U.S. patent application number 11/450696 was filed with the patent office on 2007-02-08 for methods and compositions for treating and diagnosing diabetes and related diseases involving beta-trp.
This patent application is currently assigned to Metabolex, Inc.. Invention is credited to Jeffrey D. Johnson, Yun-Ping Zhou.
Application Number | 20070031897 11/450696 |
Document ID | / |
Family ID | 32962734 |
Filed Date | 2007-02-08 |
United States Patent
Application |
20070031897 |
Kind Code |
A1 |
Johnson; Jeffrey D. ; et
al. |
February 8, 2007 |
Methods and compositions for treating and diagnosing diabetes and
related diseases involving beta-TRP
Abstract
Expression of beta-TRP is enriched in islet cells. Introduction
of expression cassettes encoding beta-TRP into diabetic islet cells
improved glucose-stimulated insulin production. Therefore, the
invention provides methods of identifying beta-TRP modulators for
treating diabetic individuals and introducing beta-TRP into islet
cells
Inventors: |
Johnson; Jeffrey D.;
(Moraga, CA) ; Zhou; Yun-Ping; (San Ramon,
CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Metabolex, Inc.
Hayward
CA
94545
|
Family ID: |
32962734 |
Appl. No.: |
11/450696 |
Filed: |
June 9, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10794897 |
Mar 4, 2004 |
7087394 |
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11450696 |
Jun 9, 2006 |
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60452596 |
Mar 5, 2003 |
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Current U.S.
Class: |
435/7.2 ;
435/320.1; 435/325; 435/69.4; 530/399; 536/23.5 |
Current CPC
Class: |
G01N 33/6872 20130101;
G01N 33/507 20130101; A61K 48/00 20130101; C07K 14/705 20130101;
G01N 2333/62 20130101; G01N 33/5088 20130101; A61P 3/10 20180101;
G01N 33/74 20130101; A61P 5/50 20180101 |
Class at
Publication: |
435/007.2 ;
435/069.4; 435/320.1; 435/325; 530/399; 536/023.5 |
International
Class: |
G01N 33/567 20060101
G01N033/567; C07H 21/04 20060101 C07H021/04; C07K 14/575 20070101
C07K014/575 |
Claims
1. A method of identifying an agent that induces glucose-stimulated
insulin production in an animal, the method comprising the steps
of: (i) contacting an agent to a polypeptide comprising at least 20
contiguous amino acids of SEQ ID NO:2; (ii) selecting an agent that
binds to the polypeptide or enhances the expression or activity of
the polypeptide, and (iii) determining the effect of the selected
agent on glucose-stimulated insulin secretion, thereby identifying
an agent that induces glucose-stimulated insulin production in an
animal.
2. The method of claim 1, wherein step (ii) comprises selecting an
agent that enhances the expression of the polypeptide.
3. The method of claim I, wherein step (ii) comprises selecting an
agent that enhances the activity of the polypeptide.
4. The method of claim 1, wherein step (ii) comprises selecting an
agent that binds to the polypeptide.
5. The method of claim 1, wherein the polypeptide comprises SEQ ID
NO:2.
6. The method of claim 1, wherein the polypeptide is expressed in a
cell and the contacting step comprises contacting the agent to the
cell.
7. The method of claim 6, wherein an agent is selected that
enhances polypeptide activity and the activity of the polypeptide
is determined by a step comprising measuring a change in calcium
flux in the cell.
8. The method of claim 6, wherein an agent is selected that
enhances polypeptide activity and the activity of the polypeptide
is determined by a step comprising measuring a change in membrane
potential of the cell.
9. The method of claim 8, wherein the membrane potential of the
cell is measured be detecting a change in fluorescence of a dye
whose fluorescence is dependent on cell depolarization and wherein
the change in fluorescence is detected with a device sufficient for
high throughput screening.
10. The method of claim 6, wherein the cell is an insulin-secreting
cell.
11. The method of claim 6, wherein the cell is a pancreatic .beta.
cell.
12. The method of claim 6, wherein the polypeptide is recombinantly
expressed in the cell.
13. The method of claim 6, wherein the cell is a Chinese hamster
ovary (CHO) cell.
14. The method of claim 1, the method further comprising
administering the agent to a diabetic animal and testing the animal
for increased glucose-stimulated insulin secretion.
15. The method of claim 14, wherein the animal is selected from a
ZDF rat and a db/db mouse.
16. The method of claim 1, wherein the polypeptide comprises at
least 100 amino acids.
17. The method of claim 1, wherein the polypeptide is at least 80%
identical to SEQ ID NO:2.
18. A method of inducing glucose-stimulated insulin production in
an animal, the method comprising administering a therapeutically
effective amount of the agent selected in claim 1.
19. The method of claim 18, wherein the animal is a human.
20. The method of claim 19, wherein the human has Type 2
diabetes.
21. The method of claim 19, wherein the human is predisposed for
Type 2 diabetes.
22. A method of expressing .beta.TRP in a pancreatic islet cell,
the method comprising introducing into an islet cell a
polynucleotide encoding a polypeptide comprising at least 20
contiguous amino acids of SEQ ID NO:2.
23. The method of claim 22, wherein the islet cell is defective for
glucose-stimulated insulin secretion.
24. The method of claim 22, wherein the polynucleotide is
introduced in vitro.
25. The method of claim 22, wherein the polynucleotide is
introduced in vivo.
26. The method of claim 22, wherein the polypeptide comprises at
least 100 amino acids.
27. The method of claim 22, wherein the polypeptide is at least 80%
identical to SEQ ID NO:2.
28. The method of claim 22, wherein the polypeptide comprises SEQ
ID NO:2.
29. The method of claim 22, wherein the polynucleotide comprises
SEQ ID NO:1.
30. The method of claim 22, wherein the islet cell is a .beta.
cell.
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] The present application claims benefit of priority to U.S.
Provisional Patent Application No 60/452,596, filed Mar. 5, 2003,
which is incorporated by reference in its entirety for any
purpose.
BACKGROUND OF THE INVENTION
[0002] Diabetes mellitus can be divided into two clinical
syndromes, Type 1 and Type 2 diabetes mellitus. Type 1, or
insulin-dependent diabetes mellitus (IDDM), is a chronic autoimmune
disease characterized by the extensive loss of beta cells in the
pancreatic Islets of Langerhans, which produce insulin. As these
cells are progressively destroyed, the amount of secreted insulin
decreases, eventually leading to hyperglycemia (abnormally high
level of glucose in the blood) when the amount of secreted insulin
drops below the level required for euglycemia (normal blood glucose
level). Although the exact trigger for this immune response is not
known, patients with IDDM have high levels of antibodies against
pancreatic beta cells. However, not all patients with high levels
of these antibodies develop IDDM.
[0003] Type 2 diabetes (also referred to as non-insulin dependent
diabetes mellitus (NIDDM)) develops when muscle, fat and liver
cells fail to respond normally to insulin. This failure to respond
(called insulin resistance) may be due to reduced numbers of
insulin receptors on these cells, or a dysfunction of signaling
pathways within the cells, or both. The beta cells initially
compensate for this insulin resistance by increasing their insulin
output. Over time, these cells become unable to produce enough
insulin to maintain normal glucose levels, indicating progression
to Type 2 diabetes.
[0004] Type 2 diabetes is brought on by a combination of poorly
understood genetic and acquired risk factors--including a high-fat
diet, lack of exercise, and aging. Worldwide, Type 2 diabetes has
become an epidemic, driven by increases in obesity and a sedentary
lifestyle, widespread adoption of western dietary habits, and the
general aging of the populations in many countries. In 1985, an
estimated 30 million people worldwide had diabetes--by 2000, this
figure had increased 5-fold, to an estimated 154 million people.
The number of people with diabetes is expected to double between
now and 2025, to about 300 million.
[0005] Type 2 diabetes is a complex disease characterized by
defects in glucose and lipid metabolism. Typically there are
perturbations in many metabolic parameters including increases in
fasting plasma glucose levels, free fatty acid levels and
triglyceride levels, as well as a decrease in the ratio of HDL/LDL.
As discussed above, one of the principal underlying causes of
diabetes is the inability of beta cells to produce sufficient
insulin to maintain glucose levels. Therefore, an important
therapeutic goal in the treatment of diabetes is therefore to
increase insulin production. The present invention addresses this
and other problems.
BRIEF SUMMARY OF THE INVENTION
[0006] The present invention provides methods of identifying an
agent that induces glucose-stimulated insulin production in an
animal. In some embodiments, the method comprising the steps of:
(i) contacting an agent to a polypeptide comprising at least 20
contiguous amino acids of SEQ ID NO:2; (ii)selecting an agent that
binds to the polypeptide or enhances the expression or activity of
the polypeptide, and (iii) determining the effect of the selected
agent on glucose-stimulated insulin secretion, thereby identifying
an agent that induces glucose-stimulated insulin production in an
animal.
[0007] In some embodiments, step (ii) comprises selecting an agent
that enhances the expression of the polypeptide. In some
embodiments, step (ii) comprises selecting an agent that enhances
the activity of the polypeptide. In some embodiments, step (ii)
comprises selecting an agent that binds to the polypeptide.
[0008] In some embodiments, the polypeptide comprises SEQ ID
NO:2.
[0009] In some embodiments, the polypeptide is expressed in a cell
and the contacting step comprises contacting the agent to the cell.
In some embodiments, an agent is selected that enhances polypeptide
activity and the activity of the polypeptide is determined by a
step comprising measuring a change in calcium flux in the cell. In
some embodiments, an agent is selected that enhances polypeptide
activity and the activity of the polypeptide is determined by a
step comprising measuring a change in membrane potential of the
cell.
[0010] In some embodiments, the membrane potential of the cell is
measured be detecting a change in fluorescence of a dye whose
fluorescence is dependent on cell depolarization and wherein the
change in fluorescence is detected with a device sufficient for
high throughput screening. In some embodiments, the cell is an
insulin-secreting cell. In some embodiments, the cell is a
pancreatic .beta. cell.
[0011] In some embodiments, the polypeptide is recombinantly
expressed in the cell. In some embodiments, the cell is a Chinese
hamster ovary (CHO) cell.
[0012] In some embodiments, the method further comprising
administering the agent to a diabetic animal and testing the animal
for increased glucose-stimulated insulin secretion. In some
embodiments, the animal is selected from a ZDF rat and a db/db
mouse.
[0013] In some embodiments, the polypeptide comprises at least 100
amino acids. In some embodiments, the polypeptide is at least 80%
identical to SEQ ID NO:2.
[0014] The present invention also provides methods of inducing
glucose-stimulated insulin production in an animal. In some
embodiments, the methods comprise administering a therapeutically
effective amount of the agent selected in a method comprising the
steps of: (i) contacting an agent to a polypeptide comprising at
least 20 contiguous amino acids of SEQ ID NO:2; (ii)selecting an
agent that binds to the polypeptide or enhances the expression or
activity of the polypeptide, and (iii) determining the effect of
the selected agent on glucose-stimulated insulin secretion. In some
embodiments, the animal is a human. In some embodiments, the human
has Type 2 diabetes. In some embodiments, the human is predisposed
for Type 2 diabetes.
[0015] The present invention also provides methods of expressing
.beta.TRP in a pancreatic islet cell. In some embodiments, the
method comprises introducing into an islet. cell a polynucleotide
encoding a polypeptide comprising at least 20 contiguous amino
acids of SEQ ID NO:2. In some embodiments, the islet cell is
defective for glucose-stimulated insulin secretion.
[0016] In some embodiments, the polynucleotide is introduced in
vitro. In some embodiments, the polynucleotide is introduced in
vivo.
[0017] In some embodiments, the polypeptide comprises at least 100
amino acids. In some embodiments, the polypeptide is at least 80%
identical to SEQ ID NO:2. In some embodiments, the polypeptide
comprises SEQ ID NO:2. In some embodiments, the polynucleotide
comprises SEQ ID NO:1.
[0018] In some embodiments, the islet cell is a .beta. cell.
DEFINITIONS
[0019] A person is "predisposed for diabetes" when the person is at
high risk for developing diabetes. A number of risk factors are
known to those of skill in the art and include: genetic factors
(e.g., carrying alleles that result in a higher occurrence of
diabetes than in the average population or having parents or
siblings with diabetes); overweight (e.g., body mass index (BMI)
greater or equal to 25 kg/m.sup.2); habitual physical inactivity,
race/ethnicity (e.g., African-American, Hispanic-American, Native
Americans, Asian-Americans, Pacific Islanders); previously
identified impaired fasting glucose or impaired glucose tolerance,
hypertension (e.g., greater or equal to 140/90 mmHg in adults); HDL
cholesterol greater or equal to 35 mg/dl; triglyceride levels
greater or equal to 250 mg/dl; a history of gestational diabetes or
delivery of a baby over nine pounds; and/or polycystic ovary
syndrome. See, e.g., "Report of the Expert Committee on the
Diagnosis and Classification of Diabetes Mellitus" and "Screening
for Diabetes" Diabetes Care 25(1): S5-S24 (2002).
[0020] A ".beta.TRP" or "beta-TRP" polypeptide refers to a calcium
channel that is substantially identical to SEQ ID NO:2, SEQ ID
NO:4, or SEQ ID NO:6. .beta.TRP polypeptides, when inserted into a
membrane or expressed in a cell, typically form a functional cation
channel. In some embodiments, amino acid residues conserved between
mouse and human (see, FIG. 7), rat and human, rat and mouse or
between all three sequences are present in .beta.TRP sequences of
the invention. In some cases, .beta.TRP comprises a glutamine
(e.g., encoded by the codon CAG) or an arginine (e.g., encoded by
the codon CGG) at position 579. .beta.TRP polypeptides typically
have a "TRP" motif and transmembrane domains. See, e.g., FIG. 8
illustrating these motifs and domains in the human .beta.TRP
sequence.
[0021] ".beta.TRP activity," as used herein, refers to the ability
of a protein to set or modulate electrical potential of the plasma
membrane of a cell. One can use fluorescent dyes or fluorescent
resonance energy transfer (FRET) reagents that are sensitive to
membrane potential to detect the activity of a channel in a cell.
See, e.g., Miller et al., Eur JPharmacol. 370(2): 179-85 (1999);
Fedida, et aL, Prog Biophys Mol Biol. 75(3): 165-99 (2000).
Alternatively, calcium flux assays using calcium-dependent
fluorescent dyes can be used to detect channel activity. Activity
can also be measured, for example, using patch-clamp techniques.
Patch-clamp analysis generally involves formation of a high
resistance seal between the cell membrane and the glass wall of a
micropipette. Current passing through the ion channels in the
membrane is then measured.
[0022] An "activator of .beta.TRP" refers to an agent that opens,
stimulates, sensitizes or up regulates the activity or expression
of .beta.TRP. "Enhanced .beta.TRP activity" refers to activity of a
.beta.TRP channel that is opened, stimulated, sensitized or
up-regulated compared to a control (e.g., a sample not containing a
potential .beta.TRP modulator).
[0023] "A dye whose fluorescence is dependent on cell
depolarization" refers to dyes or probes that exhibit
potential-dependent changes in their transmembrane distribution
that are accompanied by a fluorescence change. The magnitude of
their optical responses can be about 1% fluorescence change per mV.
These dyes, sometimes referred to as "slow-response probes,"
include, e.g., cationic carbocyanines and rhodamines and anionic
oxonols, as well as proprietary dyes available for the Molecular
Devices (Sunnyvale, CA) FLEXstation and FLIPR systems. Dyes that
fluoresce in response to changes in membrane potential and cell
depolarization are described in, e.g., Zochowski M, et al. Biol
Bull 198, 1-21 (2000); Shapiro, H M, Methods 21, 271-279 (2000);
Nicholls D G, et al. Trends Neurosci 23, 166-174 (2000); Loew L M.
Cell Biology: A Laboratory Handbook, 2nd Ed., Vol. 3, Celis J E,
Ed. pp. 375-379 (1998); Plasek J, et al. J Photochem Photobiol B
33, 101-124 (1996); Loew L M. Pure Appl Chem 68,1405 (1996); Loew L
M. Adv Chem Ser 235,151 (1994); and Smith J C. Biochim Biophys Acta
1016, 1-28 (1990).
[0024] Increases or decreases in membrane potential are also
referred to as "membrane hyperpolarization" and "membrane
depolarization," respectively.
[0025] "A device sufficient for high throughput screening" refers
to a device that can be used by one person to analyze a large
number of samples (e.g., at least 96 and sometimes at least 200,
384, 500 or even 1000 samples on a daily basis). Examples of high
throughput devices for use in measuring cell depolarization and
membrane potential changes include, e.g., the FLIPR and FLEXstation
devices from Molecular Devices (Sunnyvale, Calif.). Methods such as
patch-clamping are not practical for high throughput analyses.
[0026] "Antibody" refers to a polypeptide substantially encoded by
an immunoglobulin gene or immunoglobulin genes, or fragments
thereof which specifically bind and recognize an analyte (antigen).
The recognized immunoglobulin genes include the kappa, lambda,
alpha, gamma, delta, epsilon and mu constant region genes, as well
as the myriad immunoglobulin variable region genes. Light chains
are classified as either kappa or lambda. Heavy chains are
classified as gamma, mu, alpha, delta, or epsilon, which in turn
define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,
respectively.
[0027] An exemplary immunoglobulin (antibody) structural unit
comprises a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each
chain defines a variable region of about 100 to 110 or more amino
acids primarily responsible for antigen recognition. The terms
variable light chain (V.sub.L) and variable heavy chain (V.sub.H)
refer to these light and heavy chains respectively.
[0028] Antibodies exist, e.g., as intact immunoglobulins or as a
number of well-characterized fragments produced by digestion with
various peptidases. Thus, for example, pepsin digests an antibody
below the disulfide linkages in the hinge region to produce
F(ab)'.sub.2 a dimer of Fab which itself is a light chain joined to
V.sub.H-C.sub.HI by a disulfide bond. The F(ab)'.sub.2 may be
reduced under mild conditions to break the disulfide linkage in the
hinge region, thereby converting the F(ab)'.sub.2 dimer into an
Fab' monomer. The Fab' monomer is essentially an Fab with part of
the hinge region (see, Paul (Ed.) Fundamental Immunology, Third
Edition, Raven Press, NY (1993)). While various antibody fragments
are defined in terms of the digestion of an intact antibody, one of
skill will appreciate that such fragments may be synthesized de
novo either chemically or by utilizing recombinant DNA methodology.
Thus, the term antibody, as used herein, also includes antibody
fragments either produced by the modification of whole antibodies
or those synthesized de novo using recombinant DNA methodologies
(e.g., single chain Fv).
[0029] The term "gene" means the segment of DNA involved in
producing a polypeptide chain; it includes regions preceding and
following the coding region (leader and trailer) as well as
intervening sequences (introns) between individual coding segments
(exons).
[0030] The term "isolated," when applied to a nucleic acid or
protein, denotes that the nucleic acid or protein is essentially
free of other cellular components with which it is associated in
the natural state. It is preferably in a homogeneous state although
it can be in either a dry or aqueous solution. Purity and
homogeneity are typically determined using analytical chemistry
techniques such as polyacrylamide gel electrophoresis or high
performance liquid chromatography. A protein that is the
predominant species present in a preparation is substantially
purified. In particular, an isolated gene is separated from open
reading frames that flank the gene and encode a protein other than
the gene of interest. The term "purified" denotes that a nucleic
acid or protein gives rise to essentially one band in an
electrophoretic gel. Particularly, it means that the nucleic acid
or protein is at least 85% pure, more preferably at least 95% pure,
and most preferably at least 99% pure.
[0031] The term "nucleic acid" or "polynucleotide" refers to
deoxyribonucleotides or ribonucleotides and polymers thereof in
either single- or double-stranded form. Unless specifically
limited, the term encompasses nucleic acids containing known
analogues of natural nucleotides that have similar binding
properties as the reference nucleic acid and are metabolized in a
manner similar to naturally occurring nucleotides. Unless otherwise
indicated, a particular nucleic acid sequence also implicitly
encompasses conservatively modified variants thereof (e.g.,
degenerate codon substitutions) and complementary sequences as well
as the sequence explicitly indicated. Specifically, degenerate
codon substitutions may be achieved by generating sequences in
which the third position of one or more selected (or all) codons is
substituted with mixed-base and/or deoxyinosine residues (Batzer et
al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol.
Chem. 260:2605-2608 (1985); and Cassol et al. (1992); Rossolini et
al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is
used interchangeably with gene, cDNA, and mRNA encoded by a
gene.
[0032] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers and non-naturally occurring
amino acid polymers. As used herein, the terms encompass amino acid
chains of any length, including full-length proteins (i.e.,
antigens), wherein the amino acid residues are linked by covalent
peptide bonds.
[0033] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally occurring amino acids are those
encoded by the genetic code, as well as those amino acids that are
later modified, e.g., hydroxyproline, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an ct carbon that is bound to a hydrogen, a carboxyl
group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. "Amino acid mimetics" refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but which functions in
a manner similar to a naturally occurring amino acid.
[0034] Amino acids may be referred to herein by either the commonly
known three letter symbols or by the one-letter symbols recommended
by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides,
likewise, may be referred to by their commonly accepted
single-letter codes.
[0035] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, "conservatively modified variants" refers to those
nucleic acids which encode identical or essentially identical amino
acid sequences, or where the nucleic acid does not encode an amino
acid sequence, to essentially identical sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every nucleic acid sequence
herein that encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of skill will recognize
that each codon in a nucleic acid (except AUG, which is ordinarily
the only codon for methionine, and TGG, which is ordinarily the
only codon for tryptophan) can be modified to yield a functionally
identical molecule. Accordingly, each silent variation of a nucleic
acid that encodes a polypeptide is implicit in each described
sequence.
[0036] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. Such conservatively modified variants are in addition to and
do not exclude polymorphic variants, interspecies homologs, and
alleles of the invention.
[0037] The following eight groups each contain amino acids that are
conservative substitutions for one another: [0038] 1) Alanine (A),
Glycine (G); [0039] 2) Aspartic acid (D), Glutamic acid (E); [0040]
3) Asparagine (N), Glutamine (Q); [0041] 4) Arginine (R), Lysine
(K); [0042] 5) Isoleucine (1), Leucine (L), Methionine (M), Valine
(V); [0043] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
[0044] 7) Serine (S), Threonine (T); and [0045] 8) Cysteine (C),
Methionine (M) (see, e.g., Creighton, Proteins (1984)).
[0046] "Percentage of sequence identity" is determined by comparing
two optimally aligned sequences over a comparison window, wherein
the portion of the polynucleotide sequence in the comparison window
may comprise additions or deletions (i.e., gaps) as compared to the
reference sequence (which does not comprise additions or deletions)
for optimal alignment of the two sequences. The percentage is
calculated by determining the number of positions at which the
identical nucleic acid base or amino acid residue occurs in both
sequences to yield the number of matched positions, dividing the
number of matched positions by the total number of positions in the
window of comparison and multiplying the result by 100 to yield the
percentage of sequence identity.
