U.S. patent application number 10/303204 was filed with the patent office on 2003-09-04 for growth hormone secretagogue receptor family.
This patent application is currently assigned to Merck & Co., Inc.. Invention is credited to Arena, Joseph P., Cully, Doris F., Feighner, Scott D., Howard, Andrew D., Liberator, Paul A., Schaeffer, James M., Van Der Ploeg, Leonardus H. T..
Application Number | 20030166144 10/303204 |
Document ID | / |
Family ID | 22139436 |
Filed Date | 2003-09-04 |
United States Patent
Application |
20030166144 |
Kind Code |
A1 |
Arena, Joseph P. ; et
al. |
September 4, 2003 |
Growth hormone secretagogue receptor family
Abstract
Human, swine and rat growth hormone secretagogue receptors have
been isolated, cloned and sequenced. Growth hormone secretagogue
receptors are new members of the G-protein family of receptors. The
growth hormone secretagogue receptors may be used to screen and
identify compounds which bind to the growth hormone secretagogue
receptor. Such compounds may be used in the treatment of conditions
which occur when there is a shortage of growth hormone, such as
observed in growth hormone deficient children, elderly patients
with musculoskeletal impairment and recovering from hip fracture
and osteoporosis.
Inventors: |
Arena, Joseph P.;
(Eagleville, PA) ; Cully, Doris F.; (Scotch
Plains, NJ) ; Feighner, Scott D.; (Highlands, NJ)
; Howard, Andrew D.; (Park Ridge, NJ) ; Liberator,
Paul A.; (Holmdel, NJ) ; Schaeffer, James M.;
(Westfield, NJ) ; Van Der Ploeg, Leonardus H. T.;
(Scotch Plains, NJ) |
Correspondence
Address: |
MERCK AND CO INC
P O BOX 2000
RAHWAY
NJ
070650907
|
Assignee: |
Merck & Co., Inc.
Rahway
NJ
07065
|
Family ID: |
22139436 |
Appl. No.: |
10/303204 |
Filed: |
November 25, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10303204 |
Nov 25, 2002 |
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09077674 |
Jun 3, 1998 |
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6531314 |
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09077674 |
Jun 3, 1998 |
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PCT/US96/19445 |
Dec 10, 1996 |
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60008582 |
Dec 13, 1995 |
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60018962 |
Jun 6, 1996 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/353; 530/350; 536/23.5 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/723 20130101; C07B 59/002 20130101 |
Class at
Publication: |
435/69.1 ;
435/320.1; 435/353; 530/350; 536/23.5 |
International
Class: |
C12P 021/02; C07K
014/72; C07H 021/04; C12N 005/06 |
Claims
What is claimed is:
1. A receptor which is a member of the growth hormone family of
receptors, free-from receptor-associated proteins.
2. Growth hormone secretagogue receptor, free from
receptor-associated proteins.
3. A growth hormone secretagogue receptor according to claim 2
which is human.
4. A growth hormone secretagogue receptor according to claim 2
which is from swine.
5. A growth hormone secretagogue receptor according to claim 2
which is from rat.
6. Growth hormone secretagogue related receptor, free from
receptor-associated proteins.
7. Isolated growth hormone secretagogue receptor.
8. A growth hormone secretagogue receptor according to claim 7
which is human.
9. A growth hormone secretagogue receptor according to claim 7
which is from swine.
10. A growth hormone secretagogue receptor according to claim 7
which is from rat.
11. A receptor according to claim 4 or 9 which comprises a full
length receptor or which comprises the amino acid sequence as shown
in any one of FIGS. 3 or 5.
12. A receptor according to claim 3 or 8 which comprises the amino
acid sequence as shown in any one of FIGS. 7, 8, 10 or 22.
13. A receptor according to claim 5 or 10 which comprises the amino
acid sequence shown in FIG. 25.
14. A functional equivalent of a receptor of claim 1.
15. A functional equivalent of a receptor of claim 2.
16. A functional equivalent of a receptor of claim 6.
17. A nucleic acid which encodes a receptor that is a member of the
growth hormone secretagogue family of receptors, said nucleic acid
being free from associated nucleic acids.
18. A nucleic acid which encodes a growth hormone secretagogue
receptor or a functional equivalent, said nucleic acid being free
from associated nucleic acids.
19. A nucleic acid according to claim 18 which encodes human growth
hormone secretagogue receptor, or a functional equivalent.
20. A nucleic acid according to Claim 18 which encodes swine growth
hormone secretagogue receptor, or a functional equivalent.
21. A nucleic acid according to claim 18 which encodes rat growth
hormone secretagogue receptor, or a functional equivalent.
