp
Ala Val Gln Asn Phe Ser Ser Glu Arg 945 950 955 960 Asn Pro Cys Asn
Ser Ser Val Pro Arg Asp Leu Arg Arg Pro Ala Ser 965 970 975 Leu Ile
Gly Gln Glu Val Val Leu Val Phe Phe Leu Leu Leu Leu Leu 980 985 990
Val Trp Phe Leu Asn Arg Glu Phe Glu Val Ser Tyr Arg Leu His Tyr 995
1000 1005 His Gly Asp Val Glu Ala Asp Leu His Arg Thr Lys Ile Gln
Ser 1010 1015 1020 Met Arg Asp Gln Ala Asp Trp Leu Leu Arg Asn Ile
Ile Pro Tyr 1025 1030 1035 His Val Ala Glu Gln Leu Lys Val Ser Gln
Thr Tyr Ser Lys Asn 1040 1045 1050 His Asp Ser Gly Gly Val Ile Phe
Ala Ser Ile Val Asn Phe Ser 1055 1060 1065 Glu Phe Tyr Glu Glu Asn
Tyr Glu Gly Gly Lys Glu Cys Tyr Arg 1070 1075 1080 Val Leu Asn Glu
Leu Ile Gly Asp Phe Asp Glu Leu Leu Ser Lys 1085 1090 1095 Pro Asp
Tyr Ser Ser Ile Glu Lys Ile Lys Thr Ile Gly Ala Thr 1100 1105 1110
Tyr Met Ala Ala Ser Gly Leu Asn Thr Ala Gln Ala Gln Asp Gly 1115
1120 1125 Ser His Pro Gln Glu His Leu Gln Ile Leu Phe Glu Phe Ala
Lys 1130 1135 1140 Glu Met Met Arg Val Val Asp Asp Phe Asn Ser Asn
Met Leu Trp 1145 1150 1155 Phe Asn Phe Lys Leu Arg Val Gly Phe Asn
His Gly Pro Leu Thr 1160 1165 1170 Ala Gly Val Ile Gly Thr Thr Lys
Leu Leu Tyr Asp Ile Trp Gly 1175 1180 1185 Asp Thr Val Asn Ile Ala
Ser Arg Met Asp Thr Thr Gly Val Glu 1190 1195 1200 Cys Arg Ile Gln
Val Ser Glu Glu Ser Tyr Arg Val Leu Ser Lys 1205 1210 1215 Met Gly
Tyr Asp Phe Asp Tyr Arg Gly Thr Val Asn Val Lys Gly 1220 1225 1230
Lys Gly Gln Met Lys Thr Tyr Leu Tyr Pro Lys Cys Thr Asp His 1235
1240 1245 Arg Val Ile Pro Gln His Gln Leu Ser Ile Ser Pro Asp Ile
Arg 1250 1255 1260 Val Gln Val Asp Gly Ser Ile Gly Arg Ser Pro Thr
Asp Glu Ile 1265 1270 1275 Ala Asn Leu Val Pro Ser Val Gln Tyr Val
Asp Lys Thr Ser Leu 1280 1285 1290 Gly Ser Asp Ser Ser Thr Gln Ala
Lys Asp Ala His Leu Ser Arg 1295 1300 1305 Lys Arg Pro Trp Lys Glu
Pro Val Lys Ala Glu Glu Arg Gly Arg 1310 1315 1320 Phe Gly Lys Ala
Ile Glu Lys Asp Asp Cys Asp Glu Thr Gly Ile 1325 1330 1335 Glu Glu
Ala Asn Glu Leu Thr Lys Leu Asn Val Ser Lys Ser Val 1340 1345 1350
100 26 DNA Artificial sequence Primer 100 gacctttggc taccatttcc
gggatg 26 101 25 DNA Artificial sequence Primer 101 cttcgctcac
ctggatgcgg cactc 25 102 19 DNA Artificial sequence primer based on
mouse adenyl cyclase 9 102 ggagcgctgc tttccgcag 19 103 25 DNA
Artificial sequence Primer based on mouse adenyl cyclase 9 103
catgttcacc atctctttct tctcc 25 104 29 DNA Artificial sequence
Primer based on mouse adenyl cyclase 9 104 gtggccgtga gagtatgatt
ggagctgtc 29
* * * * *
References