[0047] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same. The term
"substantially identical" refers to two or more sequences that have
a specified percentage of amino acid residues or nucleotides that
are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%,
85%, 90%, or 95% identity over a specified region), when compared
and aligned for maximum correspondence over a comparison window, or
designated region as measured using one of the following sequence
comparison algorithms or by manual alignment and visual inspection.
Optionally, the identity exists over a region that is at least
about 50 nucleotides in length, or more preferably over a region
that is 100 to 500 or 1000 or more nucleotides in length. The
present invention provides polynucleotides and polypeptides
substantially identical to SEQ ID NOs: 1, 2, 3, 4, 5, and 6.
[0048] The term "similarity," or percent "similarity," in the
context of two or more polypeptide sequences, refer to two or more
sequences or subsequences that have a specified percentage of amino
acid residues that are either the same or similar as defined in the
8 conservative amino acid substitutions defined above (i.e., 60%,
optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% similar over a
specified region), when compared and aligned for maximum
correspondence over a comparison window, or designated region as
measured using one of the following sequence comparison algorithms
or by manual alignment and visual inspection. Such sequences are
then said to be "substantially similar." Optionally, this identity
exists over a region that is at least about 50 amino acids in
length, or more preferably over a region that is at least about 100
to 500 or 1000 or more amino acids in length. The present invention
provides polypeptides substantially similar to SEQ ID NOs: 2, 4,
and 6.
[0049] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
[0050] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are well known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith and
Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment
algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by
the search for similarity method of Pearson and Lipman (1988) Proc.
Nat'L Acad. Sci. USA 85:2444, by computerized implementations of
these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, 575 Science
Dr., Madison, Wis.), or by manual alignment and visual inspection
(see, e.g., Ausubel et al., Current Protocols in Molecular Biology
(1995 supplement)).
[0051] An example of an algorithm that is suitable for determining
percent sequence identity and sequence similarity are the BLAST and
BLAST 2.0 algorithms, which are described in Altschul et al. (1977)
Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol.
Biol. 215:403-410, respectively. Software
forperformingBLASTanalysesis publicly available through the
National Center for Biotechnology Information
(http://www.ncbi.nlm.nih.gov/). This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence, which either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold (Altschul et al., supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
extended in both directions along each sequence for as far as the
cumulative alignment score can be increased. Cumulative scores are
calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always>0) and N
(penalty score for mismatching residues; always<0). For amino
acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction are
halted when: the cumulative aligrnnent score falls off by the
quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T, and X determine
the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation (E) or 10, M=5, N=-4 and a comparison of both strands.
For amino acid sequences, the BLASTP program uses as defaults a
wordlength of 3, and expectation (E) of 10, and the BLOSUM62
scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad.
Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10,
M=5, N=-4, and a comparison of both strands.
[0052] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin and
Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One
measure of similarity provided by the BLAST algorithm is the
smallest sum probability (P(N)), which provides an indication of
the probability by which a match between two nucleotide or amino
acid sequences would occur by chance. For example, a nucleic acid
is considered similar to a reference sequence if the smallest sum
probability in a comparison of the test nucleic acid to the
reference nucleic acid is less than about 0.2, more preferably less
than about 0.01, and most preferably less than about 0.001.
[0053] An indication that two nucleic acid sequences or
polypeptides are substantially identical is that the polypeptide
encoded by the first nucleic acid is immunologically cross reactive
with the antibodies raised against the polypeptide encoded by the
second nucleic acid, as described below. Thus, a polypeptide is
typically substantially identical to a second polypeptide, for
example, where the two peptides differ only by conservative
substitutions. Another indication that two nucleic acid sequences
are substantially identical is that the two molecules or their
complements hybridize to each other under stringent conditions, as
described below. Yet another indication that two nucleic acid
sequences are substantially identical is that the same primers can
be used to amplify the sequence.
[0054] The phrase "selectively (or specifically) hybridizes to"
refers to the binding, duplexing, or hybridizing of a molecule only
to a particular nucleotide sequence under stringent hybridization
conditions when that sequence is present in a complex mixture
(e.g., total cellular or library DNA or RNA).
[0055] The phrase "stringent hybridization conditions" refers to
conditions under which a probe will hybridize to its target
subsequence, typically in a complex mixture of nucleic acid, but to
no other sequences. Stringent conditions are sequence-dependent and
will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures. An extensive guide
to the hybridization of nucleic acids is found in Tijssen,
Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic Probes, "Overview of principles of hybridization and
the strategy of nucleic acid assays" (1993). Generally, stringent
conditions are selected to be about 5-10.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence at a
defined ionic strength pH. The T.sub.m is the temperature (under
defined ionic strength, pH, and nucleic concentration) at which 50%
of the probes complementary to the target hybridize to the target
sequence at equilibrium (as the target sequences are present in
excess, at T.sub.m, 50% of the probes are occupied at equilibrium).
Stringent conditions will be those in which the salt concentration
is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M
sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the
temperature is at least about 30.degree. C. for short probes (e.g.,
10 to 50 nucleotides) and at least about 60.degree. C. for long
probes (e.g., greater than 50 nucleotides). Stringent conditions
may also be achieved with the addition of destabilizing agents such
as formamide. For selective or specific hybridization, a positive
signal is at least two times background, optionally 10 times
background hybridization. Exemplary stringent hybridization
conditions can be as following: 50% formamide, 5.times. SSC, and 1%
SDS, incubating at 42.degree. C., or 5.times. SSC, 1% SDS,
incubating at 65.degree. C., with wash in 0.2.times. SSC, and 0.1%
SDS at 65.degree. C. Such washes can be performed for 5, 15, 30,
60, 120, or more minutes.
[0056] Nucleic acids that do not hybridize to each other under
stringent conditions are still substantially identical if the
polypeptides that they encode are substantially identical. This
occurs, for example, when a copy of a nucleic acid is created using
the maximum codon degeneracy permitted by the genetic code. In such
cases, the nucleic acids typically hybridize under moderately
stringent hybridization conditions. Exemplary "moderately stringent
hybridization conditions" include a hybridization in a buffer of
40% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in
1.times. SSC at 45.degree. C. Such washes can be performed for 5,
15, 30, 60, 120, or more minutes. A positive hybridization is at
least twice background. Those of ordinary skill will readily
recognize that alternative hybridization and wash conditions can be
utilized to provide conditions of similar stringency.
[0057] The phrase "a nucleic acid sequence encoding" refers to a
nucleic acid which contains sequence information for a structural
RNA such as rRNA, a tRNA, or the primary amino acid sequence of a
specific protein or peptide, or a binding site for a trans-acting
regulatory agent. This phrase specifically encompasses degenerate
codons (i.e., different codons which encode a single amino acid) of
the native sequence or sequences that may be introduced to conform
with codon preference in a specific host cell.
[0058] The term "recombinant" when used with reference, e.g., to a
cell, or nucleic acid, protein, or vector, indicates that the cell,
nucleic acid, protein or vector, has been modified by the
introduction of a heterologous nucleic acid or protein or the
alteration of a native nucleic acid or protein, or that the cell is
derived from a cell so modified. Thus, for example, recombinant
cells express genes that are not found within the native
(nonrecombinant) form of the cell or express native genes that are
otherwise abnormally expressed, under-expressed or not expressed at
all.
[0059] The term "heterologous" when used with reference to portions
of a nucleic acid indicates that the nucleic acid comprises two or
more subsequences that are not found in the same relationship to
each other in nature. For instance, the nucleic acid is typically
recombinantly produced, having two or more sequences from unrelated
genes arranged to make a new functional nucleic acid, e.g., a
promoter from one source and a coding region from another source.
Similarly, a heterologous protein indicates that the protein
comprises two or more subsequences that are not found in the same
relationship to each other in nature (e.g., a fusion protein).
[0060] An "expression vector" is a nucleic acid construct,
generated recombinantly or synthetically, with a series of
specified nucleic acid elements that permit transcription of a
particular nucleic acid in a host cell. The expression vector can
be part of a plasmid, virus, or nucleic acid fragment. Typically,
the expression vector includes a nucleic acid to be transcribed
operably linked to a promoter.
[0061] The phrase "specifically (or selectively) binds to an
antibody" or "specifically (or selectively) immunoreactive with",
when referring to a protein or peptide, refers to a binding
reaction which is determinative of the presence of the protein in
the presence of a heterogeneous population of proteins and other
biologics. Thus, under designated immunoassay conditions, the
specified antibodies bind to a particular protein and do not bind
in a significant amount to other proteins present in the sample.
Specific binding to an antibody under such conditions may require
an antibody that is selected for its specificity for a particular
protein. For example, antibodies raised against a protein having an
amino acid sequence encoded by any of the polynucleotides of the
invention can be selected to obtain antibodies specifically
immunoreactive with that protein and not with other proteins,
except for polymorphic variants. A variety of immunoassay formats
may be used to select antibodies specifically immunoreactive with a
particular protein. For example, solid-phase ELISA immunoassays,
Western blots, or immunohistochemistry are routinely used to select
monoclonal antibodies specifically immunoreactive with a protein.
See, Harlow and Lane Antibodies, A Laboratory Manual, Cold Spring
Harbor Publications, NY (1988) for a description of immunoassay
formats and conditions that can be used to determine specific
immunoreactivity. Typically, a specific or selective reaction will
be at least twice the background signal or noise and more typically
more than 10 to 100 times background.
[0062] "Inhibitors," "activators," and "modulators" of .beta.TRP
expression or of .beta.TRP activity are used to refer to
inhibitory, activating, or modulating molecules, respectively,
identified using in vitro and in vivo assays for .beta.TRP
expression or .beta.TRP activity, e.g., ligands, agonists,
antagonists, and their homologs and mimetics. The term "modulator"
includes inhibitors and activators. Inhibitors are agents that,
e.g., inhibit expression of STRP or bind to, partially or totally
block stimulation or enzymatic activity, decrease, prevent, delay
activation, inactivate, desensitize, or down regulate the activity
of .beta.TRP, e.g., antagonists. Activators are agents that, e.g.,
induce or activate the expression of .beta.TRP or bind to,
stimulate, increase, open, activate, facilitate, enhance activation
or enzymatic activity, sensitize or up regulate the activity of
.beta.TRP, e.g., agonists. Modulators include naturally occurring
and synthetic ligands, antagonists, agonists, small chemical
molecules and the like. Such assays for inhibitors and activators
include, e.g., applying putative modulator compounds to pancreatic
cells or other cells expressing .beta.TRP, in the presence or
absence of .beta.TRP modulators and then determining the functional
effects on .beta.TRP activity, as described above. Samples or
assays comprising .beta.TRP that are treated with a potential
activator, inhibitor, or modulator are compared to control samples
without the inhibitor, activator, or modulator to examine the
extent of effect. Control samples (untreated with modulators) are
assigned a relative .beta.TRP activity value of 100%. Inhibition of
.beta.TRP is achieved when the .beta.TRP activity value relative to
the control is about 80%, optionally 50% or 25-1%. Activation of
.beta.TRP is achieved when the .beta.TRP activity value relative to
the control is 10%, optionally 150%, optionally 200-500%, or
1000-3000% higher.
BRIEF DESCRIPTION OF THE DRAWINGS
[0063] FIG. 1 displays array expression data for .beta.TRP. FIG. 1A
displays a custom mouse islet oligonucleotide array analysis of
probe set MBXMUSISL08907. Average difference values reflect the
relative abundance of .beta.TRP in mouse islets and the insulin
secreting mouse cell line betaHC9. FIG. 1B displays how the rat
islet custom oligonucleotide array was used to survey gene changes
in animal models of diabetes. .beta.TRP mRNA (probe set
MBXRATISL12881) is decreased 2-3 fold (p=0.002) in ZDF female rats
relative to lean control animals and is substantially restored by
concomitant treatment with troglitazone.
[0064] FIG. 2 illustrates insulin secretion in ZDF islets infected
with Ad-.beta.TRP virus. Islets from male ZDF rats were infected
with an adenovirus expressing .beta.TRP or eGFP and insulin
responses to 16 mM glucose was determined by perifusion in
Krebs-Ringers bicarbonate medium. Over-expression of .beta.TRP in
the ZDF islets enhanced both phases of the insulin secretion
stimulated by glucose.
[0065] FIG. 3 displays intracellular free calcium [Ca.sup.2+].sub.i
and membrane potential responses to ATP in CHO-K1 cells stably
transfected with .beta.TRP. Control and .beta.TRP-CHO cells were
plated in 96-well plates 2-d before the assay. [Ca.sup.2+].sub.i
and membrane potential (MP) responses to ATP (30 .mu.M added at 20
sec) were measured with FLEXStation.RTM. and correspondent dyes
from Molecular Devices (Sunnyvale, Calif.).
[0066] FIG. 4 illustrates MP responses to ATP and MTX at different
Ca.sup.2+ levels in .beta.TRP-CHO cells. MP responses to Maitotoxin
(MTX) were monitored with the Flexstation.RTM. as described in FIG.
3. The regular assay buffer contains 1.26 mM Ca.sup.2+. To raise
the calcium concentration to 2.5 or 5 mM, extra amount of
CaCl.sub.2 was added at same time as other testing reagents.
[0067] FIG. 5 illustrates dose dependent effects of ATP and
calcimycin on membrane potential in .beta.TRP-CHO cells. MP was
measured with Flexstation.RTM. as in FIG. 3. No effect was observed
in control cells for either compound.
[0068] FIG. 6 illustrated the effects of known TRP blockers on
ATP-induced depolarization in .beta.TRP-CHO cells. MP was measured
with Flexstation.RTM. as in FIG. 3. The inhibitors were added
either simultaneously (right panel) or prior to ATP stimulation
(left panel).
[0069] FIGS. 7A-D provide an alignment of the human and mouse
.beta.TRP amino acid sequences.
[0070] FIG. 8 depicts the .beta.TRP amino acid sequence.
Transmembrane domains are underlined. The TRP motif is in bold.
DETAILED DESCRIPTION OF THE INVENTION
I. Introduction
[0071] The present application demonstrates that, surprisingly,
.beta.TRP expression in pancreatic islet cells effects insulin
secretion. Expression of .beta.TRP is relatively islet cell
specific. In ZDF rats (an animal model for diabetes), .beta.TRP
expression is reduced compared to wild type rats. Significantly,
when .beta.TRP is expressed in ZDF islet cells, glucose stimulated
insulin secretion is increased compared to empty vector controls.
These results demonstrate that enhancing expression or activity of
.beta.TRP in insulin secreting cells increases glucose-stimulated
insulin production. Therefore, the present application provides
methods for identifying agents that increase .beta.TRP expression
or activity in insulin secreting cells, as well as the use of such
agents to treat diabetic or pre-diabetic individuals. The
application also provides methods for introducing
.beta.TRP-encoding polynucleotides into pancreatic .beta. cells for
the expression of .beta.TRP.
II. General Recombinant Nucleic Acid Methods for Use With the
Invention
[0072] The nucleic acid compositions used in the subject invention
may encode all or a part, usually at least substantially all, of
the .beta.TRP polypeptides as appropriate. Fragments may be
obtained of the DNA sequence by chemically synthesizing
oligonucleotides in accordance with conventional methods, by
restriction enzyme digestion, by PCR amplification, etc. For the
most part, DNA fragments will be of at least about ten contiguous
nucleotides, usually at least about 15 nucleotides, more usually at
least about 18 nucleotides to about 20 nucleotides, more usually at
least about 25 nucleotides to about 50 nucleotides. Such small DNA
fragments are useful as primers for PCR, hybridization screening,
siRNA, etc. Larger DNA fragments, i.e. greater than 100 nucleotides
are useful for production of the encoded polypeptide. For use in
amplification reactions, such as PCR, a pair of primers will be
used. The exact composition of the primer sequences is not critical
to the invention, but for most applications the primers will
hybridize to the subject sequence under stringent conditions, as
known in the art or as described herein. In some embodiments, a
pair of primers is chosen that will generate an amplification
product of at least about 50 nucleotides or at least about 100
nucleotides. Algorithms for the selection of primer sequences are
generally known, and are available in commercial software packages.
Amplification primers hybridize to complementary strands of DNA,
and will prime towards each other.
[0073] The .beta.TRP-encoding nucleic acids are isolated and
obtained in substantial purity, generally as other than an intact
mammalian chromosome. Usually, the DNA will be obtained
substantially free of other nucleic acid sequences can be typically
"recombinant", i.e. flanked by one or more nucleotides with which
it is not normally associated on a naturally occurring
chromosome.
[0074] The sequence of .beta.TRP polypeptides (or polynucleotide
coding regions or flanking promoter regions) can be mutated in
various ways known in the art to generate targeted changes in
promoter strength, sequence of the encoded protein, etc. The DNA
sequence or product of such a mutation will be substantially
similar to the sequences provided herein, i.e. will differ by at
least one nucleotide or amino acid, respectively, and can differ by
at least two, or by at least about ten or more nucleotides or amino
acids. In general, the sequence changes can be substitutions,
insertions or deletions. Deletions can further include larger
changes, such as deletions of a domain or exon. It should be noted
that TRP channel sequences are conserved mainly within the
transmembrane domain, and regions outside this domain therefore are
more likely targets for mutagenesis without affecting function. For
example, FIG. 7 illustrates the alignment of mouse and human
sequences. In some embodiments, the .beta.TRP nucleic acids of the
invention encode polypeptides, or fragments thereof, comprising the
amino acids conserved between mouse and human sequences. In other
embodiments, the .beta.TRP nucleic acids encode polypeptides, or
fragments thereof, comprising amino acids conserved between human
and rat or between rat, mouse and human sequences.
[0075] Techniques for in vitro mutagenesis of cloned genes are
known. Examples of protocols for scanning mutations may be found in
Gustin et al., Biotechniques 14:22 (1993); Barany, Gene 37:111-23
(1985); Colicelli et al., Mol Gen Genet 199:537-9 (1985); and
Prentki et al., Gene 29:303-13 (1984). Methods for site specific
mutagenesis can be found in Sambrook et al., Molecular Cloning: A
Laboratory Manual (CSH Press, 1989), pp. 15.3-15.108; Weiner et
al., Gene 126:35-41 (1993); Sayers et al., Biotechniques 13:592-6
(1992); Jones and Winistorfer, Biotechniques 12:528-30 (1992);
Barton et al., Nucleic Acids Res 18:7349-55 (1990); Marotti and
Tomich, Gene Anal Tech 6:67-70 (1989); and Zhu, Anal Biochem
177:120-4 (1989).
[0076] In numerous embodiments of the present invention, nucleic
acids encoding a .beta.TRP of interest will be isolated and cloned
using recombinant methods. Such embodiments are used, e.g., to
isolate .beta.TRP polynucleotides (e.g., SEQ ID NO: 1, SEQ ID NO:3,
or SEQ ID NO:5) for protein expression or during the generation of
variants, derivatives, expression cassettes, or other sequences
encoding a .beta.TRP polypeptide (e.g., SEQ ID NO:2, SEQ ID NO:4,
or SEQ ID NO:6), to monitor .beta.TRP gene expression, for the
isolation or detection of .beta.TRP sequences in different species,
for diagnostic purposes in a patient, e.g., to detect mutations in
STRP or to detect expression levels of .beta.TRP nucleic acids or
.beta.TRP polypeptides. In some embodiments, the sequences encoding
the .beta.TRP of the invention are operably linked to a
heterologous promoter. In one embodiment, the nucleic acids of the
invention are from any mammal, including, in particular, e.g., a
human, a mouse, a rat, etc.
[0077] This invention relies on routine techniques in the field of
recombinant genetics. Basic texts disclosing the general methods of
use in this invention include Sambrook et al., Molecular Cloning, A
Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer and
Expression: A Laboratory Manual (1990); and Current Protocols in
Molecular Biology (Ausubel et al., eds., 1994)).
[0078] In general, the nucleic acids encoding the subject proteins
are cloned from DNA sequence libraries that are made to encode cDNA
or genomic DNA. The particular sequences can be located by
hybridizing with an oligonucleotide probe, the sequence of which
can be derived from the sequences (e.g., SEQ ID NO: 1) encoding
.beta.TRP that provide a reference for PCR primers and defines
suitable regions for isolating .beta.TRP-specific probes.
Alternatively, where the sequence is cloned into an expression
library, the expressed recombinant protein can be detected
immunologically with antisera or purified antibodies made against
the .beta.TRP of interest.
[0079] Methods for making and screening genomic and cDNA libraries
are well known to those of skill in the art (see, e.g., Gubler and
Hoffinan Gene 25:263-269 (1983); Benton and Davis Science,
196:180-182 (1977); and Sambrook, supra). Pancreatic cells are an
example of suitable cells to isolate .beta.TRP RNA and cDNA.
[0080] Briefly, to make the cDNA library, one should choose a
source that is rich in mRNA. The mRNA can then be made into cDNA,
ligated into a recombinant vector, and transfected into a
recombinant host for propagation, screening and cloning. For a
genomic library, the DNA is extracted from a suitable tissue and
either mechanically sheared or enzymatically digested to yield
fragments of preferably about 5-100 kb. The fragments are then
separated by gradient centrifugation from undesired sizes and are
constructed in bacteriophage lambda vectors. These vectors and
phage are packaged in vitro, and the recombinant phages are
analyzed by plaque hybridization. Colony hybridization is carried
out as generally described in Grunstein et al., Proc. Natl. Acad.
Sci. USA., 72:3961-3965 (1975).
[0081] An alternative method combines the use of synthetic
oligonucleotide primers with polymerase extension on an mRNA or DNA
template. Suitable primers can be designed from specific .beta.TRP
sequences, e.g., the sequences set forth in SEQ ID NO:I. This
polymerase chain reaction (PCR) method amplifies the nucleic acids
encoding the protein of interest directly from mRNA, cDNA, genomic
libraries or cDNA libraries. Restriction endonuclease sites can be
incorporated into the primers. Polymerase chain reaction or other
in vitro amplification methods may also be useful, for example, to
clone nucleic acids encoding specific proteins and express said
proteins, to synthesize nucleic acids that will be used as probes
for detecting the presence of mRNA encoding a .beta.TRP polypeptide
of the invention in physiological samples, for nucleic acid
sequencing, or for other purposes (see, U.S. Pat. Nos. 4,683,195
and 4,683,202). Genes amplified by a PCR reaction can be purified
from agarose gels and cloned into an appropnate vector.
[0082] Appropriate primers and probes for identifying the genes
encoding a .beta.TRP polypeptide of the invention from mammalian
tissues can be derived from the sequences provided herein, such as
SEQ ID NO:1, or encoding amino acid sequences within .beta.TRP
polypeptides, e.g., SEQ ID NO:2. For a general overview of PCR,
see, Innis et al. PCR Protocols: A Guide to Methods and
Applications, Academic Press, San Diego (1990).
[0083] Synthetic oligonucleotides can be used to construct genes.