22. A nucleic acid according to claim 17 which encodes a growth
hormone secretagogue related to receptor.
23. A nucleic acid according to claim 18 which is a DNA.
24. A nucleic acid according to claim 23 which is shown in any one
of FIGS. 1 or 4.
25. A nucleic acid according to claim 23 which is shown in any-one
of FIGS. 6, 9 or 11.
26. A nucleic acid according to claim 23 which is shown in any one
of FIGS. 23 or 24.
27. A nucleic acid according to claim 18 which is an RNA.
28. A vector comprising a nucleic acid which encodes a receptor
which is a member of the growth hormone secretagogue family of
receptors.
29. A vector comprising a nucleic acid which encodes a growth
hormone secretagogue receptor, or a functional equivalent.
30. A vector according to claim 29 which is selected from the group
consisting of: plasmids, modified viruses, yeast artificial
chromosomes, bacteriophages, cosmids and transposable elements.
31. A vector according to claim 29 wherein the nucleic acid encodes
human growth hormone secretagogue receptor or a functional
equivalent.
32. A vector according to claim 29 wherein the nucleic acid encodes
swine growth hormone secretagogue receptor, or a functional
equivalent.
33. A vector according to claim 29 wherein the nucleic acid encodes
rat growth hormone secretagogue receptor, or a functional
equivalent.
34. A vector according to claim 28 wherein the nucleic acid encodes
a growth hormone secretagogue related receptor.
35. A host cell comprising a vector according to claim 28.
36. A host cell comprising a vector according to claim 28.
37. A host cell according to claim 36 wherein the nucleic acid
encodes human growth hormone secretagogue receptor, or a functional
equivalent.
38. A host cell according to claim 36 wherein the nucleic acid
encodes swine growth hormone secretagogue receptor, or a functional
equivalent.
39. A host cell according to claim 36 wherein the nucleic acid
encodes rat growth hormone secretagogue receptor, or a functional
equivalent.
40. A nucleic acid encoding a GPCR clone that belongs to the GHSR
family and that hybridizes with a nucleotide which encodes either
human, swine or rat GHSR under reduced stringency of hybridization.
Description
FIELD OF THE INVENTION
[0001] This invention relates to a new family of receptors, which
includes the growth hormone secretagogue receptors (GHSRs) and
growth hormone secretagogue-related receptors (GHSRRs), nucleic
acids encoding these receptors; and to the use of a GHSR to
identify growth hormone secretagogues and compounds that modulate
GHSR function.
BACKGROUND OF THE INVENTION
[0002] Growth hormone (GH) is an anabolic hormone capable of
promoting linear growth, weight gain and whole body nitrogen
retention. Classically, GH is thought to be released primarily from
the somatotroph cells of the anterior pituitary under the
coordinate regulation of two hypothalamic hormones, growth hormone
releasing factor (GHRF or GRF) and somatostatin. Both GHRF
stimulation and somatostatin inhibition of the release of GH occurs
by the specific engagement of receptors on the cell membrane of the
somatotroph.
[0003] Recent evidence has been mounting which suggests that GH
release is also stimulated by a group of short peptides, the growth
hormone releasing peptides (GHRP; GHRP-6, GHRP-2 [hexarelin]) which
are described, for example, in U.S. Pat. No. 4,411,890, PCT Patent
Pub. No. WO 89/07110, PCT Patent Pub. No. WO 89/07111, PCT Patent
Pub. No. WO 93/04081, and J. Endocrinol Invest., 15 (Suppl 4), 45
(1992). These peptides function by selectively binding to distinct
somatotroph cell membrane receptor, the growth hormone secretagogue
receptor(s) (GHSRs). A medicinal chemical approach has resulted in
the design of several classes of orally-active, low molecular
weight, non-peptidyl compounds which bind specifically to this
receptor and result in the pulsatile release of GH. Such compounds
possessing growth hormone secretagogue activity are disclosed, for
example, in the following: U.S. Pat. Nos. 3,239,345; 4,036,979;
4,411,890; 5,206,235; 5,283,241; 5,284,841; 5,310,737; 5,317,017;
5,374,721; 5,430,144; 5,434,261; 5,438,136; 5,494,919; 5,494,920;
5,492,916; EPO Patent Pub. No. 0,144,230; EPO Patent Pub. No.
0,513,974; PCT Patent Pub. No. WO 94/07486; PCT Patent Pub. No. WO
94/08583; PCT Patent Pub. No. WO 94/11012; PCT Patent Pub. No. WO
94/13696; PCT Patent Pub. No. WO 94/19367; PCT Patent Pub. No. WO
95/03289; PCT Patent Pub. No. WO 95/03290; PCT Patent Pub. No. WO
95/09633; PCT Patent Pub. No. WO 95/11029; PCT Patent Pub. No. WO
95/12598; PCT Patent Pub. No. WO 95/13069; PCT Patent Pub. No. WO
95/14666; PCT Patent Pub. No. WO 95/16675; PCT Patent Pub. No. WO
95/16692; PCT Patent Pub. No. WO 95/17422; PCT Patent Pub. No. WO
95/17423; PCT Patent Pub. No. WO 95/34311; PCT Patent Pub. No. WO
96/02530; Science, 260, 1640-1643 (Jun. 11, 1993); Ann. Rep. Med.
Chem., 28, 177-186 (1993); Bioorg. Med. Chem. Ltrs., 4(22),
2709-2714 (1994); and Proc. Natl. Acad. Sci. USA 92, 7001-7005
(July 1995).
[0004] The use of orally-active agents which stimulate the
pulsatile release of GH would be a significant advance in the
treatment of growth hormone deficiency in children and adults as
well as provide substantial benefit under circumstances where the
anabolic effects of GH might be exploited clinically (e.g. post-hip
fracture rehabilitation, the frail elderly and in post-operative
recovery patients).
[0005] It would also be desirable to know the molecular structure
of growth hormone secretagogue receptors in order to analyze this
new receptor family and understand its normal physiological role in
concert with the actions of GHRF and somatostatin. This could lead
to a better understanding of the in vivo processes which occur upon
ligand-receptor binding. Further, it would be desirable to use
cloned-growth hormone secretagogue receptors as essential
components of an assay system which can identify new growth hormone
secretagogues.
DETAILED DESCRIPTION OF THE INVENTION
[0006] This invention relates to a novel family of receptors which
includes growth hormone secretagogue receptors (GHSRs) and growth
hormone secretagogue-related receptors (GHSRRs).
[0007] A first aspect of this invention are the growth hormone
secretagogue receptors, which are free from receptor associated
proteins. GHSRs may be from any species, and in further embodiments
may be isolated or purified. One embodiment of this invention is
human growth hormone secretagogue receptor (hGHSR), free from
receptor-associated proteins. A further aspect of this invention is
hGHSR which is isolated or purified.
[0008] Another aspect of this invention is swine growth hormone
secretagogue receptor (sGHSR), free from receptor-associated
proteins. A further aspect of this invention is sGHSR which is
isolated or purified.
[0009] Another aspect of this invention is rat growth hormone
secretagogue receptor (rGHSR), free from receptor-associated
proteins. A further aspect of this invention is RGHSR which is
isolated or purified.
[0010] Another aspect of this invention are human, swine and rat
GHSRs which are encoded by substantially the same nucleic acid
sequences, but which have undergone changes in splicing or other
RNA processing-derived modifications or mutagenesis induced
changes, so that the expressed protein has a homologous, but
different amino acid sequence from the native forms. These variant
forms may have different and/or additional functions in human and
animal physiology or in vitro in cell based assays.
[0011] Another aspect of this invention are the growth hormone
secretagogue-related receptors, free from associated receptor
proteins. A further embodiment are GHSRRs which are isolated or
purified. These may be from any species, including human, mouse,
rat and swine.
[0012] Growth hormone secretagogue receptors are proteins
containing various functional domains, including one or more
domains which anchor the receptor in the cell membrane, and at
least one ligand binding domain. As with many receptor proteins, it
is possible to modify many of the amino acids, particularly those
which are not found in the ligand binding domain, and still retain
at least a percentage of the biological activity of the original
receptor. In accordance with this invention, it has been shown that
the N-terminal portions of the GHSR are not essential for its
activation by the Growth Hormone Secretagogues (GHSs). Thus this
invention specifically includes modified functionally equivalent
GHSRs which have deleted, truncated, or mutated N-terminal
portions. This invention also specifically includes modified
functionally equivalent GHSRs which contain modified and/or
deletions in other domains, which are not accompanied by a loss of
functional activity.
[0013] Additionally, it is possible to modify other functional
domains such as those that interact with second messenger effector
systems, by altering binding specificity and/or selectivity. Such
functionally equivalent mutant receptors are also within the scope
of this invention.
[0014] A further aspect of this invention are nucleic acids which
encode a growth hormone secretagogue receptor or a functional
equivalent from swine, human, rat or other species. These nucleic
acids may be free from associated nucleic acids, or they may be
isolated or purified. For most cloning purposes, cDNA is a
preferred nucleic acid, but this invention specifically includes
other forms of DNA as well as RNAs which encode a GHSR or a
functional equivalent.