This is done using a series of overlapping oligonucleotides,
usually 40-120 bp in length, representing both the sense and
anti-sense strands of the gene. These DNA fragments are then
annealed, ligated and cloned.
[0084] A gene encoding a .beta.TRP polypeptide of the invention can
be cloned using intermediate vectors before transformation into
mammalian cells for expression. These intermediate vectors are
typically prokaryote vectors or shuttle vectors. The proteins can
be expressed in either prokaryotes, using standard methods well
known to those of skill in the art, or eukaryotes as described
infra.
III. Introduction of Polynucleotides Encoding .beta.TRP into
Cells
[0085] Where the .beta.TRP nucleic acid to be delivered into a cell
is DNA, any construct having a promoter (e.g., a promoter that is
functional in a eukaryotic cell) operably linked to a .beta.TRP DNA
of interest, or allowing for linkage to an endogenous promoter upon
introduction into a genome, can be used in the invention. The
constructs containing the .beta.TRP DNA sequence (or the
corresponding RNA sequence) can be any eukaryotic expression
construct containing the .beta.TRP DNA or the RNA sequence of
interest. For example, a plasmid or viral construct (e.g.
adenovirus) can be cleaved to provide linear DNA having ligatable
termini. These termini are bound to exogenous DNA having
complementary-like ligatable termini to provide a biologically
functional recombinant DNA molecule having an intact replicon and a
desired phenotypic property. Preferably the construct is capable of
replication in eukaryotic and/or prokaryotic hosts (viruses in
eukaryotic, plasmids in prokaryotic), which constructs are known in
the art and are commercially available.
[0086] The constructs can be prepared using techniques well known
in the art. Likewise, techniques for obtaining expression of
exogenous DNA or RNA sequences in a genetically altered host cell
are known in the art (see, for example, Kormal et al., Proc. Natl.
Acad. Sci. USA, 84:2150-2154 (1987); Sambrook et al Molecular
Cloning: a Laboratory Manual, 2nd Ed., 1989, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y).
[0087] In some embodiments, the DNA construct contains a promoter
to facilitate expression of the DNA of interest within a pancreatic
(e.g., an islet) cell. The promoter can be a strong, viral promoter
that functions in eukaryotic cells such as a promoter from
cytomegalovirus (CMV), mouse mammary tumor virus (MMTV), Rous
sarcoma virus (RSV), or adenovirus. More specifically, exemplary
promoters include the promoter from the immediate early gene of
human CMV (Boshart et al., Cell 41:521-530 (1985)) and the promoter
from the long terminal repeat (LTR) of RSV (Gorman et al., Proc.
Natl. Acad. Sci. USA 79:6777-6781 (1982)).
[0088] Alternatively, the promoter used can be a strong general
eukaryotic promoter such as the actin gene promoter. In one
embodiment, the promoter used can be a tissue-specific promoter.
For example, the promoter used in the construct can be a
pancreas-specific promoter, a duct cell specific promoter or a stem
cell-specific promoter. Exemplary .beta. cell-specific promoters
include the insulin and amylin promoters. The constructs of the
invention can also include sequences (e.g., enhancers) in addition
to promoters which enhance expression in the target cells
[0089] In another embodiment, the promoter is a regulated promoter,
such as a tetracycline-regulated promoter, expression from which
can be regulated by exposure to an exogenous substance (e.g.,
tetracycline.).
[0090] Other components such as a marker (e.g., an antibiotic
resistance gene (such as an ampicillin resistance gene) or
P-galactosidase) aid in selection or identification of cells
containing and/or expressing the construct, an origin of
replication for stable replication of the construct in a bacterial
cell (preferably, a high copy number origin of replication), or
other elements which facilitate production of the DNA construct,
the protein encoded thereby, or both.
[0091] For eukaryotic expression, the construct should contain at a
minimum a eukaryotic promoter operably linked to a DNA of interest,
which is in turn operably linked to a polyadenylation signal
sequence. The polyadenylation signal sequence may be selected from
any of a variety of polyadenylation signal sequences known in the
art. An exemplary polyadenylation signal sequence is the SV40 early
polyadenylation signal sequence. The construct may also include one
or more introns, where appropriate, which can increase levels of
expression of the DNA of interest, particularly where the DNA of
interest is a cDNA (e.g., contains no introns of the
naturally-occurring sequence). Any of a variety of introns known in
the art may be used.
[0092] In an alternative embodiment, the nucleic acid delivered to
the cell is an RNA encoding .beta.TRP. In this embodiment, the RNA
is adapted for expression (i.e., translation of the RNA) in a
target cell. Methods for production of RNA (e.g., mRNA) encoding a
protein of interest are well known in the art, and can be readily
applied to the product of RNA encoding .beta.TRP useful in the
present invention.
[0093] A. Delivery of .beta.TRP-encoding Nucleic Acid
[0094] Delivery of .beta.TRP-encoding nucleic acids can be
accomplished using any means known in the art. For example,
delivery can be accomplished using a viral or a non-viral vector.
In some embodiments, the nucleic acid is delivered within a viral
particle, such as an adenovirus. In another embodiment, the nucleic
acid is delivered in a formulation comprising naked DNA admixed
with an adjuvant such as viral particles (e.g., adenovirus) or
cationic lipids or liposomes. An "adjuvant" is a substance that
does not by itself produce the desired effect, but acts to enhance
or otherwise improve the action of the active compound. The precise
vector and vector formulation used will depend upon several
factors, such as the size of the DNA to be transferred, the
delivery protocol to be used, and the like. Exemplary non-viral and
viral vectors are described in more detail below.
[0095] 1. Viral Vectors
[0096] In general, viral vectors used in accordance with the
invention are composed of a viral particle derived from a
naturally-occurring virus which has been genetically altered to
render the virus replication-defective and to deliver a recombinant
gene of interest for expression in a target cell in accordance with
the invention. Numerous viral vectors are well known in the art,
including, for example, retrovirus, adenovirus, adeno-associated
virus, herpes simplex virus (HSV), cytomegalovirus (CMV), vaccinia
and poliovirus vectors. The viral vector can be selected according
to its preferential infection of the cells targeted
[0097] Where a replication-deficient virus is used as the viral
vector, the production of infectious virus particles containing
either DNA or RNA corresponding to the DNA of interest can be
achieved by introducing the viral construct into a recombinant cell
line which provides the missing components essential for viral
replication. In one embodiment, transformation of the recombinant
cell line with the recombinant viral vector will not result in
production or substantial production of replication-competent
viruses, e.g., by homologous recombination of the viral sequences
of the recombinant cell line into the introduced viral vector.
Methods for production of replication-deficient viral particles
containing a nucleic acid of interest are well known in the art and
are described in, for example, Rosenfeld et al., Science
252:431-434 (1991) and Rosenfeld et al., Cell 68:143-155 (1992)
(adenovirus); U.S. Pat. No. 5,139,941 (adeno-associated virus);
U.S. Pat. No. 4,861,719 (retrovirus); and U.S. Pat. No. 5,356,806
(vaccinia virus). Methods and materials for manipulation of the
mumps virus genome, characterization of mumps virus genes
responsible for viral fusion and viral replication, and the
structure and sequence of the mumps viral genome are described in
Tanabayashi et al., J. Virol. 67:2928-2931 (1993); Takeuchi et al.,
Archiv. Virol., 128:177-183 (1993); Tanabayashi et al., Virol.
187:801-804 (1992); Kawano et al., Virol., 179:857-861 (1990);
Elango et al., J Gen. Virol. 69:2893-28900 (1988).
[0098] 2. Non-Viral Vectors
[0099] The nucleic acids of interest can be introduced into a cell
using a non-viral vector. "Non-viral vector" as used herein is
meant to include naked DNA (e.g., DNA not contained within a viral
particle, and free of a carrier molecules such as lipids), chemical
formulations comprising naked nucleic acid (e.g., a formulation of
DNA (and/or RNA) and cationic compounds (e.g., dextran sulfate,
cationic lipids)), and naked nucleic acid mixed with an adjuvant
such as a viral particle (e.g., the DNA of interest is not
contained within the viral particle, but the formulation is
composed of both naked DNA and viral particles (e.g., adenovirus
particles) (see, e.g., Curiel et al., Am. J Respir. Cell Mol. Biol.
6:247-52 (1992)).
[0100] In some embodiments, the formulation comprises viral
particles which are mixed with the naked DNA construct prior to
administration. In some embodiments, the viral particles are
adenovirus particles. See, e.g., Curiel et al., Am. J. Respir. Cell
Mol. Biol. 6:247-52 (1992)).
[0101] Alternatively or in addition, the nucleic acid can be
complexed with polycationic substances such as poly-L-lysine or
DEAC-dextran, targeting ligands, and/or DNA binding proteins (e.g.,
histones). DNA- or RNA-liposome complex formulations comprise a
mixture of lipids which bind to genetic material (DNA or RNA) and
facilitate delivery of the nucleic acid into the cell. Liposomes
which can be used in accordance with the invention include DOPE
(dioleyl phosphatidyl ethanol amine), CUDMEDA
(N-(5-cholestrum-3-.beta.-ol 3-urethanyl)-N',N'-dimethylethylene
diamine).
[0102] For example, the naked DNA can be administered in a solution
containing Lipofectin.TM. (LTI/BRL) at concentrations ranging from
about 2.5% to 15% volume:volume, e.g., about 6% to 12%
volume:volume. Exemplary methods and compositions for formulation
of DNA for delivery according to the method of the invention are
described in U.S. Pat. No. 5,527,928.
[0103] The nucleic acid of interest can also be administered as a
chemical formulation of DNA or RNA coupled to a carrier molecule
(e.g., an antibody or a receptor ligand) which facilitates delivery
to host cells. By the term "chemical formulations" is meant
modifications of nucleic acids which allow coupling of the nucleic
acid compounds to a carrier molecule such as a protein or lipid, or
derivative thereof. Exemplary protein carrier molecules include
antibodies specific to the cells of a targeted pancreatic cell or
receptor ligands, e.g., molecules capable of interacting with
receptors associated with a cell of a targeted pancreatic cell.
[0104] B. Introduction of .beta.TRP Nucleic Acids Into Pancreatic
Cells In Vitro
[0105] Nucleic acids encoding .beta.TRP can be introduced into a
cell in vitro to accomplish expression in the cell to provide for
at least transient expression. The cells into which the nucleic
acid is introduced can be differentiated epithelial cells (e.g.,
pancreatic cells (including, e.g., islet cells such as
.beta.-cells) , gut cells, hepatic cells or duct cells),
pluripotent adult or embryonic stem cells, or any mammalian cell
capable of developing into .beta. cells or cells capable of
expression of insulin in vitro. The cell can be subsequently
implanted into a subject having a disorder characterized by a
deficiency in insulin (e.g., type 1 or 2 diabetes), which disorder
is amenable to treatment by islet cell replacement therapy. In some
embodiments, the host cell in which .beta.TRP expression is
provided and which is implanted in the subject is derived from the
individual who will receive the transplant (e.g., to provide an
autologous transplant). Alternatively, cells from another subject
(the "donor") could be modified to express .beta.TRP, and the cells
subsequently implanted in the affected subject to provide for
insulin production.
[0106] Introduction of nucleic acid into the cell in vitro can be
accomplished according to methods well known in the art (e.g.,
through use of electroporation, microinjection, lipofection
infection with a recombinant (preferably replication-deficient)
virus, and other means well known in the art). The nucleic acid is
generally operably linked to a promoter that facilitates a desired
level of polypeptide expression (e.g., a promoter derived from CMV,
SV40, adenovirus, or a tissue-specific or cell type-specific
promoter). Transformed cells containing the recombinant nucleic
acid can be selected and/or enriched via, for example, expression
of a selectable marker gene present in the introduced construct or
that is present on a nucleic acid that is co-transfected with the
construct. Typically selectable markers provide for resistance to
antibiotics such as tetracycline, hygromycin, neomycin, and the
like. Other markers can include thymidine kinase and the like.
Other markers can include markers that can be used to identify
.beta.TRP-expressing cells, such as .beta.-galactosidase or green
florescent protein.
[0107] Expression of the introduced nucleic acid in the transformed
cell can be assessed by various methods known in the art. For
example, expression of the introduced gene can be examined by
northern blot to detect mRNA which hybridizes with a DNA probe
derived from the relevant gene. Those cells that express the
desired gene can be further isolated and expanded in-in vitro
culture using methods well known in the art. The host cells
selected for transformation will vary with the purpose of ex vivo
therapy (e.g., insulin production), the site of implantation of the
cells, and other factors that will vary with a variety of factors
that will be appreciated by the ordinarily skilled artisan.
[0108] The transformed cell can also be examined for insulin
production. For example, expression of insulin could be detected by
PCR, northern blot, immunocytochemistry, western blot or ELISA.
Alternatively a marker gene such as green florescent protein or an
antibiotic resistance gene operatively linked to an islet specific
promoter such as the insulin gene promoter could be used for
identification or selection of transformed islet cells.
[0109] Methods for engineering a host cell for expression of a
desired gene product(s) and implantation or transplantation of the
engineered cells (e.g., ex vivo therapy) are known in the art (see,
e.g., Gilbert et al., Transplantation 56:423-427 (1993)). For
expression of a desired gene in exogenous or autologous cells and
implantation of the cells (e.g., islet cells) into pancreas, see,
e.g., Docherty, Clin Sci (Colch) 92:321-330 (1997); Hegre et al.
Acta Endocrinol Suppl (Copenh) 205:257-281 (1976); Sandler et al.,
Transplantation 63:1712-1718 (1997); Calafiore, Diabetes Care
20:889-896 (1997); Kenyon et al., Diabetes Metab Rev 12:361-372
(1996); Chick et al., Science 197:780-782 (1977). In general, the
cells can be implanted into the pancreas, or to any practical or
convenient site, e.g., subcutaneous site, liver, peritoneum.
[0110] Methods for transplanting islets cells are well known in the
art, see, e.g., Hegre et al. Acta Endocrinol Suppl (Copenh)
205:257-281 (1976); Sandler et al. Transplantation 63:1712-1718
(1997); Calafiore, Diabetes Care 20:889-896 (1997); Kenyon et al.,
Diabetes Metab Rev 12:361-372 (1996); Chick et al., Science
197:780-782 (1977).
[0111] In general, after expansion of the transformed cells in
vitro, the cells can be implanted into the mammalian subject by
methods well known in the art. The number of cells implanted is a
number of cells sufficient to provide for expression of levels of
insulin sufficient to lower blood glucose levels. The number of
cells to be transplanted can be determined based upon such factors
as the levels of polypeptide expression achieved in vitro, and/or
the number of cells that survive implantation. The transformed
cells are implanted in an area of dense vascularization such as the
liver, and in a manner that minimizes surgical intervention in the
subject. The engraftment of the implant of transformed cells is
monitored by examining the mammalian subject for classic signs of
graft rejection, i.e., inflammation and/or exfoliation at the site
of implantation, and fever, and by monitoring blood glucose
levels.
[0112] C. Introduction of 6TRP Nucleic Acids Into Pancreatic Cells
In Vivo
[0113] .beta.TRP nucleic acids can be delivered directly to a
subject to provide for .beta.TRP expression in a target cell (e.g.,
a pancreatic islet cell), thereby promoting glucose-stimulated
insulin production. Methods for in vivo delivery of a nucleic acid
of interest for expression in a target cell are known in the art.
For example, in vivo methods of gene delivery normally employ
either a biological means of introducing the DNA into the target
cells (e.g., a virus containing the DNA of interest) or a
mechanical means to introduce the DNA into the target cells (e.g.,
direct injection of DNA into the cells, liposome fusion, or
pneumatic injection using a gene gun).
[0114] In general, the transformed cells expressing the protein
encoded by the DNA of interest produce a therapeutically effective
amount of .beta.TRP to produce islet cells, in particular
.beta.-cells in the mammalian patient capable of glucose-stimulated
insulin production. In some embodiments, the introduced DNA also
encodes an islet-specific transcription factor or other polypeptide
that controls or stimulates insulin production in islet cells.
[0115] In general terms, the delivery method comprises introducing
the nucleic of interest-containing vector into a pancreatic cell.
By way of example, a .beta.TRP DNA-containing vector may comprise
either a viral or non-viral vector (including naked DNA), which is
introduced into the pancreas in vivo via the duct system.
Intraductal administration can be accomplished by cannulation by,
for example, insertion of the cannula through a lumen of the
gastrointestinal tract, by insertion of the cannula through an
external orifice, or insertion of the cannula through the common
bile duct. Retrograde ductal administration may be accomplished in
the pancreas by endoscopic retrograde chalangio-pancreatography
(ECRP). Exemplary methods for accomplishing intraductal delivery to
the pancreas are described in U.S. Pat. No. 6,004,944.
[0116] The precise amount of .beta.TRP-encoding nucleic acid
administered will vary greatly according to a number of factors
including the susceptibility of the target cells to transformation,
the size and weight of the subject, the levels of protein
expression desired, and the condition to be treated. The amount of
nucleic acid and/or the number of infectious viral particles
effective to infect the targeted tissue, transform a sufficient
number of cells, and provide for production of a desired level of
insulin can be readily determined based upon such factors as the
efficiency of the transformation in vitro and the susceptibility of
the targeted cells to transformation. For example, the amount of
DNA introduced into the pancreatic duct of a human is, for example,
generally from about 1 .mu.g to about 750 mg, e.g., from about 500
pg to about 500 mg, e.g., from about 10 mg to about 200 mg, e.g.,
about 100 mg. Generally, the amounts of DNA can be extrapolated
from the amounts of DNA effective for delivery and expression of
the desired gene in an animal model. For example, the amount of DNA
for delivery in a human is roughly 100 times the amount of DNA
effective in a rat.
[0117] Pancreatic cells modified according to the invention can
facilitate sufficiently high levels of expression of a nucleic acid
of interest, e.g., where the nucleic acid delivered is DNA and the
DNA of interest is operably linked to a strong eukaryotic promoter
(e.g., CMV, MMTV). The expressed protein can induce
glucose-stimulated insulin production in islet cells. Thus the
methods of the invention are useful in treating a mammalian subject
having a variety of insulin related conditions.
[0118] The actual number of transformed pancreatic cells required
to achieve therapeutic levels of the protein of interest will vary
according to several factors including the protein to be expressed,
the level of expression of the protein by the transformed cells,
the rate in which the protein induces insulin production, and the
condition to be treated.
[0119] Regardless of whether the islet transcription
factor-encoding nucleic acid is introduced in vivo or ex vivo, the
nucleic acid (or islet cells produced in vitro or recombinant cells
expressing the .beta.TRP nucleic acid that are to be transplanted
for development into islet cells in vivo post-transplantation) can
be administered in combination with other genes and other
agents.
[0120] D. Assessnient of Therapy
[0121] The effects of ex vivo or in vivo therapy according to the
methods of the invention can be monitored in a variety of ways.
Generally, a sample of blood from the subject can be assayed for,
for example, levels of glucose, proinsulin, c-peptide, and insulin.
Appropriate assays for detecting proinsulin, c-peptide, insulin and
glucose in blood samples are well known in the art.
IV. Identification of Modulators of .beta.TRP
[0122] Modulators of .beta.TRP, i.e. agonists or antagonists of
.beta.TRP activity or .beta.TRP polypeptide or polynucleotide
expression, are useful for treating a number of human diseases,
including diabetes. Administration of .beta.TRP activators can be
used to treat diabetic (e.g., Type 2) individuals.
[0123] A. Agents that Modulate .beta.TRP
[0124] The agents tested as modulators of .beta.TRP can be any
small chemical compound, or a biological entity, such as a protein,
sugar, nucleic acid or lipid. Typically, test compounds will be
small chemical molecules and peptides. Essentially any chemical
compound can be used as a potential modulator or ligand in the
assays of the invention, although most often compounds that can be
dissolved in aqueous or organic (especially DMSO-based) solutions
are used. The assays are designed to screen large chemical
libraries by automating the assay steps and providing compounds
from any convenient source to assays, which are typically run in
parallel (e.g., in microtiter formats on microtiter plates in
robotic assays). Activators will include molecules that directly
activate (open) .beta.TRP as well as molecules that activate
regulators (GPCRs, G-proteins, etc.) that subsequently activate
.beta.TRP. Modulators also include agents designed to reduce the
level of STRP mRNA (e.g. antisense molecules, ribozymes, DNAzymes,
small inhibitory RNAs (siRNAs) and the like) or the level of
translation from an mRNA (e.g., translation blockers such as an
antisense molecules that are complementary to translation start or
other sequences on an mRNA molecule). It will be appreciated that
there are many suppliers of chemical compounds, including Sigma
(St. Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St.
Louis, Mo.), Fluka Chemika-Biochemica Analytika (Buchs,
Switzerland) and the like. Generally, the compounds to be tested
are present in the range from 1 .mu.M to 100 mM.
[0125] In some embodiments, high throughput screening methods
involve providing a combinatorial chemical or peptide library
containing a large number of potential therapeutic compounds
(potential modulator compounds). Such "combinatorial chemical
libraries" or "ligand libraries" are then screened in one or more
assays, as described herein, to identify those library members
(particular chemical species or subclasses) that display a desired
characteristic activity. The compounds thus identified can serve as
conventional "lead compounds" or can themselves be used as
potential or actual therapeutics.
[0126] A combinatorial chemical library is a collection of diverse
chemical compounds generated by either chemical synthesis or
biological synthesis, by combining a number of chemical "building
blocks" such as reagents. For example, a linear combinatorial
chemical library such as a polypeptide library is formed by
combining a set of chemical building blocks (amino acids) in every
possible way for a given compound length (i.e., the number of amino
acids in a polypeptide compound). Millions of chemical compounds
can be synthesized through such combinatorial mixing of chemical
building blocks.
[0127] Preparation and screening of combinatorial chemical
libraries is well known to those of skill in the art. Such
combinatorial chemical libraries include, but are not limited to,
peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, Int.
J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al., Nature
354:84-88 (1991)). Other chemistries for generating chemical
diversity libraries can also be used. Such chemistries include, but
are not limited to: peptoids (e.g., PCT Publication No. WO
91/19735), encoded peptides (e.g., PCT Publication WO 93/20242),
random bio-oligomers (e.g., PCT Publication No. WO 92/00091),
benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such
as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc.
Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides
(Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal
peptidomimetics with glucose scaffolding (Hirschmann et al., J.
Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses
of small compound libraries (Chen et al., J Amer. Chem. Soc.
116:2661 (1994)), oligocarbamates (Cho et al., Science 261:1303
(1993)), and/or peptidyl phosphonates (Campbell et al., J. Org.
Chem. 59:658 (1994)), nucleic acid libraries (see Ausubel, Berger
and Sambrook, all supra), peptide nucleic acid libraries (see,
e.g., U.S. Pat. No. 5,539,083), antibody libraries (see, e.g.,
Vaughn et al., Nature Biotechnology, 14(3):309-314 (1996) and
PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al.,
Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), small
organic molecule libraries (see, e.g., benzodiazepines, Baum
C&EN, January 18, page 33 (1993); isoprenoids, U.S. Pat. No.