[0015] Yet another aspect of this invention relates to vectors
which comprise nucleic acids encoding a GHSR or a functional
equivalent. These vectors may be comprised of DNA or RNA; for most
cloning purposes DNA vectors are preferred. Typical vectors include
plasmids, modified viruses, bacteriophage and cosmids, yeast
artificial chromosomes and other forms of episomal or integrated
DNA that can encode a GHSR. It is well within the skill of the
ordinary artisan to determine an appropriate vector for a
particular gene transfer or other use.
[0016] A further aspect of this invention are host cells which are
transformed with a gene which encodes a growth hormone secretagogue
receptor or a functional equivalent. The host cell may or may not
naturally express a GHSR on the cell membrane. Preferably, once
transformed, the host cells are able to express the growth hormone
secretagogue receptor or a functional equivalent on the cell
membrane. Depending on the host cell, it may be desirable to adapt
the DNA so that particular codons are used in order to optimize
expression. Such adaptations are known in the art, and these
nucleic acids are also included within the scope of this invention.
Generally, mammalian cell lines, such as COS, HEK-293, CHO, HeLa,
NS/0, CV-1, GC, GH3 or VERO cells are preferred host cells, but
other cells and cell lines such as Xenopus oocytes or insect cells,
may also be used.
[0017] Growth hormone secretagogue related receptors are related to
GHRS, but are encoded by a distinct gene. The GHRR genes may be
identified by hybridization (using relaxed or moderate stringency
post-hybridizational washing conditions) of cDNA of GHR DNA to
genonic DNA. These sequences have a high degree of similarity to
GHR.
[0018] Another aspect of this invention is a process for
identifying nucleic acids encoding growth hormone secretagogue
related receptors comprising hybridizing a first nucleic acid
encoding a growth hormone secretagogue receptor with a second
nucleic acid suspected of comprising nucleic acids encoding a
growth hormone secretagogue, wherein the hybridizing takes place
under relaxed or moderate post hybridizational washing conditions;
and identify areas of the second nucleic acid where hybridization
occurred.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 1 is the DNA of Swine GHSR (type I) contained in Clone
7-3.
[0020] FIG. 2 is the amino acid sequence of swine GHSR encoded by
the DNA of FIG. 1.
[0021] FIG. 3 is the entire open reading frame of the type I clone
of FIG. 1.
[0022] FIG. 4 is the DNA of Swine GHSR (type II) contained in Clone
1375.
[0023] FIG. 5 is the amino acid sequence of swine GHSR (type II)
encoded by the DNA of FIG. 4.
[0024] FIG. 6 is the DNA for human GHSR (Type I) contained in Clone
1146.
[0025] FIG. 7 is the amino acid sequence of human GHSR (type 1)
encoded by the DNA of FIG. 6.
[0026] FIG. 8 is the entire open reading frame of Type I GHSR,
encoded by the DNA sequence of FIG. 6.
[0027] FIG. 9 is the DNA for human GHSR (type II) contained in
Clone 1141.
[0028] FIG. 10 is the amino acid sequence of human GHSR (Type II)
encoded by Clone 1141.
[0029] FIG. 11 is the DNA for human GHSR (Type I) contained in
Clone 1143.
[0030] FIG. 12 is the amino acid sequence of human GHSR (Type I)
encoded by Clone 1143.
[0031] FIG. 13 compares the ORF of swine Type I (lacking the MET
initiator of the full length GHSR and lacking 12 additional amino
acids) to the homologous domain of swine Type II receptors.
[0032] FIG. 14 compares the homologous domain of human Type I and
Type II receptors (the amino terminal sequence lacks the MET
initiator and four additional amino acids).
[0033] FIG. 15 compares the ORFs of swine Type I and human Type I
receptors (the amino terminal sequence lacks the MET initiator and
12 additional amino acids).
[0034] FIG. 16 compares full length swine Type II and human Type II
receptors.
[0035] FIG. 17 is a schematic diagram depicting the physical map of
swine and human growth hormone secretagogue receptor cDNA
clones.
[0036] FIG. 18 is a graph demonstrating the pharmacology of the
expressed swine and human growth hormone secretagogue receptors in
Xenopus oocytes using the aequorin bioluminescence assay.
[0037] FIG. 19 is a table demonstrating the pharmacology of the
expressed swine and human growth hormone secretagogue receptors in
Xenopus oocytes using the aequorin bioluminescence assay and
various secretagogues.
[0038] FIG. 20 is a graph representing the pharmacology of the pure
expressed swine growth hormone secretagogue receptor in COS-7 cells
using the .sup.35S-labeled Compound A binding assay.
[0039] FIG. 21 is a table representing the competition analysis
with the pure expressed swine growth hormone secretagogue receptor
in COS-7 cells using the .sup.35S-labeled Compound A binding assay
and various secretagogues and other G-protein coupled-receptors
(GPC-Receptors) ligands in a competition assay.
[0040] FIG. 22 is the amino acid sequence of the full length human
GHSR (type I) encoded by clone 11304.
[0041] FIG. 23 is the rat GHSR DNA sequence from the Met Initiation
codon to the Stop codon. This sequence includes an intron.
[0042] FIG. 24 is the open reading frame only of the rat GHSR of
FIG. 23.
[0043] FIG. 25 is the deduced amino acid sequence of the ORF of
FIG. 24.
[0044] FIG. 26 shows the expression of functional rat GHSR in
transfected HEK-293 cells.
[0045] As used throughout the specification and claims, the
following definitions shall apply:
[0046] Growth Hormone Secretagogue--any compound or agent that
directly or indirectly stimulates or increases the release of
growth hormone in an animal.
[0047] Ligands--any molecule which binds to GHSR of this invention.
These ligands can have either agonist, partial agonist, partial
antagonist or antagonist activity.
[0048] Free from receptor-associated proteins--the receptor protein
is not in a mixture or solution with other membrane receptor
proteins.
[0049] Free from associated nucleic acids--the nucleic acid is not
covalently linked to DNA which it is naturally covalently linked in
the organism's chromosome.
[0050] Isolated receptor--the protein is not in a mixture or
solution with any other proteins.
[0051] Isolated nucleic acid--the nucleic acid is not in a mixture
or solution with any other nucleic acid.
[0052] Functional equivalent--a receptor which does not have the
exact same amino acid sequence of a naturally occurring growth
hormone secretagogue receptor, due to alternative splicing,
deletions, mutations, or additions, but retains at least 1%,
preferably 10%, and more preferably 25% of the biological activity
of the naturally occurring receptor. Such derivatives will have a
significant homology with a natural GHSR and can be detected by
reduced stringency hybridization with a DNA sequence obtained from
a GHSR. The nucleic acid encoding a functional equivalent has at
least about 50% homology at the nucleotide level to a naturally
occurring receptor nucleic acid.
[0053] Purified receptor--the receptor is at least about 95%
pure.
[0054] Purified nucleic acid--the nucleic acid is at least about
95% pure.
[0055] Compound
A--(N-[1(R)-[(1,2-dihydro-1-methane-sulfonylspiro[3H-indol-
e-3,4'-piperidin]-1'-yl)carbonyl]-2-(phenyl-methyloxy)ethyl]-2-amino-2-met-
hyl propanamide, described in Patchett, 1995 Proc. Natl. Acad. Sci.
92:7001-7005.
[0056] Compound
B--3-amino-3-methyl-N-(2,3,4,5-tetra-hydro-2-oxo-1-{[2'-1H-
-tetrazol-5-yl)(1,1'-biphenyl)-4-yl]methyl}-1H-benzazepin-3(R)yl-butanamid-
e, described in Patchett, 1995 Proc. Natl. Acad. Sci.
92:7001-7005.
[0057] Compound
C--3-amino-3-methyl-N-(2,3,4,5-tetrahydro-2-oxo-1{[2'-1H-t-
etrazol-5-yl)(1,1'-biphenyl)-4-yl]methyl}-1H-benzazepin-3(S)yl-butanamide,
described in U.S. Pat. No. 5,206,235.
[0058] Standard or high stringency post hybridizational washing
conditions--6.times.SSC at 55.degree. C.
[0059] Moderate post hybridizational washing
conditions--6.times.SSC at 45.degree. C.
[0060] Relaxed post hybridizational washing conditions--6.times.SSC
at 30.degree. C.
[0061] The proteins of this invention were found to have structural
features which are typical of the 7-transmembrane domain (TM)
containing G-protein linked receptor superfamily (GPC-R's or 7-TM
receptors). Thus growth hormone secretagogue family of receptors
make up new members of the GPC-R family of receptors. The intact
GHSRs of this invention were found to have the general features of
GPC-R's, including seven transmembrane regions, three intra- and
extracellular loops, and the GPC-R protein signature sequence. The
TM domains and GPC-R protein signature sequence are noted in the
protein sequences of the Type I GHS receptor in FIGS. 3 and 8. Not
all regions are required for functioning, and therefore this
invention also comprises functional receptors which lack one or
more non-essential domains.
[0062] The GHSRs of this invention share some sequence homology
with previously cloned GPC-receptors including the rat and human
neurotensin receptor (approximately 32% identity) and the rat and
human TRH receptor (approximately 30% identity).