5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No.
5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134;
morpholino compounds, U.S. Pat. No. 5,506,337; benzodiazepines,
U.S. Pat. No. 5,288,514, and the like).
[0128] Devices for the preparation of combinatorial libraries are
commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem
Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied
Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford,
Mass.). In addition, numerous combinatorial libraries are
themselves commercially available (see, e.g., ComGenex, Princeton,
N.J., Tripos, Inc., St. Louis, Mo., 3D Pharmaceuticals, Exton, Pa.,
Martek Biosciences, Columbia, Md., etc.).
[0129] B. Methods of Screening for Modulators of .beta.TRP
[0130] A number of different screening protocols can be utilized to
identify agents that modulate the level of expression or activity
of .beta.TRP in cells, particularly mammalian cells, and especially
human cells. In general terms, the screening methods involve
screening a plurality of agents to identify an agent that modulates
the activity of .beta.TRP by, e.g., binding to a .beta.TRP
polypeptide, preventing an inhibitor or activator from binding to
.beta.TRP, increasing association of an inhibitor or activator with
.beta.TRP, or activating or inhibiting expression or activity of
.beta.TRP.
[0131] In some embodiments, different TRP polypeptides (e.g.,
TRPC1, TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6, TRPC7, TRPV1,
TRPV2, TRPV3, TRPV4, TRPV5, TRPV6, TRPM1, TRPM2, TRPM3, TRPM4,
TRPM5, TRPM6, TRPM7, and TRPM8) are screened in parallel to
identify an agent that modulates .beta.TRP but not at least one
other TRP channel.
[0132] 1. .beta.TRP Binding Assays
[0133] Preliminary screens can be conducted by screening for agents
capable of binding to .beta.TRP, as at least some of the agents so
identified are likely .beta.TRP modulators. Binding assays are also
useful, e.g., for identifying endogenous proteins that interact
with .beta.TRP. For example, antibodies, receptors or other
molecules that bind .beta.TRP can be identified in binding
assays.
[0134] Binding assays usually involve contacting a pTRP protein
with one or more test agents and allowing sufficient time for the
protein and test agents to form a binding complex. Any binding
complexes formed can be detected using any of a number of
established analytical techniques. Protein binding assays include,
but are not limited to, methods that measure co-precipitation or
co-migration on non-denaturing SDS-polyacrylamide gels, and
co-migration on western blots (see, e.g., Bennet, J. P. and
Yamamura, H. I. (1985) "Neurotransmitter, Hormone or Drug Receptor
Binding Methods," in Neurotransmitter Receptor Binding (Yamamura,
H. I., et al., eds.), pp. 61-89. Other binding assays involve the
use of mass spectrometry or NMR techniques to identify molecules
bound to .beta.TRP or displacement of labeled substrates. The
.beta.TRP protein utilized in such assays can be naturally
expressed, cloned or synthesized.
[0135] In addition, mammalian or yeast two-hybrid approaches (see,
e.g., Bartel, P.l. et. al. Methods Enzymol, 254:241 (1995)) can be
used to identify polypeptides or other molecules that interact or
bind when expressed together in a cell.
[0136] 2. Expression Assays
[0137] Screening for a compound that modulates the expression of
TRP are also provided. Screening methods generally involve
conducting cell-based assays in which test compounds are contacted
with one or more cells expressing .beta.TRP, and then detecting an
increase or decrease in .beta.TRP expression (either transcript,
translation product). Assays can be performed with cells that
naturally express .beta.TRP or in cells recombinantly altered to
express .beta.TRP.
[0138] .beta.TRP expression can be detected in a number of
different ways. As described infra, the expression level of
.beta.TRP in a cell can be determined by probing the mRNA expressed
in a cell with a probe that specifically hybridizes with a
transcript (or complementary nucleic acid derived therefrom) of
.beta.TRP. Probing can be conducted by lysing the cells and
conducting northern blots or without lysing the cells using in
situ-hybridization techniques. Alternatively, .beta.TRP protein can
be detected using immunological methods in which a cell lysate is
probed with antibodies that specifically bind to .beta.TRP.
[0139] Other cell-based assays involve reporter assays conducted
with cells using standard reporter gene assays. These assays can be
performed in either cells that do, or do not, express .beta.TRP.
Some of these assays are conducted with a heterologous nucleic acid
construct that includes a .beta.TRP promoter that is operably
linked to a reporter gene that encodes a detectable product. A
number of different reporter genes can be utilized. Some reporters
are inherently detectable. An example of such a reporter is green
fluorescent protein that emits fluorescence that can be detected
with a fluorescence detector. Other reporters generate a detectable
product. Often such reporters are enzymes. Exemplary enzyme
reporters include, but are not limited to, .beta.-glucuronidase,
CAT (chloramphenicol acetyl transferase; Alton and Vapnek (1979)
Nature 282:864-869), luciferase, .beta.-galactosidase and alkaline
phosphatase (Toh, et al. (1980) Eur. J. Biochem. 182:231-238; and
Hall et al. (1983) J. Mol. Appl. Gen. 2:101).
[0140] In these assays, cells harboring the reporter construct are
contacted with a test compound. Modulated promoter expression is
monitored by detecting the level of a detectable reporter. A number
of different kinds of .beta.TRP modulators can be identified in
this assay. For example, a test compound that inhibits the promoter
by binding to it, inhibits the promoter by binding to transcription
factors or other regulatory factors, binds to their promoter or
triggers a cascade that produces a molecule that inhibits the
promoter can be identified. Similarly a test compound that, e.g.,
activates the promoter by binding to it, activates the promoter by
binding to transcription factors or other regulatory factors, binds
to their promoter or triggers a cascade that produces a molecule
that activates the promoter can also be identified.
[0141] The level of expression or activity can be compared to a
baseline value. The baseline value can be a value for a control
sample or a statistical value that is representative of .beta.TRP
expression levels for a control population (e.g., lean individuals
not having or at risk for Type 2 diabetes) or cells (e.g., tissue
culture cells not exposed to a .beta.TRP modulator). Expression
levels can also be determined for cells that do not express
.beta.TRP as a negative control. Such cells generally are otherwise
substantially genetically the same as the test cells.
[0142] A variety of different types of cells can be utilized in the
reporter assays. Cells that express an endogenous .beta.TRP
include, e.g., pancreatic cells such as islet cells, e.g., .beta.
cells. Cells that do not endogenously express .beta.TRP can be
prokaryotic or eukaryotic. The eukaryotic cells can be any of the
cells typically utilized in generating cells that harbor
recombinant nucleic acid constructs. Exemplary eukaryotic cells
include, but are not limited to, yeast, and various higher
eukaryotic cells such as the HepG2, COS, CHO and HeLa cell lines.
Xenopus oocytes can also be used.
[0143] Various controls can be conducted to ensure that an observed
activity is authentic including running parallel reactions with
cells that lack the reporter construct or by not contacting a cell
harboring the reporter construct with test compound. Compounds can
also be further validated as described below.
[0144] 3. Activity
[0145] Analysis of .beta.TRP polypeptide activity is performed
according to general biochemical procedures. Such assays include
cell-based assays as well as in vitro assays involving purified or
partially purified .beta.TRP polypeptides or crude cell lysates. In
some embodiments, the .beta.TRP polypeptide is expressed on a cell
and the cell is contacted with a test agent.
[0146] The level of .beta.TRP activity in a cell or other sample is
determined and compared to a baseline value (e.g., a control
value). Activity can be measured based on a crude extract or
partially or essentially purified .beta.TRP from a sample.
Measurement of .beta.TRP activity involves measuring cation (e.g.,
Ca.sup.2+) channel activity, for example, as described in Lesage et
al. Am. J. Physiol. Renal. Physiol. 279:F793-F801 (2000) and Girad
et al., Biochem. Biophys. Res. Commun. 282:249-256 (2001). For
example, changes in ion flux may be assessed by determining changes
in polarization (i.e., electrical potential) of the cell or
membrane expressing a .beta.TRP polypeptide. In some embodiments,
changes in cellular polarization is monitored by measuring changes
in current (thereby measuring changes in polarization) with
voltage-clamp and patch-clamp techniques, e.g., the "cell-attached"
mode, the "inside-out" mode, and the "whole cell" mode (see, e.g.,
Ackerman et al., New Engl. J. Med. 336:1575-1595 (1997)). Whole
cell currents are conveniently determined using the standard
methodology (see, e.g., Hamil et al., PFlugers. Archiv. 391:85
(1981). Other known assays include: .sup.45Ca.sup.2+ flux assays
and fluorescence assays using voltage-sensitive dyes or ion
sensitive dyes (see, e.g., Vestergarrd-Bogind et al., J. Membrane
Biol. 88:67-75 (1988); Daniel et al., J. Pharmacol. Meth.
25:185-193 (1991); Holevinsky et al., J Membrane Biology 137:59-70
(1994)). Assays for compounds capable of inhibiting or increasing
cation flux through the channel proteins comprising a .beta.TRP
polypeptide can be performed by application of the compounds to a
bath solution in contact with and comprising cells having a channel
of the present invention (see, e.g., Blatz et al., Nature
323:718-720 (1986); Park, J. Physiol. 481:555-570 (1994)).
[0147] The effects of the test compounds upon the function of the
channels can be measured by changes in the electrical currents or
ionic flux or by the consequences of changes in currents and flux.
Changes in electrical current or ionic flux are measured by either
increases or decreases in flux of ions such as calcium ions. The
ions can be measured in a variety of standard-ways. They can be
measured directly by concentration changes of the ions, e.g.,
changes in intracellular concentrations, or indirectly by membrane
potential or by radio-labeling of the ions or by using
calcium-dependent fluorescent dyes.
[0148] As illustrated in FIG. 3, membrane potential provides a
particularly clean signal:noise ratio compared to calcium flux
measurements for measuring .beta.TRP activity. Cell membrane
depolarization upon activation of .beta.TRP can be measured with
membrane potential dependent fluorescent dyes such as cationic
carbocyanines and rhodamines and anionic oxonols, as well as
proprietary dyes available for the Molecular Devices (Sunnyvale,
Calif.) FLEXstation.RTM. and FLIPR.RTM. systems. Fluorescence
triggered by cell depolarization can be detected with devices known
in the art, e.g., FLEXstation.RTM..
[0149] Consequences of the test compound on ion flux can be quite
varied. Accordingly, any suitable physiological change can be used
to assess the influence of a test compound on the channels of this
invention. The effects of a test compound can be measured by a
toxin binding assay. When the functional consequences are
determined using intact cells or animals, one can also measure a
variety of effects such as transmitter release, intracellular
calcium changes, hormone release (e.g., insulin), transcriptional
changes to both known and uncharacterized genetic markers (e.g.,
northern blots), cell volume changes (e.g., in red blood cells),
immunoresponses (e.g., T cell activation), changes in cell
metabolism such as cell growth or pH changes, changes in
intracellular second messengers such as cyclic nucleotides and
modulation (e.g., decrease) of apoptosis.
[0150] 4. Validation
[0151] Agents that are initially identified by any of the foregoing
screening methods can be further tested to validate the apparent
activity. In some embodiments, a .beta.TRP activator is selected by
any or all of the following criteria: (i) the activator induces a
depolarization response specifically in a cell expressing a
heterologous .beta.TRP polypeptide (but not in cells not expressing
.beta.TRP); (ii) the activator is not suppressible by PLC
inhibitors (i.e., is not activated by an upstream regulator of
.beta.TRP); and (iii) the activating effect of the activator is
suppressible by TRP blockers such as 2-APB.
[0152] Validation assays can include, e.g., in vitro single cell
imaging or patch clamping to confirm effects on ion flux. In vitro
insulin secretion assays using isolated islet cells (normal or
diabetic) can be performed in the presence or absence of the
candidate activator.
[0153] In some embodiments, validation studies are conducted with
suitable animal models. The basic format of such methods involves
administering a lead compound identified during an initial screen
to an animal that serves as a model for humans and then determining
if .beta.TRP is in fact modulated. The animal models utilized in
validation studies generally are mammals of any kind. Specific
examples of suitable animals include, but are not limited to,
primates, mice and rats. For example, monogenic models of diabetes
(e.g., ob/ob and db/db mice, Zucker rats and Zucker Diabetic Fatty
(ZDF) rats etc.) or polygenic models of diabetes (e.g., a high fat
fed mouse model) can be useful for validating .beta.TRP modulation
and its effect in a diabetic animal.
[0154] Ideally, .beta.TRP activation should enhance insulin
secretion only in high glucose. Therefore, in some embodiments, a
selected activator compound reduces hyperglycemia in ZDF rats and
db/db mice and does not induce hypoglycemia in either diabetic or
control animals.
[0155] C. Solid Phase and Soluble High Throughput Assays
[0156] In the high throughput assays of the invention, it is
possible to screen up to several thousand different modulators or
ligands in a single day. In particular, each well of a microtiter
plate can be used to run a separate assay against a selected
potential modulator, or, if concentration or incubation time
effects are to be observed, every 5-10 wells can test a single
modulator. Thus, a single standard microtiter plate can assay about
100 (e.g., 96) modulators. If 1536 well plates are used, then a
single plate can easily assay from about 100 to about 1500
different compounds. It is possible to assay several different
plates per day; assay screens for up to about 6,000-20,000 or more
different compounds are possible using the integrated systems of
the invention. In addition, microfluidic approaches to reagent
manipulation can be used.
[0157] The molecule of interest (e.g., .beta.TRP or fragments
thereof) can be bound to the solid state component, directly or
indirectly, via covalent or non covalent linkage, e.g., via a tag.
The tag can be any of a variety of components. In general, a
molecule that binds the tag (a tag binder) is fixed to a solid
support, and the tagged molecule of interest (e.g., .beta.TRP or
fragments thereof) is attached to the solid support by interaction
of the tag and the tag binder.
[0158] A number of tags and tag binders can be used, based upon
known molecular interactions well described in the literature. For
example, where a tag has a natural binder, for example, biotin,
protein A, or protein G, it can be used in conjunction with
appropriate tag binders (avidin, streptavidin, neutravidin, the Fc
region of an immunoglobulin, poly-His, etc.) Antibodies to
molecules with natural binders such as biotin are also widely
available and appropriate tag binders (see, SIGMA Immunochemicals
1998 catalogue SIGMA, St. Louis Mo.).
[0159] Similarly, any haptenic or antigenic compound can be used in
combination with an appropriate antibody to form a tag/tag binder
pair. Thousands of specific antibodies are commercially available
and many additional antibodies are described in the literature. For
example, in one common configuration, the tag is a first antibody
and the tag binder is a second antibody that recognizes the first
antibody. In addition to antibody-antigen interactions,
receptor-ligand interactions are also appropriate as tag and
tag-binder pairs, such as agonists and antagonists of cell membrane
receptors (e.g., cell receptor-ligand interactions such as
transferrin, c-kit, viral receptor ligands, cytokine receptors,
chemokine receptors, interleukin receptors, immunoglobulin
receptors and antibodies, the cadherin family, the integrin family,
the selectin family, and the like; see, e.g., Pigott & Power,
The Adhesion Molecule Facts Book I (1993)). Similarly, toxins and
venoms, viral epitopes, hormones (e.g., opiates, steroids, etc.),
intracellular receptors (e.g., which mediate the effects of various
small ligands, including steroids, thyroid hormone, retinoids and
vitamin D; peptides), drugs, lectins, sugars, nucleic acids (both
linear and cyclic polymer configurations), oligosaccharides,
proteins, phospholipids and antibodies can all interact with
various cell receptors.
[0160] Synthetic polymers, such as polyurethanes, polyesters,
polycarbonates, polyureas, polyamides, polyethyleneimines,
polyarylene sulfides, polysiloxanes, polyimides, and polyacetates
can also form an appropriate tag or tag binder. Many other tag/tag
binder pairs are also useful in assay systems described herein, as
would be apparent to one of skill upon review of this
disclosure.
[0161] Common linkers such as peptides, polyethers, and the like
can also serve as tags, and include polypeptide sequences, such as
poly-gly sequences of between about 5 and 200 amino acids. Such
flexible linkers are known to those of skill in the art. For
example, poly(ethelyne glycol) linkers are available from
Shearwater Polymers, Inc., Huntsville, Ala. These linkers
optionally have amide linkages, sulfhydryl linkages, or
heterofunctional linkages.
[0162] Tag binders are fixed to solid substrates using any of a
variety of methods currently available. Solid substrates are
commonly derivatized or functionalized by exposing all or a portion
of the substrate to a chemical reagent that fixes a chemical group
to the surface that is reactive with a portion of the tag binder.
For example, groups that are suitable for attachment to a longer
chain portion include amines, hydroxyl, thiol, and carboxyl groups.
Aminoalkylsilanes and hydroxyalkylsilanes can be used to
functionalize a variety of surfaces, such as glass surfaces. The
construction of such solid phase biopolymer arrays is well
described in the literature (see, e.g., Merrifield, J. Am. Chem.
Soc. 85:2149-2154 (1963) (describing solid phase synthesis of,
e.g., peptides); Geysen et al., J. Immun. Meth. 102:259-274 (1987)
(describing synthesis of solid phase components on pins); Frank and
Doring, Tetrahedron 44:60316040 (1988) (describing synthesis of
various peptide sequences on cellulose disks); Fodor et al.,
Science, 251:767-777 (1991); Sheldon et al., Clinical Chemistry
39(4):718-719 (1993); and Kozal et al., Nature Medicine 2(7):753759
(1996) (all describing arrays of biopolymers fixed to solid
substrates). Non-chemical approaches for fixing tag binders to
substrates include other common methods, such as heat,
cross-linking by UV radiation, and the like.
[0163] The invention provides in vitro assays for identifying, in a
high throughput format, compounds that can modulate the expression
or activity of .beta.TRP. Control reactions that measure .beta.TRP
activity of the cell in a reaction that does not include a
potential modulator are optional, as the assays are highly uniform.
Such optional control reactions are appropriate and increase the
reliability of the assay. Accordingly, in one embodiment, the
methods of the invention include such a control reaction. For each
of the assay formats described, "no modulator" control reactions
that do not include a modulator provide a background level-of
binding activity.
[0164] In some assays it will be desirable to have positive
controls. At least two types of positive controls are appropriate.
First, a known activator of .beta.TRP of the invention can be
incubated with one sample of the assay, and the resulting increase
in signal resulting from an increased expression level or activity
of .beta.TRP are determined according to the methods herein.
Exemplary activators include, e.g., calcimycin. Second, a known
inhibitor of .beta.TRP can be added, and the resulting decrease in
signal for the expression or activity of .beta.TRP can be similarly
detected. Exemplary inhibitors include, e.g., 2-APB or U73122, a
PLC inhibitor from Sigma Chemicals. It will be appreciated that
modulators can also be combined with activators or inhibitors to
find modulators that inhibit the increase or decrease that is
otherwise caused by the presence of the known modulator of
.beta.TRP.
[0165] D. Computer-Based Assays
[0166] Yet another assay for compounds that modulate the activity
of .beta.TRP involves computer-assisted drug design, in which a
computer system is used to generate a three-dimensional structure
of .beta.TRP based on the structural information encoded by its
amino acid sequence. The input amino acid sequence interacts
directly and actively with a pre-established algorithm in a
computer program to yield secondary, tertiary, and quaternary
structural models of the protein. The models of the protein
structure are then examined to identify regions (e.g., the active
site) of the structure that have the ability to bind ligands or
otherwise be modulated. Similar analyses can be performed on
potential receptors or binding partners of .beta.TRP and can be
used to identify regions of interaction with .beta.TRP. These
regions are then used to identify polypeptides that bind to
.beta.TRP.
[0167] Once the tertiary structure of a protein of interest has
been generated, potential modulators can be identified by the
computer system. Three-dimensional structures for potential
modulators are generated by entering chemical formulas of
compounds. The three-dimensional structure of the potential
modulator is then compared to that of .beta.TRP to identify binding
sites of .beta.TRP. Binding affinity between the protein and
modulators is determined using energy terms to determine which
ligands have an enhanced probability of binding to the protein.
V. Administration and Pharmaceutical Compositions
[0168] Modulators of .beta.TRP (e.g., antagonists or agonists) can
be administered directly to the mammalian subject for modulation of
.beta.TRP activity in vivo. Administration is by any of the routes
normally used for introducing a modulator compound into ultimate
contact with the tissue to be treated and is well known to those of
skill in the art. Although more than one route can be used to
administer a particular composition, a particular route can often
provide a more immediate and more effective reaction than another
route.
[0169] The pharmaceutical compositions of the invention may
comprise a pharmaceutically acceptable carrier. Pharmaceutically
acceptable carriers are determined in part by the particular
composition being administered, as well as by the particular method
used to administer the composition. Accordingly, there is a wide
variety of suitable formulations of pharmaceutical compositions of
the present invention (see, e.g., Remington's Pharmaceutical
Sciences, 17.sup.th ed. 1985)).
[0170] The modulators (e.g., agonists or antagonists) of the
expression or activity of .beta.TRP, alone or in combination with
other suitable components, can be prepared for injection or for use
in a pump device. Pump devices (also known as "insulin pumps") are
commonly used to administer insulin to patients and therefore can
be easily adapted to include compositions of the present invention.
Manufacturers of insulin pumps include Animas, Disetronic and
MiniMed.
[0171] The modulators (e.g., agonists or antagonists) of the
expression or activity of .beta.TRP, alone or in combination with
other suitable components, can be made into aerosol formulations
(i.e., they can be "nebulized") to be administered via inhalation.
Aerosol formulations can be placed into pressurized acceptable
propellants, such as dichlorodifluoromethane, propane, nitrogen,
and the like.
[0172] Formulations suitable for administration include aqueous and
non-aqueous solutions, isotonic sterile solutions, which can
contain antioxidants, buffers, bacteriostats, and solutes that
render the formulation isotonic, and aqueous and non-aqueous
sterile suspensions that can include suspending agents,
solubilizers, thickening agents, stabilizers, and preservatives. In
the practice of this invention, compositions can be administered,
for example, orally, nasally, topically, intravenously,
intraperitoneally, or intrathecally. The formulations of compounds
can be presented in unit-dose or multi-dose sealed containers, such
as ampoules and vials. Solutions and suspensions can be prepared
from sterile powders, granules, and tablets of the kind previously
described. The modulators can also be administered as part of a
prepared food or drug.
[0173] The dose administered to a patient, in the context of the
present invention should be sufficient to induce a beneficial
response in the subject over time. The optimal dose level for any
patient will depend on a variety of factors including the efficacy
of the specific modulator employed, the age, body weight, physical
activity, and diet of the patient, on a possible combination with
other drugs, and on the severity of the case of diabetes. It is
recommended that the daily dosage of the modulator be determined
for each individual patient by those skilled in the art in a
similar way as for known insulin compositions. The size of the dose
also will be determined by the existence, nature, and extent of any
adverse side-effects that accompany the administration of a
particular compound or vector in a particular subject.
[0174] In determining the effective amount of the modulator to be
administered a physician may evaluate circulating plasma levels of
the modulator, modulator toxicity, and the production of
anti-modulator antibodies. In general, the dose equivalent of a
modulator is from about 1 ng/kg to 10 mg/kg for a typical
subject.
[0175] For administration, .beta.TRP modulators of the present
invention can be administered at a rate determined by the LD-50 of
the modulator, and the side-effects of the modulator at various
concentrations, as applied to the mass and overall health of the
subject. Administration can be accomplished via single or divided
doses.
[0176] The compounds of the present invention can also be used
effectively in combination with one or more additional active
agents depending on the desired target therapy (see, e.g., Turner,
N. et al. Prog. Drug Res. (1998) 51: 33-94; Haffnier, S. Diabetes
Care (1998) 21: 160-178; and DeFronzo, R. et al. (eds.), Diabetes
Reviews (1997) Vol. 5 No. 4). A number of studies have investigated
the benefits of combination therapies with oral agents (see, e.g.,
Mahler, R., J. Clin. Endocrinol. Metab. (1999) 84: 1165-71; United
Kingdom Prospective Diabetes Study Group: UKPDS 28, Diabetes Care
(1998) 21: 87-92; Bardin, C. W., (ed.), Current Therapy In
Endocrinology And Metabolism, 6th Edition (Mosby--Year Book, Inc.,
St. Louis, Mo. 1997); Chiasson, J. et al., Ann. Intern. Med. (1994)
121: 928-935; Coniff, R. et al., Clin. Ther. (1997) 19: 16-26;
Coniff, R. et al., Am. J. Med. (1995) 98: 443-451; and Iwamoto, Y.
et al., Diabet. Med. (1996) 13 365-370; Kwiterovich, P. Am. J.
Cardiol (1998) 82(12A): 3U-17U). These studies indicate that
modulation of diabetes, among other diseases, can be further
improved by the addition of a second agent to the therapeutic
regimen. Combination therapy includes administration of a single
pharmaceutical dosage formulation that contains a .beta.TRP
modulator of the invention and one or more additional active
agents, as well as administration of a .beta.TRP modulator and each
active agent in its own separate pharmaceutical dosage formulation.
For example, a .beta.TRP modulator and a thiazolidinedione can be
administered to the human subject together in a single oral dosage
composition, such as a tablet or capsule, or each agent can be
administered in separate oral dosage formulations. Where separate
dosage formulations are used, a .beta.TRP modulator and one or more
additional active agents can be administered at essentially the
same time (i.e., concurrently), or at separately staggered times
(i.e., sequentially). Combination therapy is understood to include
all these regimens.
[0177] One example of combination therapy can be seen in modulating
diabetes (or treating diabetes and its related symptoms,
complications, and disorders), wherein the .beta.TRP modulators can
be effectively used in combination with, for example, sulfonylureas
(such as chlorpropamide, tolbutamide, acetohexamide, tolazamide,
glyburide, gliclazide, glynase, glimepiride, and glipizide);
biguanides (such as metformin); a PPAR beta delta agonist; a ligand
or agonist of PPAR gamma such as thiazolidinediones (such as
ciglitazone, pioglitazone (see, e.g., U.S. Pat. No. 6,218,409),
troglitazone, and rosiglitazone (see, e.g., U.S. Pat. No.
5,859,037)); PPAR alpha agonists such as clofibrate, gemfibrozil,
fenofibrate, ciprofibrate, and bezafibrate; dehydroepiandrosterone
(also referred to as DHEA or its conjugated sulphate ester,
DHEA-SO4); antiglucocorticoids; TNFa inhibitors;
.alpha.-glucosidase inhibitors (such as acarbose, miglitol, and
voglibose); amylin and amylin derivatives (such as pramlintide,
(see, also, U.S. Pat. Nos. 5,902,726; 5,124,314; 5,175,145 and
6,143,718.)); insulin secretogogues (such as repaglinide,
gliquidone, and nateglinide (see, also, U.S. Pat. Nos. 6,251,856;
6,251,865; 6,221,633; 6,174,856)), and insulin.
VI. Methods For Identifying Modulators of Polypeptides that
Regulate .beta.TRP
[0178] As illustrated in FIG. 3, activation of .beta.TRP can be
readily measured by detecting changes in membrane potential.
Membrane potential-dependent fluorescent dyes provide significant
signal that can be measured using devices useful in high throughput
screening assays. To the inventors' knowledge, no channel in the
TRP family has been described to provide such a clear, readily
measurable membrane potential/cell depolarization signal upon
activation. Moreover, it is not predictable that a calcium channel
would mediate such a large change in membrane potential so as to be
readily measured in assays other than patch clamping. Accordingly,
the present invention provides methods of identifying modulators of
polypeptides that regulate .beta.TRP by identifying agents that
induce a change in membrane potential in cells expressing
.beta.TRP.
[0179] In some embodiments, the methods comprise contacting an
agent to a cell, wherein the cell expresses .beta.TRP and an
upstream regulator of .beta.TRP; and detecting a change membrane
potential of the cell, wherein a change in the membrane potential
of the cell in the presence of the agent compared to the absence of
the agent indicates that the agent modulates activity of the
regulator. Exemplary regulators of .beta.TRP include, e.g.,
G-protein coupled receptors (GPCRs) and G-proteins. For example, in
some embodiments, the GPCR is selected from Gq, Gi and Gs
receptors. Exemplary Gq receptors include, e.g., a muscarinic or
PT2Y receptor. In embodiments involving the Gi receptors, a
promiscuous G protein such as Gqi5, Galpha16, Gqs5, Gqo5, is also
expressed in the cell to mediate signaling between the GPCR and
.beta.TRP.
[0180] The methods of the invention can comprise detecting changes
in membrane potential using a device sufficient for high throughput
screening. For instance, changes in membrane potential can be
detected using cell-based assays in the presence of dyes that
respond fluorescently to membrane potential changes. Because of the
significant change in membrane potential induced by activation of
.beta.TRP, devices for measuring the fluorescence, such as the
FLEXstationg and FLIPR.RTM. systems (Molecular Devices, Sunnyvale,
Calif.) can be used to measure .beta.TRP activation. These devices,
in contrast to patch clamping techniques, are useful devices for
high throughput screening. Thus, a significant number (e.g., at
least 96, 384, 500, or 1000 or more) of potential modulators (e.g.,
in a combinatorial library) can be assayed for an effect on a
regulator of .beta.TRP in a single day by one person. Thus, large
combinatorial libraries of compounds, as described herein can be
screened to identify small molecules that modulate regulator
activity.
[0181] In some embodiments, the regulator polypeptide and /or
.beta.TRP polypeptide are recombinantly expressed in a cell.
Exemplary cells for recombinant expression include, e.g. mammalian
cells (e.g., HEK293, CHO, Cos7), insect cells (e.g., sf21),
bacterial cells (e.g., E; coli), or yeast (e.g., Pichia or S.
cervisiae).
EXAMPLES
Example 1
[0182] This example demonstrates that .beta.TRP is expressed in
pancreatic islet cells and demonstrates that introduction of
.beta.TRP into islet cells of diabetic animals improves glucose
stimulated insulin secretion.
[0183] Custom Affymetrix.TM. oligonucletide arrays were used to
survey islet gene expression. Microarray probe set MBXRATISLI2881
was called "Present" by the Affymetrix GeneChipT.sub.m analysis
software in 5 independent rat islet mRNA samples and absent in 10
other tissues examined. The mouse probe set MBXMUSISL22609 also
demonstrated a high degree of enrichment in islets and in the
cultured beta cell line (betaHC9) mRNA samples relative to those of
other tissues (FIG. 1B). Multiple clones for the corresponding
cDNAs were found in human, rat and mouse islet libraries, and
sequencing of these revealed that the encoded protein was a
predicted TRP channel, which we named betaTRP. The human gene for
betaTRP had been sequenced as part of an examination of the
Beckwith-Wiedeman syndrome locus and had been named MTRI. See, PCT
Application WO0132693. In contrast to the statements in PCT
Application WO0132693, betaTRP ESTs are well represented in human,
rat and mouse islet libraries. Oligonucleotide array data confirmed
that pancreatic islets are highly enriched for betaTRP. In-situ
hybridization was used to determine that betaTRP mRNA is abundant
in the majority of the core cells of rat islets, which indicates
that many if not most beta cells express betaTRP.
[0184] In an effort to identify genes that are functionally
important for proper regulation of insulin secretion that are
altered in the diabetic state we used a rat model of Type II
diabetes. Custom rat islet array hybridization for the mRNA
corresponding to betaTRP (probe set MBXRATISLI2881) is reduced 2.4
fold in islets of diabetic (9 week old ZDF) rats relative to
non-diabetic (9 week old ZLC) control animals (FIG. 1B). .beta.TRP
expression was substantially restored by concomitant treatment with
troglitazone FIG. 1B). A similar result is observed in ZDF female
rats fed a high fat diet. Beta TRP mRNA is also decreased greater
than 2-fold in male ZDF rats at 9 weeks of age relative to lean
control rats (data not shown)
[0185] The islets from these diabetic animals are deficient in
glucose stimulated insulin secretion (GSIS) relative to the islets
of control animals. However, adenoviral expression of betaTRP in
the ZDF islets restored their responsiveness to glucose in a static
insulin secretion assay or in a islet perifusion experiment (FIG.
2). Expression of betaTRP did not increase basal insulin secretion
and had little effect on islets from non-diabetic animals. These
data indicate that the betaTRP deficiency found in ZDF islets is
functionally linked to the decline in GSIS is these islets.
Example 2
[0186] This example demonstrates methods for high throughput
screening of modulators of .beta.TRP.
[0187] Activation (opening) of the pancreatic .beta. cell cation
channel .beta.TRP represents a novel mechanism for enhancing
glucose-stimulated insulin secretion in individuals with type II
diabetes. Type II diabetes results when pancreatic beta cells are
unable to compensate for the increased insulin demand caused by
peripheral insulin resistance. Therapeutic agents such as
sulphonylureas and meglitinides promote insulin secretion via the
same molecular mechanism (KATP channel closure) as the major
pathway by which glucose regulates insulin secretion. Although
these agents are widely used, they can be less than ideal in that
they have intrinsic potential to cause hypoglycemia and also have
significant rates of primary and secondary failure. Activation of
mechanisms in the .beta. cell that do not in themselves trigger
insulin secretion but potentiate Ca.sup.++ influx after
glucose-dependent K.sub.ATP channel closure can enhance insulin
secretion in a more physiologically appropriate manner. The
.beta.TRP cation channel represents a component of such a
potentiator mechanism for enhancing Ca.sup.++ influx and insulin
secretion. This type of therapeutic approach also offers a novel
therapy for diabetes patients for whom sulphonylureas/meglitinides
fail.
[0188] The TRP family (of which pTRP is a member) includes a
diverse group of proteins that share a common core domain that is
similar to the channel-forming core of Drosophila TRP (Transient
Receptor Potential), a light-activated Ca.sup.2+-selective channel
of the fly visual system. TRP channels mediate the influx of
Ca.sup.2+ and/or other cations when endoplasmic reticullum (ER)
Ca.sup.2+ stores are depleted or when receptors coupled to Gq and
phospholipase-.beta. (PLC) are activated. We have demonstrated that
.beta.TRP facilitates Ca2+ influx in response to PLC activation and
ER Ca.sup.2+ store depletion when expressed in HEK293 and COS-1
cells.
[0189] The activation (opening) of .beta.TRP (TRPM5) results a
robust Ca.sup.2+ influx and membrane depolarization which can be
detected indirectly with specific fluorescent dyes for Ca.sup.2+
and membrane potential (MP). FIG. 3 displays representative
recordings of [Ca.sup.2+].sub.i and MP responses to ATP in control
and .beta.TRP-CHO cells. The MP response to ATP is present only in
.beta.TRP cells. As one option to execute high throughput screens
for .beta.TRP modulators, CHO-K1 based .beta.TRP stable cell lines
are used to screen for small molecule modulators using the
Molecular Devices' FLIPR.RTM. (Molecular Devices, Sunnyvale,
Calif.) membrane potential assay with minor modifications.
[0190] The goal of the screening is to find specific and director
activators (openers) of .beta.TRP channel. The activation of the
channel is ideally complete and able to trigger a rise in
[Ca.sup.2+] and plasma membrane depolarization of its host
cells.
[0191] The following protocol is optimized for the measurement of
membrane potential (MP) in 96-well format using Molecular Devices'
Membrane Potential Dye (Cat # R-8034) on the FLEXStation (Molecular
Devices). It is expected to be applicable or adaptable to the
384-well format assay with the FLIPR.sup.384 system.
[0192] A CHO-K1 based stable cell line expressing .beta.TRP
(designated "Line A2-18" herein) is used for .beta.TRP high
throughput screening according to the follow protocol: [0193] 1.
Seed A2-18 .beta.Trp-CHO cell and the control line (Al-5) to
96-well plates. [0194] 2. Grow the cells for 24-48 h in DMEM medium
to 85-95% confluency. 3. Prepare following reagents immediately
before the assay: [0195] i. Assay buffer: dilute Component B of the
FLIPRO assay kit and adjust pH to 7.4 with 1 N NaOH. No probenecid
was needed. [0196] ii. MP dye: suspend a vial of Component A of the
FLIPR.RTM. assay kit with 10 ml of the assay buffer. [0197] iii.
Compound solutions: dilute DMSO stocks of the testing compounds to
a 5.times. solution with the assay buffer. Raise Ca.sup.2+
concentration in the compound solutions to 12.5 mM with 1 M
CaCl.sub.2. [0198] 4. Carefully remove the culture medium from all
wells. [0199] 5. Add 50 .mu.l assay buffer and 50 .mu.l MP dye.
[0200] 6. Incubate the plates at 37.degree. C. for 30-60 minutes.
[0201] 7. Set the FLEXStation to MP assay mode and to 37.degree. C.
[0202] 8. Transfer the cell plate and compound plate to
FLEXStation. [0203] 9. Record baseline for 18 sec (Ex 530, EmS60).
[0204] 10. Add the 5.times. compound solutions to the cells and
read another 100 sec. [0205] 11. Save and analyze data. Reagent
List [0206] 1. Assay buffer: Hank's Balanced Salt Solution (HBSS)
with 20 mM Hepes, pH 7.4 [0207] 2. MP dye: Molecular Devices
Corporation, Cat#8034 (for FLIPR) [0208] 3. Positive control: ATP
10-100 .mu.M (a purinergic receptor agonist) Calcimycin (A23187)
5-10 .mu.M (a calcium ionophore) [0209] 4. Antagonist control:
[0210] i. 2-APB 75 .mu.M (from Tocris, TRP channel and IP3 receptor
blocker); [0211] ii. U73122 10 .mu.M (from Sigma Chemicals, PLC
inhibitor). Other Experimental Conditions [0212] 1. The Cells:
[0213] iii. Maintenance: DMEM with 10% FCS and 200 pg/ml G418;
Passaged 1-2 time/week with trypsin-EDTA. [0214] iv. Confluency:
85-95% by the time of assay. [0215] v. Plating: Regular or
pre-coated plates; 24-48 h before assay. [0216] vi. Passage: up to
the 10.sup.th passage. [0217] 2. Ca2+ concentration in the assay
system:
[0218] The relative low Ca.sup.2+ (1.26 mM) in Molecular Device's
original assay systems may limit the influx of extracellular
Ca.sup.2+ through TRP channel and the accompanied depolarization as
suggested by the lack of action of maitotoxin (MTX) in
.beta.TRP-CHO cells. MTX is a marine polyether toxin known to be a
direct activation of non-selective cation channels (including
.beta.TRP). Raising Ca2+ concentration to 2.5-5 mM in the assay
system significantly enhanced the ability for the MP and the
Calcium Assay Kits to detect the increase in [Ca.sup.2+], or MP
induced by MTX (FIG. 4).
Example 3
[0219] This Example demonstrates solvent dose responses and
responses to other channel modulators.
[0220] Effect of DMSO
[0221] In the FLEXStation assay, upon the addition of the testing
compound (a 25 .mu.l of compound solution was added to each well
which contains 100 .mu.l of diluted dye) the CHO-.beta.TRP cells
tend to lose some intensity of fluorescent signal. The drop in RFU
signal was indistinguishable between the assay buffer and low
concentration of DMSO (<0.5%), but was significantly exaggerated
by 2.5% DMSO (FIG. 5). On the other hand, we did not observe any
non-specific depolarization responses to DMSO.
Example 4
[0222] This Example provides positive controls useful in the assays
of the invention.
[0223] Effects ofother channel modulators on MP of the bTRP-CHO
cells
[0224] To test the specificity of the depolarization responses to
ATP in the bTRP-CHO cells, we tested the effects of 80 compounds
from the Sigma-RBI Ion Channel Modulators Ligand-Set (Sigma #L6912)
in both control (A1-5) and a .beta.TRP-CHO cell line (A2-18). The
Sigma ligand-set consists of modulators of multiple members of the
K.sup.+, Na.sup.+, Ca2+ and Cl.sup.- channels, as well as several
channel forming amino acid transpoters. Among the 80 compounds,
only A23 187 (Calcimycin) induced a .beta.TRP cell specific
depolarization similar to ATP. Calcimycin is a Ca.sup.2+ ionophore
known to be able to deplete intracellular Ca2+ stores. In addition,
the class-Ill antiarrythmic compound Clofilium also triggered
depolarization in both control and bTRP cells. The mechanism of
clofilium is not known.
[0225] Activator controlfor TRP channel
[0226] ATP and calcimycin dose-dependently induced depolarization
in .beta.TRP-CHO cells, but not in control cells, as expected (FIG.
6). The estimated EC.sub.50 of ATP and calcimycin is 11 and 0.8
.mu.M respectively. ATP activates TRP channels by generating IP3
through the Gq-coupled P2Y receptor, whereas calcimycin acts by a
direct depletion of intracellular Ca.sup.2+ stores.
Example 5
[0227] This Example provides negative (antagonist) controls useful
in the assays of the invention.
[0228] The MP responses to ATP and calcimycin were suppressed over
50% by 2-APB when it was added to the cells at the-same time as the
two stimuli. 2-APB is known to blocker the IP3 receptor on the ER
(ligand-gated Ca.sup.2+ channel) and TRP channels in the plasma
membrane. The effect of ATP on MP in .beta.TRP-CHO cells was also
inhibited by a 30 minute pre-incubation of the cells with the PLC
inhibitor U73122 (FIG. 6).