[0063] The GHSRs of this invention were isolated and characterized
using expression cloning techniques in Xenopus oocytes. The cloning
was made difficult by three factors. First, prior to this
invention, there was very little information available about the
biochemical characteristics and intracellular signaling/effector
pathways of the proteins. Thus, cloning approaches which depended
on the use of protein sequence information for the design of
degenerate oligonucleotides to screen cDNA libraries or utilize PCR
could not be effectively utilized. In accordance with this
invention, therefore, receptor bioactivity needed to be
determined.
[0064] Secondly, the growth hormone secretagogue receptor does not
occur in abundance--it is present on the cell membrane in about 10
fold less concentration than most other membrane receptors. In
order to successfully clone the receptors in accordance with this
invention, exhaustive precautions had be taken to ensure that the
GHSR was represented in a cDNA library to be screened. This
required isolation of intact, undegraded and pure poly (A)+ mRNA,
and optimization of cDNA synthesis to maximize the production of
full-length molecules. In addition, a library of larger size than
normal needed to be screened (approximately 0.5 to 1.times.10.sup.7
clones) to increase the probability that a functional cDNA clone
may be obtained.
[0065] Thirdly, no permanent cell line which expresses this
receptor is known. Therefore, primary pituitary tissue had to be
used as a source for mRNA or protein. This posed an additional
obstacle because most primary tissues express lower amounts of a
given receptor than an immortalized cell line that may be
maintained in tissue culture or some tumor materials. Further, the
surgical removal of a pig pituitary and extraction of
biologically-active intact mRNA for the construction of a cDNA
expression library is considerably more difficult than the
extraction of mRNA from a tissue culture cell line. Along with the
need to obtain fresh tissue continuously, there are problems
associated with its intrinsic inter-animal and inter-preparation
variability. The development of cell lines expressing a receptor of
this invention is therefore a significant aspect of this
invention.
[0066] Yet another aspect of this invention is the development of
an extremely sensitive, robust, reliable and high-throughput
screening assay which could be used to identify portions of a cDNA
library containing the receptor. This assay is described and
claimed in copending patent applications Ser. No. 60/008,584, filed
Dec. 13, 1995, and Attorney Docket No. 19590PV2 filed herewith.
[0067] Briefly, the ability to identify cDNAs which encode growth
hormone secretagogue receptors depended upon two discoveries made
in accordance with this invention: 1) that growth hormone
secretagogue receptor-ligand binding events are transduced through
G proteins; and 2) that a particular G protein subunit, G.sub.a11,
must be present in the cells
[0068] in order to detect receptor activity. Only when these two
discoveries were made could an assay be devised to detect the
presence of GHSR-encoding DNA sequences.
[0069] When the GHSR is bound by ligand (a growth hormone
secretagogue), the G-proteins present in the cell activate
phosphatidylinositol-specific phospholipase C (PI-PLC), an enzyme
which releases intracellular signaling molecules (diacylglycerol
and inositol triphosphate), which in turn start a cascade of
biochemical events that promote calcium mobilization. This can be
used as the basis of an assay. A detector molecule which can
respond to changes in calcium concentrations, such as aequorin, a
jellyfish photoprotein, is introduced into a cell along with a
complex pool of up to 10,000 individual RNAs from a cDNA expression
library, at least one of which may encode a GHSR. The cell is then
exposed to a known growth hormone secretagogue, such as Compound A
or Compound B. If one or more RNAs encodes a GHSR, then the
secretagogue ligand will bind the receptor, G-protein will be
activated, the calcium level will fluctuate, and the aequorin will
produce measurable bioluminescence. Once a positive result is
found, the procedure can be repeated with a sub-division of the RNA
pool (for example, approximately 1,000, then approximately 500,
then approximately 50, and then pure clones) until a single clone
is identified from which RNA can be generated which encodes a
GHSR.
[0070] Using this general protocol in Xenopus oocytes with a swine
cDNA expression library, Clone 7-3 was identified as containing
nucleic acid encoding a swine GHSR. The insert of the cDNA clone is
approximately 1.5 kb in size, and downstream from the presumed
initiator methionine (MET), contains an open reading frame (ORF)
encoding 302 amino acids (M.sub.r=34,516). The DNA and deduced
amino acid sequence are given in FIGS. 1 and 2. When hydropathy
analysis (e.g. Kyte-Doolittle; Eisenberg, Schwartz, Komaron and
Wall) is performed on the protein sequence of clone 7-3, only 6
predicted transmembrane domains are present downstream of the
presumed MET initiator. Translation of the longest ORF encoded in
clone 7-3 encodes a protein of 353 amino acids (M.sub.r=39,787);
however an apparent MET initiator cannot be identified for this
longer reading frame (FIG. 3). This longer reading frame is
significant since 7 transmembrane segments are encoded in the 353
amino acids protein in which a MET translation initiation codon
located upstream of TM1 is absent. In addition, this longer protein
also shares homology with known G-protein coupled receptors in its
predicted TM1 domain (FIG. 3 and next sections). Thus, clone 7-3
while truncated at its amino terminus, is fully functional,
demonstrating that clone 7-3 is but one embodiment of a functional
equivalent of a native GHSR.