[0229] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be sugested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
Sequence CWU 1
1
9 1 3931 DNA Homo sapiens human betaTRP (MTR1) calcium channel 1
gaggccacca tgcaggatgt ccaaggcccc cgtcccggaa gccccgggga tgctgaagac
60 cggcgggagc tgggcttgca caggggcgag gtcaactttg gagggtctgg
gaagaagcga 120 ggcaagtttg tacgggtgcc gagcggagtg gccccgtctg
tgctctttga cctgctgctt 180 gctgagtggc acctgccggc ccccaacctg
gtggtgtccc tggtgggtga ggagcagcct 240 ttcgccatga agtcctggct
gcgggatgtg ctgcgcaagg ggctggtgaa ggcggctcag 300 agcacaggag
cctggatcct gaccagtgcc ctccgcgtgg gcctggccag gcatgtcggg 360
caggccgtgc gcgaccactc gctggccagc acgtccacca aggtccgtgt ggttgctgtc
420 ggcatggcct cgctgggccg cgtcctgcac cgccgcattc tggaggaggc
ccaggtgcac 480 gaggattttc ctgtccacta ccctgaggat gacggcggca
gccagggccc cctctgttca 540 ctggacagca acctctccca cttcatcctg
gtggagccag gccccccggg gaagggcgat 600 gggctgacgg agctgcggct
gaggctggag aagcacatct cggagcagag ggcgggctac 660 gggggcactg
gcagcatcga gatccctgtc ctctgcttgc tggtcaatgg tgatcccaac 720
accttggaga ggatctccag ggccgtggag caggctgccc cgtggctgat cctggtaggc
780 tcggggggca tcgccgatgt gcttgctgcc ctagtgaacc agccccacct
cctggtgccc 840 aaggtggccg agaagcagtt taaggagaag ttccccagca
agcatttctc ttgggaggac 900 atcgtgcgct ggaccaagct gctgcagaac
atcacctcac accagcacct gctcaccgtg 960 tatgacttcg agcaggaggg
ctccgaggag ctggacacgg tcatcctgaa ggcgctggtg 1020 aaagcctgca
agagccacag ccaggagcct caggactatc tggatgagct caagctggcc 1080
gtggcctggg accgcgtgga catcgccaag agtgagatct tcaatgggga cgtggagtgg
1140 aagtcctgtg acctggagga ggtgatggtg gacgccctgg tcagcaacaa
gcccgagttt 1200 gtgcgcctct ttgtggacaa cggcgcagac gtggccgact
tcctgacgta tgggcggctg 1260 caggagctct accgctccgt gtcacgcaag
agcctgctct tcgacctgct gcagcggaag 1320 caggaggagg cccggctgac
gctggccggc ctgggcaccc agcaggcccg ggagccaccc 1380 gcggggccac
cggccttctc cctgcacgag gtctcccgcg tactcaagga cttcctgcag 1440
gacgcctgcc gaggcttcta ccaggacggc cggccagggg accgcaggag ggcggagaag
1500 ggcccggcca agcggcccac gggccagaag tggctgctgg acctgaacca
gaagagcgag 1560 aacccctggc gggacctgtt cctgtgggcc gtgctgcaga
accgccacga gatggccacc 1620 tacttctggg ccatgggcca ggaaggtgtg
gcagccgcac tggccgcctg caaaatcctc 1680 aaagagatgt cgcacctgga
gacggaggcc gaggcggccc gagccacgcg cgaggcgaaa 1740 tacgagcagc
tggccctcga cctcttctcc gagtgctaca gcaacagtga ggcccgcgcc 1800
ttcgccctgc tggtgcgccg gaaccgctgc tggagcaaga ccacctgcct gcacctggcc
1860 accgaggctg acgccaaggc cttctttgcc cacgacggcg ttcaggcctt
cctgaccagg 1920 atctggtggg gggacatggc cgcaggcacg cccatcctgc
ggctgctagg agccttcctc 1980 tgccccgccc tcgtctatac caacctcatc
accttcagtg aggaagctcc cctgaggaca 2040 ggcctggagg acctgcagga
cctggacagc ctggacacgg agaagagccc gctgtatggc 2100 ctgcagagcc
gggtggagga gctggtggag gcgccgaggg ctcagggtga ccgaggccca 2160
cgtgctgtct tcctgctcac acgctggcgg aaattctggg gcgctcccgt gactgtgttc
2220 ctggggaacg tggtcatgta cttcgccttc ctcttcctgt tcacctacgt
cctgctggtg 2280 gacttcaggc cgccccccca gggcccctca gggcccgagg
tcaccctcta cttctgggtc 2340 tttacgctgg tgctggagga aatccggcag
ggcttcttca cagacgagga cacacacctg 2400 gtgaagaagt tcacactgta
tgtgggggac aactggaaca agtgtgacat ggtggccatc 2460 ttcctgttca
tcgtgggtgt cacctgcagg atgctgccgt cggcgtttga ggctggccgc 2520
acagtcctcg ccatggactt catggtgttc acgctgcggc tgatccatat ctttgccata
2580 cacaagcagc tgggccccaa gatcatcgtg gtagagcgca tgatgaagga
cgtcttcttc 2640 ttcctcttct ttctgagcgt gtggctcgtg gcctacggtg
tcaccaccca ggcgctgctg 2700 cacccccatg acggccgcct ggagtggatc
ttccgccggg tgctctaccg gccctacctg 2760 cagatcttcg gccagatccc
actggacgag attgatgaag cccgtgtgaa ctgctccacc 2820 cacccactgc
tgctggagga ctcaccatcc tgccccagcc tctatgccaa ctggctggtc 2880
atcctcctgc tggtcacctt cctgttggtc accaatgtgc tgctcatgaa cctgctcatc
2940 gccatgttca gctacacgtt ccaggtggtg cagggcaacg cagacatgtt
ctggaagttc 3000 cagcgctaca acctgattgt ggagtaccac gagcgccccg
ccctggcccc gcccttcatc 3060 ctgctcagcc acctgagcct gacgctccgc
cgggtcttca agaaggaggc tgagcacaag 3120 cgggagcacc tggagagaga
cctgccagac cccctggacc agaaggtcgt cacctgggag 3180 acagtccaga
aggagaactt cctgagcaag atggagaagc ggaggaggga cagcgagggg 3240
gaggtgctgc ggaaaaccgc ccacagagtg gacttcattg ccaagtacct cgggggtctg
3300 agagagcaag aaaagcgcat caagtgtctg gagtcacaga tcaactactg
ctcggtgctc 3360 gtgtcctccg tggctgacgt gctggcccag ggtggcggcc
cccggagctc tcagcactgt 3420 ggcgagggaa gccagctggt ggctgctgac
cacagaggtg gtttagatgg ctgggaacaa 3480 cccggggctg gccagcctcc
ctcggacacc tgagctgctt ggcctgccac gtgtggggcc 3540 acctctcctc
agctggccac cctgcacgtt gtgcactgac ctttgccgac ctccagcgga 3600
accccccagg gggcaccagc cccccagcag acaatggccc tcctggtgcc tcaccacaga
3660 ccctcaccca aaggaaccgc tccttgtccc tcctggcctc cccggaggca
cagcagtgtc 3720 atggggctgt ctcccctgac aggcacaact ccccgggcag
aaaacgtgcc ccaccggcat 3780 ccctacctgg aaactgacca gcctgcactg
tggaaaagct ggccctgtgg cgtgacgggg 3840 gagcaccccc atccagactg
cgaagctgct ctggggtctg cacccacccc tgccctgact 3900 tgtgttgcct
gacaagagac tcatcttttt t 3931 2 1167 PRT Homo sapiens human betaTRP
(MTR1) calcium channel 2 Met Gln Asp Val Gln Gly Pro Arg Pro Gly
Ser Pro Gly Asp Ala Glu 1 5 10 15 Asp Arg Arg Glu Leu Gly Leu His
Arg Gly Glu Val Asn Phe Gly Gly 20 25 30 Ser Gly Lys Lys Arg Gly
Lys Phe Val Arg Val Pro Ser Gly Val Ala 35 40 45 Pro Ser Val Leu
Phe Asp Leu Leu Leu Ala Glu Trp His Leu Pro Ala 50 55 60 Pro Asn
Leu Val Val Ser Leu Val Gly Glu Glu Gln Pro Phe Ala Met 65 70 75 80
Lys Ser Trp Leu Arg Asp Val Leu Arg Lys Gly Leu Val Lys Ala Ala 85
90 95 Gln Ser Thr Gly Ala Trp Ile Leu Thr Ser Ala Leu Arg Val Gly
Leu 100 105 110 Ala Arg His Val Gly Gln Ala Val Arg Asp His Ser Leu
Ala Ser Thr 115 120 125 Ser Thr Lys Val Arg Val Val Ala Val Gly Met
Ala Ser Leu Gly Arg 130 135 140 Val Leu His Arg Arg Ile Leu Glu Glu
Ala Gln Val His Glu Asp Phe 145 150 155 160 Pro Val His Tyr Pro Glu
Asp Asp Gly Gly Ser Gln Gly Pro Leu Cys 165 170 175 Ser Leu Asp Ser
Asn Leu Ser His Phe Ile Leu Val Glu Pro Gly Pro 180 185 190 Pro Gly
Lys Gly Asp Gly Leu Thr Glu Leu Arg Leu Arg Leu Glu Lys 195 200 205
His Ile Ser Glu Gln Arg Ala Gly Tyr Gly Gly Thr Gly Ser Ile Glu 210
215 220 Ile Pro Val Leu Cys Leu Leu Val Asn Gly Asp Pro Asn Thr Leu
Glu 225 230 235 240 Arg Ile Ser Arg Ala Val Glu Gln Ala Ala Pro Trp
Leu Ile Leu Val 245 250 255 Gly Ser Gly Gly Ile Ala Asp Val Leu Ala
Ala Leu Val Asn Gln Pro 260 265 270 His Leu Leu Val Pro Lys Val Ala
Glu Lys Gln Phe Lys Glu Lys Phe 275 280 285 Pro Ser Lys His Phe Ser
Trp Glu Asp Ile Val Arg Trp Thr Lys Leu 290 295 300 Leu Gln Asn Ile
Thr Ser His Gln His Leu Leu Thr Val Tyr Asp Phe 305 310 315 320 Glu
Gln Glu Gly Ser Glu Glu Leu Asp Thr Val Ile Leu Lys Ala Leu 325 330
335 Val Lys Ala Cys Lys Ser His Ser Gln Glu Pro Gln Asp Tyr Leu Asp
340 345 350 Glu Leu Lys Leu Ala Val Ala Trp Asp Arg Val Asp Ile Ala
Lys Ser 355 360 365 Glu Ile Phe Asn Gly Asp Val Glu Trp Lys Ser Cys
Asp Leu Glu Glu 370 375 380 Val Met Val Asp Ala Leu Val Ser Asn Lys
Pro Glu Phe Val Arg Leu 385 390 395 400 Phe Val Asp Asn Gly Ala Asp
Val Ala Asp Phe Leu Thr Tyr Gly Arg 405 410 415 Leu Gln Glu Leu Tyr
Arg Ser Val Ser Arg Lys Ser Leu Leu Phe Asp 420 425 430 Leu Leu Gln
Arg Lys Gln Glu Glu Ala Arg Leu Thr Leu Ala Gly Leu 435 440 445 Gly
Thr Gln Gln Ala Arg Glu Pro Pro Ala Gly Pro Pro Ala Phe Ser 450 455
460 Leu His Glu Val Ser Arg Val Leu Lys Asp Phe Leu Gln Asp Ala Cys
465 470 475 480 Arg Gly Phe Tyr Gln Asp Gly Arg Pro Gly Asp Arg Arg
Arg Ala Glu 485 490 495 Lys Gly Pro Ala Lys Arg Pro Thr Gly Gln Lys
Trp Leu Leu Asp Leu 500 505 510 Asn Gln Lys Ser Glu Asn Pro Trp Arg
Asp Leu Phe Leu Trp Ala Val 515 520 525 Leu Gln Asn Arg His Glu Met
Ala Thr Tyr Phe Trp Ala Met Gly Gln 530 535 540 Glu Gly Val Ala Ala
Ala Leu Ala Ala Cys Lys Ile Leu Lys Glu Met 545 550 555 560 Ser His
Leu Glu Thr Glu Ala Glu Ala Ala Arg Ala Thr Arg Glu Ala 565 570 575
Lys Tyr Glu Gln Leu Ala Leu Asp Leu Phe Ser Glu Cys Tyr Ser Asn 580
585 590 Ser Glu Ala Arg Ala Phe Ala Leu Leu Val Arg Arg Asn Arg Cys
Trp 595 600 605 Ser Lys Thr Thr Cys Leu His Leu Ala Thr Glu Ala Asp
Ala Lys Ala 610 615 620 Phe Phe Ala His Asp Gly Val Gln Ala Phe Leu
Thr Arg Ile Trp Trp 625 630 635 640 Gly Asp Met Ala Ala Gly Thr Pro
Ile Leu Arg Leu Leu Gly Ala Phe 645 650 655 Leu Cys Pro Ala Leu Val
Tyr Thr Asn Leu Ile Thr Phe Ser Glu Glu 660 665 670 Ala Pro Leu Arg
Thr Gly Leu Glu Asp Leu Gln Asp Leu Asp Ser Leu 675 680 685 Asp Thr
Glu Lys Ser Pro Leu Tyr Gly Leu Gln Ser Arg Val Glu Glu 690 695 700
Leu Val Glu Ala Pro Arg Ala Gln Gly Asp Arg Gly Pro Arg Ala Val 705
710 715 720 Phe Leu Leu Thr Arg Trp Arg Lys Phe Trp Gly Ala Pro Val
Thr Val 725 730 735 Phe Leu Gly Asn Val Val Met Tyr Phe Ala Phe Leu
Phe Leu Phe Thr 740 745 750 Tyr Val Leu Leu Val Asp Phe Arg Pro Pro
Pro Gln Gly Pro Ser Gly 755 760 765 Pro Glu Val Thr Leu Tyr Phe Trp
Val Phe Thr Leu Val Leu Glu Glu 770 775 780 Ile Arg Gln Gly Phe Phe
Thr Asp Glu Asp Thr His Leu Val Lys Lys 785 790 795 800 Phe Thr Leu
Tyr Val Gly Asp Asn Trp Asn Lys Cys Asp Met Val Ala 805 810 815 Ile
Phe Leu Phe Ile Val Gly Val Thr Cys Arg Met Leu Pro Ser Ala 820 825
830 Phe Glu Ala Gly Arg Thr Val Leu Ala Met Asp Phe Met Val Phe Thr
835 840 845 Leu Arg Leu Ile His Ile Phe Ala Ile His Lys Gln Leu Gly
Pro Lys 850 855 860 Ile Ile Val Val Glu Arg Met Met Lys Asp Val Phe
Phe Phe Leu Phe 865 870 875 880 Phe Leu Ser Val Trp Leu Val Ala Tyr
Gly Val Thr Thr Gln Ala Leu 885 890 895 Leu His Pro His Asp Gly Arg
Leu Glu Trp Ile Phe Arg Arg Val Leu 900 905 910 Tyr Arg Pro Tyr Leu
Gln Ile Phe Gly Gln Ile Pro Leu Asp Glu Ile 915 920 925 Asp Glu Ala
Arg Val Asn Cys Ser Thr His Pro Leu Leu Leu Glu Asp 930 935 940 Ser
Pro Ser Cys Pro Ser Leu Tyr Ala Asn Trp Leu Val Ile Leu Leu 945 950
955 960 Leu Val Thr Phe Leu Leu Val Thr Asn Val Leu Leu Met Asn Leu
Leu 965 970 975 Ile Ala Met Phe Ser Tyr Thr Phe Gln Val Val Gln Gly
Asn Ala Asp 980 985 990 Met Phe Trp Lys Phe Gln Arg Tyr Asn Leu Ile
Val Glu Tyr His Glu 995 1000 1005 Arg Pro Ala Leu Ala Pro Pro Phe
Ile Leu Leu Ser His Leu Ser Leu 1010 1015 1020 Thr Leu Arg Arg Val
Phe Lys Lys Glu Ala Glu His Lys Arg Glu His 1025 1030 1035 1040 Leu
Glu Arg Asp Leu Pro Asp Pro Leu Asp Gln Lys Val Val Thr Trp 1045
1050 1055 Glu Thr Val Gln Lys Glu Asn Phe Leu Ser Lys Met Glu Lys
Arg Arg 1060 1065 1070 Arg Asp Ser Glu Gly Glu Val Leu Arg Lys Thr
Ala His Arg Val Asp 1075 1080 1085 Phe Ile Ala Lys Tyr Leu Gly Gly
Leu Arg Glu Gln Glu Lys Arg Ile 1090 1095 1100 Lys Cys Leu Glu Ser
Gln Ile Asn Tyr Cys Ser Val Leu Val Ser Ser 1105 1110 1115 1120 Val
Ala Asp Val Leu Ala Gln Gly Gly Gly Pro Arg Ser Ser Gln His 1125
1130 1135 Cys Gly Glu Gly Ser Gln Leu Val Ala Ala Asp His Arg Gly
Gly Leu 1140 1145 1150 Asp Gly Trp Glu Gln Pro Gly Ala Gly Gln Pro
Pro Ser Asp Thr 1155 1160 1165 3 4608 DNA Mus musculus mouse
betaTRP (TRPM5) calcium channel 3 atgcaaacaa cccagagctc ctgccccggc
agccccccag atactgagga tggctgggag 60 cccatcctat gcaggggaga
gatcaacttc ggagggtctg ggaagaagcg aggcaagttt 120 gtgaaggtgc
caagcagtgt ggccccctcc gtgctttttg aactcctgct caccgagtgg 180
cacctgccag cccccaacct ggtggtgtcc ctggtgggtg aggaacgacc tttggctatg
240 aagtcgtggc ttcgggatgt cctgcgcaag gggctggtga aagcagctca
gagcacaggt 300 gcctggatcc tgaccagtgc cctccacgtg ggcctggccc
gccatgttgg acaagctgta 360 cgtgatcact ctctggctag cacatccacc
aagatccgtg tagtggccat cggaatggcc 420 tctctggatc gaatccttca
ccgtcaactt ctagatggtg tccaccaaaa ggaggatact 480 cccatccact
acccagcaga tgagggcaac attcagggac ccctctgccc cctggacagc 540
aatctctccc acttcatcct tgtggagtca ggcgcccttg ggagtgggaa cgacgggctg
600 acagagctgc agctgagcct ggagaagcac atctctcagc agaggacagg
ttatgggggc 660 accagctgca tccagatacc tgtcctttgc ctgttggtca
atggtgaccc caacacccta 720 gagaggattt ccagggcagt ggagcaggct
gccccatggc tgatcctggc aggttctggt 780 ggcattgctg atgtactcgc
tgccctggtg agccagcctc atctcctggt gccccaggtg 840 gctgagaagc
agttcagaga gaaattcccc agcgagtgtt tctcttggga agccattgta 900
cactggacag agctgttaca gaacattgct gcacaccccc acctgctcac agtatatgac
960 ttcgagcagg agggttcgga ggacctggac actgtcatcc tcaaggcact
tgtgaaagcc 1020 tgcaagagcc acagccaaga agcccaagac tacctagatg
agctcaagtt agcagtggcc 1080 tgggatcgcg tggacattgc caagagtgaa
atcttcaatg gggacgtgga atggaagtcc 1140 tgtgacttgg aagaggtgat
gacagatgcc ctcgtgagca acaagcctga ctttgtccgc 1200 ctctttgtgg
acagcggtgc tgacatggcc gagttcttga cctatgggcg gctgcagcag 1260
ctttaccatt ctgtgtcccc caagagcctc ctctttgaac tgctgcagcg taagcatgag
1320 gagggtaggc tgacactggc cggcctgggt gcccagcagg ctcgggagct
gcccattggt 1380 ctgcctgcct tctcactcca cgaggtctcc cgcgtactca
aagacttcct gcatgacgcc 1440 tgccgtggct tctaccagga cgggcgcagg
atggaggaga gagggccacc taagcggccc 1500 gcaggccaga agtggctgcc
agacctcagt aggaagagtg aagacccttg gagggacctg 1560 ttcctctggg
ctgtgctgca gaatcgttat gagatggcca catacttctg ggccatgggc 1620
cgggagggtg tggctgctgc tctggctgcc tgcaagatca taaaggaaat gtcccacctg
1680 gagaaagagg cagaggtggc ccgcaccatg cgtgaggcca agtatgagca
gctggccctg 1740 gatcttttct cagagtgcta cggcaacagt gaggaccgtg
cctttgccct gctggtgcga 1800 aggaaccaca gctggagcag gaccacgtgc
ctgcacctgg ccactgaagc tgatgccaag 1860 gccttctttg cccatgacgg
tgtgcaagca ttcctgacca agatctggtg gggagacatg 1920 gccacaggca
cacccatcct acggcttctg ggtgccttca cctgcccagc cctcatctac 1980
acaaacctca tctccttcag tgaggatgcc ccgcagagga tggacctaga agatctgcag
2040 gagccagaca gcttggatat ggaaaagagc ttcctatgca gccggggtgg
ccaattggag 2100 aagctaacag aggcaccaag ggctccaggc gatctaggcc
cacaagctgc cttcctgctc 2160 acacggtgga ggaagttctg gggcgctcct
gtgactgtgt tcctggggaa tgtggtcatg 2220 tacttcgcat tcctcttcct
gttcacctat gtcctgctgg tggacttcag gccaccaccc 2280 caggggccgt
ctggatccga ggttaccctc tatttctggg tgttcacact ggtgctggag 2340
gaaatccgac agggcttctt cacagatgag gacacgcacc tggtgaagaa attcactctg
2400 tatgtggaag acaactggaa caagtgtgac atggtggcca tcttcctgtt
cattgtggga 2460 gtcacctgta gaatggtgcc ctcggtgttt gaggctggca
ggaccgttct ggccattgac 2520 ttcatggtgt tcacacttcg gctcatccac
atctttgcta ttcacaagca gttgggtcct 2580 aagatcatca ttgtagagcg
aatgatgaag gatgtcttct ttttcctctt cttcctgagc 2640 gtatggcttg
tggcctatgg tgtgaccact caggccctgc tgcatcccca tgatggccgt 2700
ttggagtgga ttttccgccg tgtgctatac aggccttacc tgcagatctt tgggcaaatc
2760 cctctggatg aaattgatga ggctcgtgtg aactgttctc ttcaccctct
gctgctggaa 2820 agctcggctt cctgccctaa tctctatgcc aactggctgg
tcattctcct gctggttacc 2880 ttcctgcttg tcactaatgt gctgctcatg
aaccttctga tcgccatgtt cagctacaca 2940 ttccaggtgg tgcaaggcaa
tgcagacatg ttctggaagt ttcaacgcta ccacctcatc 3000 gttgaatacc
atggaagacc agctctggcc ccgcccttca tcctgctcag ccacctgagc 3060
ctggtgctca agcaggtctt caggaaggaa gcccagcata agcgacaaca tctggagaga
3120 gacttgcctg accccttgga ccagaagatc attacctggg aaacggttca
aaaggagaac 3180 ttcctgagta ccatggagaa acggaggagg gacagcgagg
gggaggtgct gaggaaaacg 3240 gcacacagag tggacttgat tgccaaatac
atcggggggc tgagagagca agaaaagagg 3300 atcaagtgtc tggaatcaca
ggccaactac tgtatgctcc tcttgtcctc tatgacggat 3360 acactggctc
caggaggcac ctactcaagc tctcagaact gtggttgcag gagtcagcca 3420
gcctctgcta gagacaggga gtacctagag tctggcttgc caccctctga cacctgaaat
3480 ggagaaacca cttgctctag agccccagac ctggccacat cgagtttttg
gggcacatca 3540 accttccccc actcccagca gccccaagaa atggtcttca
aggccttgct acagatcact 3600 tcttggacat cccttcctaa gagaatgaaa
ctcatgtctt tggcatctat tcgggagcct 3660 cagaagtatc ctctccagca
gggcaagatt tttcatgtcc cactaaagct ttcactggct 3720 tggactggac
agctggatct ggccaagtcc tacataggac accatctgcc tggatggggc 3780
tatttaggtc taacccctgt cttaccctga gttcctaaga agccaacctc ttaaacacta
3840 ggtttctttc tgacccctga cccactcatt agctgaccag ctcctagagg
gcaggactca 3900 gatctattgt aattacctcc catctttcac cccccacagc
attatctgtc tgatcattct 3960 ggcagaaacc ccaagatatt gctcaagggt
acccaatgct actttacttt ctataaagcc 4020 tgtagaccac ctcaaatcag
ctaaactggg ccacaatggt ggctaaacgg gacatttcaa 4080 acacccgggg
aatatggagg attgtctgac ctagtgaaag gcatctccgt tccttccact 4140
gctcctcaaa ttaaatgacc atccaggtcc tttttagagg aactcagaga atggggacta
4200 cagaggctgg ggcagacctg ggtcttagca ggtctagcta acttggtcca
agtccctggc 4260 ctccacagga aacattcgct catgcatcct ccctgcatcc
ttctcttctt ctctggctca 4320 gcttcagtgg aatgacccag catcagctgt
gtcttacaca cacaagtcct ggagacacag 4380 ccagacacca accatatgct
gcttcacaac tcatcctctt cctataacct gtggtctgta 4440 caggcccagt
gctggggctg cattggtttc tgggggtggg ggtgggggtg ggttgtgcag 4500
attatgctca ttcactacca tcaggggcac aaggctgaac acagctaaga gcccagtccc
4560 tctgggtagt ttcatcagtg acaaatgtaa atgaccatgg caaacctt 4608 4
1158 PRT Mus musculus mouse betaTRP (TRPM5) calcium channel 4 Met
Gln Thr Thr Gln Ser Ser Cys Pro Gly Ser Pro Pro Asp Thr Glu 1 5 10
15 Asp Gly Trp Glu Pro Ile Leu Cys Arg Gly Glu Ile Asn Phe Gly Gly
20 25 30 Ser Gly Lys Lys Arg Gly Lys Phe Val Lys Val Pro Ser Ser
Val Ala 35 40 45 Pro Ser Val Leu Phe Glu Leu Leu Leu Thr Glu Trp
His Leu Pro Ala 50 55 60 Pro Asn Leu Val Val Ser Leu Val Gly Glu
Glu Arg Pro Leu Ala Met 65 70 75 80 Lys Ser Trp Leu Arg Asp Val Leu
Arg Lys Gly Leu Val Lys Ala Ala 85 90 95 Gln Ser Thr Gly Ala Trp
Ile Leu Thr Ser Ala Leu His Val Gly Leu 100 105 110 Ala Arg His Val
Gly Gln Ala Val Arg Asp His Ser Leu Ala Ser Thr 115 120 125 Ser Thr
Lys Ile Arg Val Val Ala Ile Gly Met Ala Ser Leu Asp Arg 130 135 140
Ile Leu His Arg Gln Leu Leu Asp Gly Val His Gln Lys Glu Asp Thr 145
150 155 160 Pro Ile His Tyr Pro Ala Asp Glu Gly Asn Ile Gln Gly Pro
Leu Cys 165 170 175 Pro Leu Asp Ser Asn Leu Ser His Phe Ile Leu Val
Glu Ser Gly Ala 180 185 190 Leu Gly Ser Gly Asn Asp Gly Leu Thr Glu
Leu Gln Leu Ser Leu Glu 195 200 205 Lys His Ile Ser Gln Gln Arg Thr
Gly Tyr Gly Gly Thr Ser Cys Ile 210 215 220 Gln Ile Pro Val Leu Cys
Leu Leu Val Asn Gly Asp Pro Asn Thr Leu 225 230 235 240 Glu Arg Ile
Ser Arg Ala Val Glu Gln Ala Ala Pro Trp Leu Ile Leu 245 250 255 Ala
Gly Ser Gly Gly Ile Ala Asp Val Leu Ala Ala Leu Val Ser Gln 260 265
270 Pro His Leu Leu Val Pro Gln Val Ala Glu Lys Gln Phe Arg Glu Lys
275 280 285 Phe Pro Ser Glu Cys Phe Ser Trp Glu Ala Ile Val His Trp
Thr Glu 290 295 300 Leu Leu Gln Asn Ile Ala Ala His Pro His Leu Leu
Thr Val Tyr Asp 305 310 315 320 Phe Glu Gln Glu Gly Ser Glu Asp Leu
Asp Thr Val Ile Leu Lys Ala 325 330 335 Leu Val Lys Ala Cys Lys Ser
His Ser Gln Glu Ala Gln Asp Tyr Leu 340 345 350 Asp Glu Leu Lys Leu
Ala Val Ala Trp Asp Arg Val Asp Ile Ala Lys 355 360 365 Ser Glu Ile
Phe Asn Gly Asp Val Glu Trp Lys Ser Cys Asp Leu Glu 370 375 380 Glu
Val Met Thr Asp Ala Leu Val Ser Asn Lys Pro Asp Phe Val Arg 385 390
395 400 Leu Phe Val Asp Ser Gly Ala Asp Met Ala Glu Phe Leu Thr Tyr
Gly 405 410 415 Arg Leu Gln Gln Leu Tyr His Ser Val Ser Pro Lys Ser
Leu Leu Phe 420 425 430 Glu Leu Leu Gln Arg Lys His Glu Glu Gly Arg
Leu Thr Leu Ala Gly 435 440 445 Leu Gly Ala Gln Gln Ala Arg Glu Leu
Pro Ile Gly Leu Pro Ala Phe 450 455 460 Ser Leu His Glu Val Ser Arg
Val Leu Lys Asp Phe Leu His Asp Ala 465 470 475 480 Cys Arg Gly Phe
Tyr Gln Asp Gly Arg Arg Met Glu Glu Arg Gly Pro 485 490 495 Pro Lys
Arg Pro Ala Gly Gln Lys Trp Leu Pro Asp Leu Ser Arg Lys 500 505 510
Ser Glu Asp Pro Trp Arg Asp Leu Phe Leu Trp Ala Val Leu Gln Asn 515
520 525 Arg Tyr Glu Met Ala Thr Tyr Phe Trp Ala Met Gly Arg Glu Gly
Val 530 535 540 Ala Ala Ala Leu Ala Ala Cys Lys Ile Ile Lys Glu Met
Ser His Leu 545 550 555 560 Glu Lys Glu Ala Glu Val Ala Arg Thr Met
Arg Glu Ala Lys Tyr Glu 565 570 575 Gln Leu Ala Leu Asp Leu Phe Ser
Glu Cys Tyr Gly Asn Ser Glu Asp 580 585 590 Arg Ala Phe Ala Leu Leu
Val Arg Arg Asn His Ser Trp Ser Arg Thr 595 600 605 Thr Cys Leu His
Leu Ala Thr Glu Ala Asp Ala Lys Ala Phe Phe Ala 610 615 620 His Asp
Gly Val Gln Ala Phe Leu Thr Lys Ile Trp Trp Gly Asp Met 625 630 635
640 Ala Thr Gly Thr Pro Ile Leu Arg Leu Leu Gly Ala Phe Thr Cys Pro
645 650 655 Ala Leu Ile Tyr Thr Asn Leu Ile Ser Phe Ser Glu Asp Ala
Pro Gln 660 665 670 Arg Met Asp Leu Glu Asp Leu Gln Glu Pro Asp Ser
Leu Asp Met Glu 675 680 685 Lys Ser Phe Leu Cys Ser Arg Gly Gly Gln
Leu Glu Lys Leu Thr Glu 690 695 700 Ala Pro Arg Ala Pro Gly Asp Leu
Gly Pro Gln Ala Ala Phe Leu Leu 705 710 715 720 Thr Arg Trp Arg Lys
Phe Trp Gly Ala Pro Val Thr Val Phe Leu Gly 725 730 735 Asn Val Val
Met Tyr Phe Ala Phe Leu Phe Leu Phe Thr Tyr Val Leu 740 745 750 Leu
Val Asp Phe Arg Pro Pro Pro Gln Gly Pro Ser Gly Ser Glu Val 755 760
765 Thr Leu Tyr Phe Trp Val Phe Thr Leu Val Leu Glu Glu Ile Arg Gln
770 775 780 Gly Phe Phe Thr Asp Glu Asp Thr His Leu Val Lys Lys Phe
Thr Leu 785 790 795 800 Tyr Val Glu Asp Asn Trp Asn Lys Cys Asp Met
Val Ala Ile Phe Leu 805 810 815 Phe Ile Val Gly Val Thr Cys Arg Met
Val Pro Ser Val Phe Glu Ala 820 825 830 Gly Arg Thr Val Leu Ala Ile
Asp Phe Met Val Phe Thr Leu Arg Leu 835 840 845 Ile His Ile Phe Ala
Ile His Lys Gln Leu Gly Pro Lys Ile Ile Ile 850 855 860 Val Glu Arg
Met Met Lys Asp Val Phe Phe Phe Leu Phe Phe Leu Ser 865 870 875 880
Val Trp Leu Val Ala Tyr Gly Val Thr Thr Gln Ala Leu Leu His Pro 885
890 895 His Asp Gly Arg Leu Glu Trp Ile Phe Arg Arg Val Leu Tyr Arg
Pro 900 905 910 Tyr Leu Gln Ile Phe Gly Gln Ile Pro Leu Asp Glu Ile
Asp Glu Ala 915 920 925 Arg Val Asn Cys Ser Leu His Pro Leu Leu Leu
Glu Ser Ser Ala Ser 930 935 940 Cys Pro Asn Leu Tyr Ala Asn Trp Leu
Val Ile Leu Leu Leu Val Thr 945 950 955 960 Phe Leu Leu Val Thr Asn
Val Leu Leu Met Asn Leu Leu Ile Ala Met 965 970 975 Phe Ser Tyr Thr
Phe Gln Val Val Gln Gly Asn Ala Asp Met Phe Trp 980 985 990 Lys Phe
Gln Arg Tyr His Leu Ile Val Glu Tyr His Gly Arg Pro Ala 995 1000
1005 Leu Ala Pro Pro Phe Ile Leu Leu Ser His Leu Ser Leu Val Leu
Lys 1010 1015 1020 Gln Val Phe Arg Lys Glu Ala Gln His Lys Arg Gln
His Leu Glu Arg 1025 1030 1035 1040 Asp Leu Pro Asp Pro Leu Asp Gln
Lys Ile Ile Thr Trp Glu Thr Val 1045 1050 1055 Gln Lys Glu Asn Phe
Leu Ser Thr Met Glu Lys Arg Arg Arg Asp Ser 1060 1065 1070 Glu Gly
Glu Val Leu Arg Lys Thr Ala His Arg Val Asp Leu Ile Ala 1075 1080
1085 Lys Tyr Ile Gly Gly Leu Arg Glu Gln Glu Lys Arg Ile Lys Cys
Leu 1090 1095 1100 Glu Ser Gln Ala Asn Tyr Cys Met Leu Leu Leu Ser
Ser Met Thr Asp 1105 1110 1115 1120 Thr Leu Ala Pro Gly Gly Thr Tyr
Ser Ser Ser Gln Asn Cys Gly Cys 1125 1130 1135 Arg Ser Gln Pro Ala
Ser Ala Arg Asp Arg Glu Tyr Leu Glu Ser Gly 1140 1145 1150 Leu Pro
Pro Ser Asp Thr 1155 5 3468 DNA Rattus sp. rat betaTRP calcium
channel 5 atgccgatgg cccagagctc ttgtcctgga agccccccag atactgggga
tggatgggag 60 ccagtcctat gcaagggaga ggtcaacttc ggagggtctg
ggaaaaagcg aagcaagttt 120 gtgaaggtgc caagcaatgt ggccccctcc
atgctctttg aactcctgct caccgagtgg 180 cacctgccag cccccaacct
ggtggtgtcc ctggtgggcg aggaacggct ttttgctatg 240 aagtcctggc
ttcgggatgt cttgcgcaag gggctggtga aagcagctca gagcacaggt 300
gcctggatcc tgaccagtgc cctccatgtg ggcctggcac gccatgttgg acaggctgta
360 cgtgatcact ctctggctag cacgtccacc aaggtccgtg tggtggccat
cggaatggcc 420 tctctggacc gaatccttca ccgccaactt ctagatggtg
tccaggagga tactcccatc 480 cactacccag cagatgaggg cagcactcag
ggacccctct gccctctgga cagcaatctc 540 tcccacttca tcctcgtgga
gccaggcacc cttgggagtg ggaacgacgg actggcagag 600 ctgcagctga
gcctggagaa gcacatctct cagcagagga caggttatgg gggtaccagc 660
agcatccaga tacctgtcct ttgcttgcta gtcaatggtg accccagcac cctagagagg
720 atgtccaggg cagtggagca ggctgcccca tggctgatcc tggcaggttc
tgggggcatt 780 gctgatgtac tcgctgccct ggtgggccag cctcatctcc
tggtgcccca ggtgaccgag 840 aagcagttca gagagaaatt cccaagcgag
tgtttctctt gggaagccat tgtacactgg 900 acagagctgc tacagaacat
tgctgcacac ccccacctgc tcacagtgta cgactttgag 960 caggagggtt
ccgaggacct ggacaccgtc atcctcaagg cacttgtgaa agcctgcaag 1020
agtcacagcc gagacgcaca agactaccta gatgagctca agttagcagt ggcctgggat
1080 cgcgtggaca ttgccaagag tgaaatcttc aatggggacg tggagtggaa
gtcctgtgac 1140 ttggaagagg tgatgacaga tgccctagtg agcaacaagc
ctgacttcgt gcgcctcttt 1200 gtggacagtg gtgctgacat ggccgagttc
ttgacctatg ggcggctgca gcagctttac 1260 cactctgtgt cccccaagag
cctcctcttt gaactgctgg agcgtaagca tgaggagggt 1320 cggctgacac
tggctggcct gggtgcccag cagacccggg agctgcccgt tggtctgcct 1380
gccttttcac tccatgaggt ctcccgagtt ctcaaagatt tcctgcatga cgcctgccgt
1440 ggcttctacc aggatgggcg caggatggag gagagagggc cacccaagcg
gcctgcaggc 1500 cagaaatggc tgccggacct cagtcggaag agtgaagacc
catggaggga cctgttcctt 1560 tgggctgtgc tgcagaaccg ttatgagatg
gccacatact tctgggccat gggccgggag 1620 ggtgtggctg ctgctctggc
ggcctgcaag atcatcaagg aaatgtccca cctggagaaa 1680 gaggcagagg
tggcccgcac tatgcgtgag gccaagtatg agcagctggc cctcgatctt 1740
ttctcagagt gctacagcaa cagtgaggac cgtgcctttg ccctgttggt gcgcaggaac
1800 cacagctgga gcaggaccac ctgcctgcac ctggccactg aggccgatgc
caaggccttc 1860 tttgcccatg atggtgtgca agcattcctg acgaagatct
ggtggggaga catggccaca 1920 ggcacaccca tcttacgact tctgggtgcc
ttcacctgcc cagccctcat ctacacaaat 1980 ctcatctcct tcagtgagga
tgccccgcag aggatggacc tggaagatct gcaggagcca 2040 gacagtttgg
atatggaaaa gagcttcctg tgcagccatg gtggccagtt ggagaagtta 2100
acagaggcgc caagggctcc tggcgatcta ggcccacaag ctgccttcct gctcacacgg
2160 tggaggaagt tctggggcgc tcctgtgact gtgttcttgg ggaatgtggt
catgtacttt 2220 gcattcctct tcctattctc ctacgtcctg ctggtggatt
tcaggccacc accccagggg 2280 ccatctgggt cggaagttac cctgtatttc
tgggtcttca cactggtgct ggaggaaatc 2340 cgacagggat tcttcacaaa
cgaggacacc cgtctggtga agaagttcac tctgtacgta 2400 gaagacaact
ggaacaaatg tgacatggtg gccatcttcc tgttcattgt tggtgtcacc 2460
tgtaggatgg tgccctccgt gtttgaggct ggccggactg ttctggccat tgacttcatg
2520 gtgttcacac ttcggctcat ccacatcttt gctattcaca agcagctggg
tcctaagatc 2580 atcattgtag agcggatgat gaaagatgtc ttcttcttcc
tcttcttcct gagcgtgtgg 2640 ctcgtggcct atggcgtgac cactcaggcc
ctgctgcacc cccacgatgg ccgtctggag 2700 tggattttcc gccgtgtgct
ctacaggcct tacctgcaga tctttgggca aatccctctg 2760 gatgaaattg
atgaggcccg tgtgaactgc tctcttcacc cgttgctgct ggacagctca 2820
gcttcctgcc ctaatctcta tgccaactgg ctggtcattc tcctgctggt taccttcctc
2880 ctcgtcacta atgtgctact tatgaacctt ctgatcgcca tgttcagcta
cacattccag 2940 gtggtgcagg gcaatgcaga catgttctgg aagtttcaac
gctaccacct catcgttgaa 3000 taccacggaa ggccggctct ggccccgccc
ttcatcctgc tcagccacct gagcctggtg 3060 ctcaagcagg tcttcaggaa
ggaagcccag cacaaacagc aacacctgga gagagacttg 3120 cctgaccccg
tggaccagaa gatcattacc tgggaaacag ttcaaaagga gaacttcctg 3180
agtaccatgg agaaacggag gagggacagt gaggaggagg tgctgaggaa aacggcacac
3240 agggtggact tgattgccaa atacatcggg ggtctgagag agcaagaaaa
gaggatcaag 3300 tgtctggagt cacaggcaaa ctactgtatg ctcctcttgt
cctccatgac tgacacactg 3360 gctcctggag gcacctactc aagttctcaa
aactgtggtc gcaggagtca gccagcctct 3420 gctagagaca gggagtacct
agaggctggc ttgccacact cagacacc 3468 6 1156 PRT Rattus sp. rat
betaTRP calcium channel 6 Met Pro Met Ala Gln Ser Ser Cys Pro Gly
Ser Pro Pro Asp Thr Gly 1 5 10 15 Asp Gly Trp Glu Pro Val Leu Cys
Lys Gly Glu Val Asn Phe Gly Gly 20 25 30 Ser Gly Lys Lys Arg Ser
Lys Phe Val Lys Val Pro Ser Asn Val Ala 35 40 45 Pro Ser Met Leu
Phe Glu Leu Leu Leu Thr Glu Trp His Leu Pro Ala 50 55 60 Pro Asn
Leu Val Val Ser Leu Val Gly Glu Glu Arg Leu Phe Ala Met 65 70 75 80
Lys Ser Trp Leu Arg Asp Val Leu Arg Lys Gly Leu Val Lys Ala Ala 85
90 95 Gln Ser Thr Gly Ala Trp Ile Leu Thr Ser Ala Leu His Val Gly
Leu 100 105 110 Ala Arg His Val Gly Gln Ala Val Arg Asp His Ser Leu
Ala Ser Thr 115 120 125 Ser Thr Lys Val Arg Val Val Ala Ile Gly Met
Ala Ser Leu Asp Arg 130 135 140 Ile Leu His Arg Gln Leu Leu Asp Gly
Val Gln Glu Asp Thr Pro Ile 145 150 155 160 His Tyr Pro Ala Asp Glu
Gly Ser Thr Gln Gly Pro Leu Cys Pro Leu 165 170 175 Asp Ser Asn Leu
Ser His Phe Ile Leu Val Glu Pro Gly Thr Leu Gly 180 185 190 Ser Gly
Asn Asp Gly Leu Ala Glu Leu Gln Leu Ser Leu Glu Lys His 195 200 205
Ile Ser Gln Gln Arg Thr Gly Tyr Gly Gly Thr Ser Ser Ile Gln Ile 210
215 220 Pro Val Leu Cys Leu Leu Val Asn Gly Asp Pro Ser Thr Leu Glu
Arg 225 230 235 240 Met Ser Arg Ala Val Glu Gln Ala Ala Pro Trp Leu
Ile Leu Ala Gly 245 250 255 Ser Gly Gly Ile Ala Asp Val Leu Ala Ala
Leu Val Gly Gln Pro His 260 265 270 Leu Leu Val Pro Gln Val Thr Glu
Lys Gln Phe Arg Glu Lys Phe Pro 275 280 285 Ser Glu Cys Phe Ser Trp
Glu Ala Ile Val His Trp Thr Glu Leu Leu 290 295 300 Gln Asn Ile Ala
Ala His Pro His Leu Leu Thr Val Tyr Asp Phe Glu 305 310 315 320 Gln
Glu Gly Ser Glu Asp Leu Asp Thr Val Ile Leu Lys Ala Leu Val 325 330
335 Lys Ala Cys Lys Ser His Ser Arg Asp Ala Gln Asp Tyr Leu Asp Glu
340 345 350 Leu Lys Leu Ala Val Ala Trp Asp Arg Val Asp Ile Ala Lys
Ser Glu 355 360 365 Ile Phe Asn Gly Asp Val Glu Trp Lys Ser Cys Asp
Leu Glu Glu Val 370 375 380 Met Thr Asp Ala Leu Val Ser Asn Lys Pro
Asp Phe Val Arg Leu Phe 385 390 395 400 Val Asp Ser Gly Ala Asp Met
Ala Glu Phe Leu Thr Tyr Gly Arg Leu 405 410 415 Gln Gln Leu Tyr His
Ser Val Ser Pro Lys Ser Leu Leu Phe Glu Leu 420 425 430 Leu Glu Arg
Lys His Glu Glu Gly Arg Leu Thr Leu Ala Gly Leu Gly 435 440 445 Ala
Gln Gln Thr Arg Glu Leu Pro Val Gly Leu Pro Ala Phe Ser Leu 450 455
460 His Glu Val Ser Arg Val Leu Lys Asp Phe Leu His Asp Ala Cys Arg
465 470 475 480 Gly Phe Tyr Gln Asp Gly Arg Arg Met Glu Glu Arg Gly
Pro Pro Lys 485 490 495 Arg Pro Ala Gly Gln Lys Trp Leu Pro Asp Leu
Ser Arg Lys Ser Glu 500 505 510 Asp Pro Trp Arg Asp Leu Phe Leu Trp
Ala Val Leu Gln Asn Arg Tyr 515 520 525 Glu Met Ala Thr Tyr Phe Trp
Ala Met Gly Arg Glu Gly Val Ala Ala 530 535 540 Ala Leu Ala Ala Cys
Lys Ile Ile Lys Glu Met Ser His Leu Glu Lys 545 550
555 560 Glu Ala Glu Val Ala Arg Thr Met Arg Glu Ala Lys Tyr Glu Gln
Leu 565 570 575 Ala Leu Asp Leu Phe Ser Glu Cys Tyr Ser Asn Ser Glu
Asp Arg Ala 580 585 590 Phe Ala Leu Leu Val Arg Arg Asn His Ser Trp
Ser Arg Thr Thr Cys 595 600 605 Leu His Leu Ala Thr Glu Ala Asp Ala
Lys Ala Phe Phe Ala His Asp 610 615 620 Gly Val Gln Ala Phe Leu Thr
Lys Ile Trp Trp Gly Asp Met Ala Thr 625 630 635 640 Gly Thr Pro Ile
Leu Arg Leu Leu Gly Ala Phe Thr Cys Pro Ala Leu 645 650 655 Ile Tyr
Thr Asn Leu Ile Ser Phe Ser Glu Asp Ala Pro Gln Arg Met 660 665 670
Asp Leu Glu Asp Leu Gln Glu Pro Asp Ser Leu Asp Met Glu Lys Ser 675
680 685 Phe Leu Cys Ser His Gly Gly Gln Leu Glu Lys Leu Thr Glu Ala
Pro 690 695 700 Arg Ala Pro Gly Asp Leu Gly Pro Gln Ala Ala Phe Leu
Leu Thr Arg 705 710 715 720 Trp Arg Lys Phe Trp Gly Ala Pro Val Thr
Val Phe Leu Gly Asn Val 725 730 735 Val Met Tyr Phe Ala Phe Leu Phe
Leu Phe Ser Tyr Val Leu Leu Val 740 745 750 Asp Phe Arg Pro Pro Pro
Gln Gly Pro Ser Gly Ser Glu Val Thr Leu 755 760 765 Tyr Phe Trp Val
Phe Thr Leu Val Leu Glu Glu Ile Arg Gln Gly Phe 770 775 780 Phe Thr
Asn Glu Asp Thr Arg Leu Val Lys Lys Phe Thr Leu Tyr Val 785 790 795
800 Glu Asp Asn Trp Asn Lys Cys Asp Met Val Ala Ile Phe Leu Phe Ile