[0071] The resultant cDNA clone (or shorter portions of, for
instance only 15 nucleotides long) may be used to probe libraries
under hybridization conditions to find other receptors which are
similar enough so that the nucleic acids can hybridize, and is
particularly useful for screening libraries from other species.
Using this procedure, additional human, swine, and rat GHSR cDNAs
have been cloned and their nucleotide sequences determined.
Further, hybridization of a cDNA to genomic DNA demonstrated that
the Type I receptor (see below) is encoded by a single gene that is
highly conserved. Human, monkey, rat, mouse, dog, cow, chicken and
invertebrate DNA all yielded a single hybridizing species at high
stringency post-hybridization conditions. Therefore, this invention
is not limited to any particular species.
[0072] A swine pituitary library, a human pituitary library, and a
rat pituitary library were hybridized with a radiolabeled cDNA
derived from the open reading frame of the swine GHSR clone 7-3. 21
positive human GHSR cDNA clones were isolated and five swine
library pools yielded a strong hybridization signal and contained
clones with inserts larger than clone 7-3, as judged by their
insert size on Southern blots. A single rat CDNA clone was also
isolated.
[0073] Nucleotide sequence analysis revealed two types of cDNAs for
both the human and swine GHSR cDNAs. The first (Type I) encodes a
protein represented by clone 7-3, encoding seven transmembrane
domains. The full length open reading frame appears to extend 13
amino acids beyond the largest predicted open reading frame of
clone 7-3 (353 amino acids). The second (type II) diverges in its
nucleotide sequence from the type I cDNA at its 3'-end, just after
the predicted second amino acid of the sixth transmembrane domain
(TM-6).
[0074] In the type II cDNAs, TM-6 is truncated and fused to a short
contiguous reading frame of only 24 amino acids, followed by a
translation stop codon. Swine clone 1375 is an example of a Type II
cDNA (FIGS. 4 and 5). These 24 amino acids beyond TM-6 are highly
conserved when compared between human and swine cDNAs. The DNA and
amino acid sequences of the human GHSR Type I and II are given in
FIGS. 6-12. A full length cDNA encoding the human Type I receptor,
that is, a molecule encoding 7-TM domains with an initiator MET in
a favorable context preceded by an inframe termination codon is
isolated, and termed clone 11304. The predicted ORF of clone 11304
for the full length Type I GHSR measures 366 amino acids
(M.sub.r=41,198; FIG. 22). The full length human Type II cDNA
encodes a polypeptide of 289 amino acids (M.sub.r=32,156; FIGS. 9
and 10).
[0075] Sequence alignments performed at both the nucleic acid and
protein levels show that Type I and II GHSR's are highly related to
each other and across species (FIGS. 13-16). The human and swine
GHSR sequences are 93% identical and 98% similar at the amino acid
level.
[0076] The nucleotide sequence encoding the missing amino terminal
extension of swine Type I clone 7-3 is derived from the predicted
full length human Type I clone and the human and swine Type II
cDNAs. The reading frame of the full length clones extended 13
amino acids beyond the amino terminal sequence of clone 7-3 and
this sequence was conserved in 12/13 amino acid residues, when
compared between human and swine. The amino terminal extension
includes a translation initiator methionine in a favorable context
according to Kosak's rule, with the reading frame further upstream
being interrupted by a stop codon. A schematic physical map of Type
I and II swine and human cDNA clones is given in FIG. 17.
[0077] The rat clone was also further investigated. Sequence
analysis revealed the presence of a non-coding intronic sequence at
nt 790 corresponding to a splice-donor site (see FIGS. 23, 24, and
25). The G/GT splice-donor site occurs two amino acids after the
completion of the predicted transmembrane domain 5 (leucine 263),
thus dividing the rGHSR into an amino-terminal segment (containing
the extracellular domain, TM-1 through TM-5, and the first two
intra- and extra-cellular loops) and a carboxy-terminal segment
(containing TM-6, TM-7, the third intra- and extra-cellular loops,
and the intra- cellular domain). The point of insertion and
flanking DNA sequence are highly conserved, and also present in
both human and swine Type I and II cDNAs.