805 810 815 Val Gly Val Thr Cys Arg Met Val Pro Ser Val Phe Glu Ala
Gly Arg 820 825 830 Thr Val Leu Ala Ile Asp Phe Met Val Phe Thr Leu
Arg Leu Ile His 835 840 845 Ile Phe Ala Ile His Lys Gln Leu Gly Pro
Lys Ile Ile Ile Val Glu 850 855 860 Arg Met Met Lys Asp Val Phe Phe
Phe Leu Phe Phe Leu Ser Val Trp 865 870 875 880 Leu Val Ala Tyr Gly
Val Thr Thr Gln Ala Leu Leu His Pro His Asp 885 890 895 Gly Arg Leu
Glu Trp Ile Phe Arg Arg Val Leu Tyr Arg Pro Tyr Leu 900 905 910 Gln
Ile Phe Gly Gln Ile Pro Leu Asp Glu Ile Asp Glu Ala Arg Val 915 920
925 Asn Cys Ser Leu His Pro Leu Leu Leu Asp Ser Ser Ala Ser Cys Pro
930 935 940 Asn Leu Tyr Ala Asn Trp Leu Val Ile Leu Leu Leu Val Thr
Phe Leu 945 950 955 960 Leu Val Thr Asn Val Leu Leu Met Asn Leu Leu
Ile Ala Met Phe Ser 965 970 975 Tyr Thr Phe Gln Val Val Gln Gly Asn
Ala Asp Met Phe Trp Lys Phe 980 985 990 Gln Arg Tyr His Leu Ile Val
Glu Tyr His Gly Arg Pro Ala Leu Ala 995 1000 1005 Pro Pro Phe Ile
Leu Leu Ser His Leu Ser Leu Val Leu Lys Gln Val 1010 1015 1020 Phe
Arg Lys Glu Ala Gln His Lys Gln Gln His Leu Glu Arg Asp Leu 1025
1030 1035 1040 Pro Asp Pro Val Asp Gln Lys Ile Ile Thr Trp Glu Thr
Val Gln Lys 1045 1050 1055 Glu Asn Phe Leu Ser Thr Met Glu Lys Arg
Arg Arg Asp Ser Glu Glu 1060 1065 1070 Glu Val Leu Arg Lys Thr Ala
His Arg Val Asp Leu Ile Ala Lys Tyr 1075 1080 1085 Ile Gly Gly Leu
Arg Glu Gln Glu Lys Arg Ile Lys Cys Leu Glu Ser 1090 1095 1100 Gln
Ala Asn Tyr Cys Met Leu Leu Leu Ser Ser Met Thr Asp Thr Leu 1105
1110 1115 1120 Ala Pro Gly Gly Thr Tyr Ser Ser Ser Gln Asn Cys Gly
Arg Arg Ser 1125 1130 1135 Gln Pro Ala Ser Ala Arg Asp Arg Glu Tyr
Leu Glu Ala Gly Leu Pro 1140 1145 1150 His Ser Asp Thr 1155 7 1165
PRT Homo sapiens human betaTRP (MBX) calcium channel 7 Met Gln Asp
Val Gln Gly Pro Arg Pro Gly Ser Pro Gly Asp Ala Glu 1 5 10 15 Asp
Arg Arg Glu Leu Gly Leu His Arg Gly Glu Val Asn Asn Gly Gly 20 25
30 Ser Gly Lys Lys Arg Gly Lys Asn Val Arg Val Pro Ser Gly Val Ala
35 40 45 Pro Ser Val Leu Asn Asp Leu Leu Leu Ala Glu Trp His Leu
Pro Ala 50 55 60 Pro Asn Leu Val Val Ser Leu Val Gly Glu Glu Gln
Pro Phe Ala Met 65 70 75 80 Lys Ser Trp Leu Arg Asp Val Leu Arg Lys
Gly Leu Val Lys Ala Ala 85 90 95 Gln Ser Thr Gly Ala Trp Ile Leu
Thr Ser Ala Leu Arg Val Gly Leu 100 105 110 Ala Arg His Val Gly Gln
Ala Val Arg Asp His Ser Leu Ala Ser Thr 115 120 125 Ser Thr Lys Val
Arg Val Val Ala Val Gly Met Ala Ser Leu Gly Arg 130 135 140 Val Leu
His Arg Arg Ile Leu Glu Glu Ala Gln Glu Asp Asn Pro Val 145 150 155
160 His Tyr Pro Glu Asp Asp Gly Gly Ser Gln Gly Pro Leu Cys Ser Leu
165 170 175 Asp Ser Asn Leu Ser His Phe Ile Leu Val Glu Pro Gly Pro
Pro Gly 180 185 190 Lys Gly Asp Gly Leu Thr Glu Leu Arg Leu Arg Leu
Glu Lys His Ile 195 200 205 Ser Glu Gln Arg Ala Gly Tyr Gly Gly Thr
Gly Ser Ile Glu Ile Pro 210 215 220 Val Leu Cys Leu Leu Val Asn Gly
Asp Pro Asn Thr Leu Glu Arg Ile 225 230 235 240 Ser Arg Ala Val Glu
Gln Ala Ala Pro Trp Leu Ile Leu Val Gly Ser 245 250 255 Gly Gly Ile
Ala Asp Val Leu Ala Ala Leu Val Asn Gln Pro His Leu 260 265 270 Leu
Val Pro Lys Val Ala Glu Lys Gln Asn Lys Glu Lys Phe Pro Ser 275 280
285 Lys His Phe Ser Trp Glu Asp Ile Val Arg Trp Thr Lys Leu Leu Gln
290 295 300 Asn Ile Thr Ser His Gln His Leu Leu Thr Val Tyr Asp Phe
Glu Gln 305 310 315 320 Glu Gly Ser Glu Glu Leu Asp Thr Val Ile Leu
Lys Ala Leu Val Lys 325 330 335 Ala Cys Lys Ser His Ser Gln Glu Pro
Gln Asp Tyr Leu Asp Glu Leu 340 345 350 Lys Leu Ala Val Ala Trp Asp
Arg Val Asp Ile Ala Lys Ser Glu Ile 355 360 365 Phe Asn Gly Asp Val
Glu Trp Lys Ser Cys Asp Leu Glu Glu Val Met 370 375 380 Val Asp Ala
Leu Val Ser Asn Lys Pro Glu Asn Val Arg Leu Asn Val 385 390 395 400
Asp Asn Gly Ala Asp Val Ala Asp Phe Leu Thr Tyr Gly Arg Leu Gln 405
410 415 Glu Leu Tyr Arg Ser Val Ser Arg Lys Ser Leu Leu Phe Asp Leu
Leu 420 425 430 Gln Arg Lys Gln Glu Glu Ala Arg Leu Thr Leu Ala Gly
Leu Gly Thr 435 440 445 Gln Gln Ala Arg Glu Pro Pro Ala Gly Pro Pro
Ala Phe Ser Leu His 450 455 460 Glu Val Ser Arg Val Leu Lys Asp Phe
Leu Gln Asp Ala Cys Arg Gly 465 470 475 480 Phe Tyr Gln Asp Gly Arg
Pro Gly Asp Arg Arg Arg Ala Glu Lys Gly 485 490 495 Pro Ala Lys Arg
Pro Thr Gly Gln Lys Trp Leu Leu Asp Leu Asn Gln 500 505 510 Lys Ser
Glu Asn Pro Trp Arg Asp Leu Phe Leu Trp Ala Val Leu Gln 515 520 525
Asn Arg His Glu Met Ala Thr Tyr Phe Trp Ala Met Gly Gln Glu Gly 530
535 540 Val Ala Ala Ala Leu Ala Ala Cys Lys Ile Leu Lys Glu Met Ser
His 545 550 555 560 Leu Glu Thr Glu Ala Glu Ala Ala Arg Ala Thr Arg
Glu Ala Lys Tyr 565 570 575 Glu Arg Leu Ala Leu Asp Leu Phe Ser Glu
Cys Tyr Ser Asn Ser Glu 580 585 590 Ala Arg Ala Phe Ala Leu Leu Val
Arg Arg Asn Arg Cys Trp Ser Lys 595 600 605 Thr Thr Cys Leu His Leu
Ala Thr Glu Ala Asp Ala Lys Ala Phe Asn 610 615 620 Ala His Asp Gly
Val Gln Ala Phe Leu Thr Arg Ile Trp Trp Gly Asp 625 630 635 640 Met
Ala Ala Gly Thr Pro Ile Leu Arg Leu Leu Gly Ala Phe Leu Cys 645 650
655 Pro Ala Leu Val Tyr Thr Asn Leu Ile Thr Phe Ser Glu Glu Ala Pro
660 665 670 Leu Arg Thr Gly Leu Glu Asp Leu Gln Asp Leu Asp Ser Leu
Asp Thr 675 680 685 Glu Lys Ser Pro Leu Tyr Gly Leu Gln Ser Arg Val
Glu Glu Leu Val 690 695 700 Glu Ala Pro Arg Ala Gln Gly Asp Arg Gly
Pro Arg Ala Val Phe Leu 705 710 715 720 Leu Thr Arg Trp Arg Lys Phe
Trp Gly Ala Pro Val Thr Val Phe Leu 725 730 735 Gly Asn Val Val Met
Tyr Phe Ala Phe Leu Phe Leu Phe Thr Tyr Val 740 745 750 Leu Leu Val
Asp Phe Arg Pro Pro Pro Gln Gly Pro Ser Gly Pro Glu 755 760 765 Val
Thr Leu Tyr Phe Trp Val Asn Thr Leu Val Leu Glu Glu Ile Arg 770 775
780 Gln Gly Phe Phe Thr Asp Glu Asp Thr His Leu Val Lys Lys Phe Thr
785 790 795 800 Leu Tyr Val Gly Asp Asn Trp Asn Lys Cys Asp Met Val
Ala Ile Phe 805 810 815 Leu Phe Ile Val Gly Val Thr Cys Arg Met Leu
Pro Ser Ala Asn Glu 820 825 830 Ala Gly Arg Thr Val Leu Ala Met Asp
Phe Met Val Phe Thr Leu Arg 835 840 845 Leu Ile His Ile Asn Ala Ile
His Lys Gln Leu Gly Pro Lys Ile Ile 850 855 860 Val Val Glu Arg Met
Met Lys Asp Val Phe Phe Phe Leu Phe Asn Leu 865 870 875 880 Ser Val
Trp Leu Val Ala Tyr Gly Val Thr Thr Gln Ala Leu Leu His 885 890 895
Pro His Asp Gly Arg Leu Glu Trp Ile Phe Arg Arg Val Leu Tyr Arg 900
905 910 Pro Tyr Leu Gln Ile Phe Gly Gln Ile Pro Leu Asp Glu Ile Asp
Glu 915 920 925 Ala Arg Val Asn Cys Ser Thr His Pro Leu Leu Leu Glu
Asp Ser Pro 930 935 940 Ser Cys Pro Ser Leu Tyr Ala Asn Trp Leu Val
Ile Leu Leu Leu Val 945 950 955 960 Thr Phe Leu Leu Val Thr Asn Val
Leu Leu Met Asn Leu Leu Ile Ala 965 970 975 Met Phe Ser Tyr Thr Phe
Gln Val Val Gln Gly Asn Ala Asp Met Phe 980 985 990 Trp Lys Phe Gln
Arg Tyr Asn Leu Ile Val Glu Tyr His Glu Arg Pro 995 1000 1005 Ala
Leu Ala Pro Pro Phe Ile Leu Leu Ser His Leu Ser Leu Thr Leu 1010
1015 1020 Arg Arg Val Phe Lys Lys Glu Ala Glu His Lys Arg Glu His
Leu Glu 1025 1030 1035 1040 Arg Asp Leu Pro Asp Pro Leu Asp Gln Lys
Val Val Thr Trp Glu Thr 1045 1050 1055 Val Gln Lys Glu Asn Phe Leu
Ser Lys Met Glu Lys Arg Arg Arg Asp 1060 1065 1070 Ser Glu Gly Glu
Val Leu Arg Lys Thr Ala His Arg Val Asp Phe Ile 1075 1080 1085 Ala
Lys Tyr Leu Gly Gly Leu Arg Glu Gln Glu Lys Arg Ile Lys Cys 1090
1095 1100 Leu Glu Ser Gln Ile Asn Tyr Cys Ser Val Leu Val Ser Ser
Val Ala 1105 1110 1115 1120 Asp Val Leu Ala Gln Gly Gly Gly Pro Arg
Ser Ser Gln His Cys Gly 1125 1130 1135 Glu Gly Ser Gln Leu Val Ala
Ala Asp His Arg Gly Gly Leu Asp Gly 1140 1145 1150 Trp Glu Gln Pro
Gly Ala Gly Gln Pro Pro Ser Asp Thr 1155 1160 1165 8 1168 PRT
Artificial Sequence Description of Artificial Sequenceconsensus
betaTRP sequence MOD_RES (3) Xaa = Met, Thr or Asp MOD_RES (4) Xaa
= Ala, Thr or Val MOD_RES (22) Xaa = Val, Ile or Gly MOD_RES (46)
Xaa = Asn, Ser or Gly MOD_RES (156) Xaa = Gln or is absent MOD_RES
(157) Xaa = Lys or is absent MOD_RES (170) Xaa = Ser, Asn or Gly
MOD_RES (171) Xaa = Thr, Ile or Ser MOD_RES (192) Xaa = Thr, Ala or
Pro MOD_RES (271) Xaa = Gly, Ser or Asn MOD_RES (459) Xaa = Val,
Ile or Ala MOD_RES (490) Xaa = Pro or is absent MOD_RES (491) Xaa =
Gly or is absent MOD_RES (492) Xaa = Asp or is absent MOD_RES (493)
Xaa = Arg or is absent MOD_RES (699) Xaa = His, Arg or Leu MOD_RES
(1140) Xaa = Arg, Cys or Glu MOD_RES (1151)..(1152) Xaa = Gly or is
absent MOD_RES (1153) Xaa = Leu or is absent MOD_RES (1154) Xaa =
Asp or is absent MOD_RES (1155) Xaa = Gly or is absent MOD_RES
(1156) Xaa = Trp or is absent 8 Met Gln Xaa Xaa Gln Ser Ser Cys Pro
Gly Ser Pro Pro Asp Thr Glu 1 5 10 15 Asp Gly Trp Glu Pro Xaa Leu
Cys Arg Gly Glu Val Asn Phe Gly Gly 20 25 30 Ser Gly Lys Lys Arg
Gly Lys Phe Val Lys Val Pro Ser Xaa Val Ala 35 40 45 Pro Ser Val
Leu Phe Glu Leu Leu Leu Thr Glu Trp His Leu Pro Ala 50 55 60 Pro
Asn Leu Val Val Ser Leu Val Gly Glu Glu Arg Pro Phe Ala Met 65 70
75 80 Lys Ser Trp Leu Arg Asp Val Leu Arg Lys Gly Leu Val Lys Ala
Ala 85 90 95 Gln Ser Thr Gly Ala Trp Ile Leu Thr Ser Ala Leu His
Val Gly Leu 100 105 110 Ala Arg His Val Gly Gln Ala Val Arg Asp His
Ser Leu Ala Ser Thr 115 120 125 Ser Thr Lys Val Arg Val Val Ala Ile
Gly Met Ala Ser Leu Asp Arg 130 135 140 Ile Leu His Arg Gln Leu Leu
Asp Gly Val Gln Xaa Xaa Glu Asp Thr 145 150 155 160 Pro Ile His Tyr
Pro Ala Asp Glu Gly Xaa Xaa Gln Gly Pro Leu Cys 165 170 175 Pro Leu
Asp Ser Asn Leu Ser His Phe Ile Leu Val Glu Pro Gly Xaa 180 185 190
Leu Gly Ser Gly Asn Asp Gly Leu Thr Glu Leu Gln Leu Ser Leu Glu 195
200 205 Lys His Ile Ser Gln Gln Arg Thr Gly Tyr Gly Gly Thr Ser Ser
Ile 210 215 220 Gln Ile Pro Val Leu Cys Leu Leu Val Asn Gly Asp Pro
Asn Thr Leu 225 230 235 240 Glu Arg Ile Ser Arg Ala Val Glu Gln Ala
Ala Pro Trp Leu Ile Leu 245 250 255 Ala Gly Ser Gly Gly Ile Ala Asp
Val Leu Ala Ala Leu Val Xaa Gln 260 265 270 Pro His Leu Leu Val Pro
Gln Val Ala Glu Lys Gln Phe Arg Glu Lys 275 280 285 Phe Pro Ser Glu
Cys Phe Ser Trp Glu Ala Ile Val His Trp Thr Glu 290 295 300 Leu Leu
Gln Asn Ile Ala Ala His Pro His Leu Leu Thr Val Tyr Asp 305 310 315
320 Phe Glu Gln Glu Gly Ser Glu Asp Leu Asp Thr Val Ile Leu Lys Ala
325 330 335 Leu Val Lys Ala Cys Lys Ser His Ser Gln Glu Ala Gln Asp
Tyr Leu 340 345 350 Asp Glu Leu Lys Leu Ala Val Ala Trp Asp Arg Val
Asp Ile Ala Lys 355 360 365 Ser Glu Ile Phe Asn Gly Asp Val Glu Trp
Lys Ser Cys Asp Leu Glu 370 375 380 Glu Val Met Thr Asp Ala Leu Val
Ser Asn Lys Pro Asp Phe Val Arg 385 390 395 400 Leu Phe Val Asp Ser
Gly Ala Asp Met Ala Glu Phe Leu Thr Tyr Gly 405 410 415 Arg Leu Gln
Gln Leu Tyr His Ser Val Ser Pro Lys Ser Leu Leu Phe 420 425 430 Glu
Leu Leu Gln Arg Lys His Glu Glu Gly Arg Leu Thr Leu Ala Gly 435 440
445 Leu Gly Ala Gln Gln Ala Arg Glu Leu Pro Xaa Gly Leu Pro Ala Phe
450 455 460 Ser Leu His Glu Val Ser Arg Val Leu Lys Asp Phe Leu His
Asp Ala 465 470 475 480 Cys Arg Gly Phe Tyr Gln Asp Gly Arg Xaa Xaa
Xaa Xaa Arg Met Glu 485 490 495 Glu Arg Gly Pro Pro Lys Arg Pro Ala
Gly Gln Lys Trp Leu Pro Asp 500 505 510 Leu Ser Arg Lys Ser Glu Asp
Pro Trp Arg Asp Leu Phe Leu Trp Ala 515 520 525 Val Leu Gln Asn Arg
Tyr Glu Met Ala Thr Tyr Phe Trp Ala Met Gly 530 535 540 Arg Glu
Gly Val Ala Ala Ala Leu Ala Ala Cys Lys Ile Ile Lys Glu 545 550 555
560 Met Ser His Leu Glu Lys Glu Ala Glu Val Ala Arg Thr Met Arg Glu
565 570 575 Ala Lys Tyr Glu Gln Leu Ala Leu Asp Leu Phe Ser Glu Cys
Tyr Ser 580 585 590 Asn Ser Glu Asp Arg Ala Phe Ala Leu Leu Val Arg
Arg Asn His Ser 595 600 605 Trp Ser Arg Thr Thr Cys Leu His Leu Ala
Thr Glu Ala Asp Ala Lys 610 615 620 Ala Phe Phe Ala His Asp Gly Val
Gln Ala Phe Leu Thr Lys Ile Trp 625 630 635 640 Trp Gly Asp Met Ala
Thr Gly Thr Pro Ile Leu Arg Leu Leu Gly Ala 645 650 655 Phe Thr Cys
Pro Ala Leu Ile Tyr Thr Asn Leu Ile Ser Phe Ser Glu 660 665 670 Asp
Ala Pro Gln Arg Met Asp Leu Glu Asp Leu Gln Glu Pro Asp Ser 675 680
685 Leu Asp Met Glu Lys Ser Phe Leu Cys Ser Xaa Gly Gly Gln Leu Glu
690 695 700 Lys Leu Thr Glu Ala Pro Arg Ala Pro Gly Asp Leu Gly Pro
Gln Ala 705 710 715 720 Ala Phe Leu Leu Thr Arg Trp Arg Lys Phe Trp
Gly Ala Pro Val Thr 725 730 735 Val Phe Leu Gly Asn Val Val Met Tyr
Phe Ala Phe Leu Phe Leu Phe 740 745 750 Thr Tyr Val Leu Leu Val Asp
Phe Arg Pro Pro Pro Gln Gly Pro Ser 755 760 765 Gly Ser Glu Val Thr
Leu Tyr Phe Trp Val Phe Thr Leu Val Leu Glu 770 775 780 Glu Ile Arg
Gln Gly Phe Phe Thr Asp Glu Asp Thr His Leu Val Lys 785 790 795 800
Lys Phe Thr Leu Tyr Val Glu Asp Asn Trp Asn Lys Cys Asp Met Val 805
810 815 Ala Ile Phe Leu Phe Ile Val Gly Val Thr Cys Arg Met Val Pro
Ser 820 825 830 Val Phe Glu Ala Gly Arg Thr Val Leu Ala Ile Asp Phe
Met Val Phe 835 840 845 Thr Leu Arg Leu Ile His Ile Phe Ala Ile His
Lys Gln Leu Gly Pro 850 855 860 Lys Ile Ile Ile Val Glu Arg Met Met
Lys Asp Val Phe Phe Phe Leu 865 870 875 880 Phe Phe Leu Ser Val Trp
Leu Val Ala Tyr Gly Val Thr Thr Gln Ala 885 890 895 Leu Leu His Pro
His Asp Gly Arg Leu Glu Trp Ile Phe Arg Arg Val 900 905 910 Leu Tyr
Arg Pro Tyr Leu Gln Ile Phe Gly Gln Ile Pro Leu Asp Glu 915 920 925
Ile Asp Glu Ala Arg Val Asn Cys Ser Leu His Pro Leu Leu Leu Glu 930
935 940 Ser Ser Ala Ser Cys Pro Asn Leu Tyr Ala Asn Trp Leu Val Ile
Leu 945 950 955 960 Leu Leu Val Thr Phe Leu Leu Val Thr Asn Val Leu
Leu Met Asn Leu 965 970 975 Leu Ile Ala Met Phe Ser Tyr Thr Phe Gln
Val Val Gln Gly Asn Ala 980 985 990 Asp Met Phe Trp Lys Phe Gln Arg
Tyr His Leu Ile Val Glu Tyr His 995 1000 1005 Gly Arg Pro Ala Leu
Ala Pro Pro Phe Ile Leu Leu Ser His Leu Ser 1010 1015 1020 Leu Val
Leu Lys Gln Val Phe Arg Lys Glu Ala Gln His Lys Arg Gln 1025 1030
1035 1040 His Leu Glu Arg Asp Leu Pro Asp Pro Leu Asp Gln Lys Ile
Ile Thr 1045 1050 1055 Trp Glu Thr Val Gln Lys Glu Asn Phe Leu Ser
Thr Met Glu Lys Arg 1060 1065 1070 Arg Arg Asp Ser Glu Gly Glu Val
Leu Arg Lys Thr Ala His Arg Val 1075 1080 1085 Asp Leu Ile Ala Lys
Tyr Ile Gly Gly Leu Arg Glu Gln Glu Lys Arg 1090 1095 1100 Ile Lys
Cys Leu Glu Ser Gln Ala Asn Tyr Cys Met Leu Leu Leu Ser 1105 1110
1115 1120 Ser Met Thr Asp Thr Leu Ala Pro Gly Gly Thr Tyr Ser Ser
Ser Gln 1125 1130 1135 Asn Cys Gly Xaa Arg Ser Gln Pro Ala Ser Ala
Arg Asp Arg Xaa Xaa 1140 1145 1150 Xaa Xaa Xaa Xaa Glu Tyr Leu Glu
Ala Gly Leu Pro Pro Ser Asp Thr 1155 1160 1165 9 200 PRT Artificial
Sequence Description of Artificial Sequencepoly-Gly flexible linker
MOD_RES (6)..(200) Gly residues from position 6 to 200 may be
present or absent 9 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly 1 5 10 15 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly 20 25 30 Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly 35 40 45 Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 50 55 60 Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 65 70 75 80 Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 85 90 95
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 100
105 110 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly 115 120 125 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly 130 135 140 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly 145 150 155 160 Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly 165 170 175 Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 180 185 190 Gly Gly Gly Gly
Gly Gly Gly Gly 195 200
* * * * *
References