[0078] Comparison of the complete open reading frame encoding the
rat GHSR protein to human and swine homologs reveals a high degree
of sequence identity (rat vs. human, 95.1%; rat vs. swine
93.4%.
[0079] The human GHSR can be assigned by fluorescent in situ
hybridization analysis [FISH; as described in Cytogenet, Cell Genet
69: 196 (1995)] to the cytogenetic band 3Q26.2. The mouse gene is
located on 3A3.
[0080] Human and swine Type I cRNAs expressed in oocytes were
functional and responded to Compound A concentrations ranging from
1 mM to as low as 0.1 nM in the aequorin bioluminescence assay.
Human or swine Type II-derived cRNAs that are truncated in TM-6
failed to give a response when injected into oocytes and these
represent a receptor subtype which may bind the GHS, but cannot
effectively activate the intracellular signal transduction pathway.
In addition the type II receptor may interact with other proteins
and thus reconstitute a functional GHSR. Proteins such as these
which may have ligand-binding activity, but are not active in
signal transduction are particularly useful for ligand-binding
assays. In these cases, one may also over-express a mutant protein
on the cell membrane and test the binding abilities of putative
labeled ligands. By using a non-signaling mutant which is
constitutively in a high affinity state, binding can be measured,
but no adverse metabolic consequences would result. Thus
non-signaling mutants are an important aspect of this
invention.
[0081] The pharmacological characterization of human, Type I swine,
Type I and rat receptors in the aequorin bioluminescence assay in
oocytes is summarized in FIGS. 18, 19, and 26. Peptidyl and
non-peptidyl bioactive GHS's were active in a similar rank order of
potency as observed for the native pituitary receptor. Independent
confirmatory evidence that the Type I GHSR (shown for swine clone
7-3) encodes a fully-functional GHSR is given by the finding that
when clone 7-3 is expressed transiently in mammalian COS-7 cells,
high affinity (K.sub.D.about.0.2 nM), saturable (B.sub.max.about.80
fmol/mg protein) and specific binding (>90% displaced by 50 nM
unlabeled Compound A) is observed for .sup.35.sub.S-Compound A
(FIGS. 20 and 21).
[0082] The GHSR receptors of this invention may be identified by
hybridization of a GHSR cDNA to genomic DNA, under relaxed or
moderate post hybridizational washing conditions. This analysis
yields a discreet number of hybridizing bands. A suitable human
genomic library which can be used in this procedure is PAC (as
described in Nature Genetics 6:84 (1994)) and a suitable mouse
genomic library is BAC (as described in Proc Natl Acad Sci USA 89:
8794 (1992).
[0083] Due to the high degree of homology to GHSRs, the GHSRs of
this invention are believed to function similarly to GHSRs and have
similar biological activity. They are useful in understanding the
biological and physiological pathways involved in an organisms
growth. They may be also used to scan for growth hormone
secretagogue agonists and antagonists; as in particular to test the
specificity of identified ligands.
[0084] Heterotrimeric G proteins, consisting of a, b and g
subunits, serve to relay information from cell surface receptors to
intracellular effectors, such as phospholipase C and adenylate
cyclase. The G-protein alpha subunit is an essential component of
the intracellular signal transduction pathway activated by
receptor-ligand interaction. In the process of ligand-induced GPCR
activation, the Ga subunit of a trimeric Gabg exchanges its bound
GDP for GTP and dissociate from the bg heterodimer. The dissociated
subunit serves as the active signal transducer, often in concert
with the bg complex, thus starting the activation of the
intracellular signal transduction pathway. By definition, cell
surface receptors which couple intracellularly through G protein
interactions are termed GPC-R's. This interaction has mainly been
characterized with respect to the type of G-alpha (G.sub.a) subunit
which is primarily involved in the signal transduction process.
G.sub.a subunits are classified into sub-families based on sequence
identity and the main type of effectors to which they are coupled
have been characterized: G.sub.s, activate adenylate cyclase;
G.sub.i/o/t, inhibit adenylate cyclase; G.sub.q/11, activate
PI-PLC; and G.sub.12/13, effector unknown.
[0085] Expression of several receptors in heterologous cells has
been shown to be increased by the co-expression of certain G.sub.a
subunits. This observation formed the basis for the rationale to
the use of G.sub.a subunits of several sub-families in conjunction
with a source of GHSR (swine poly[A+] mRNA) to test if a
GHS-induced functional response could be measured in the Xenopus
oocyte system. GHS-induced responses were detected and were found
to be strictly dependent on G.sub.a11 co-expression in this system,
an unprecedented finding outlining the specificity of the
interaction. Thus another aspect of this invention is a method of
detecting a GHS response comprising co-expressing a G.sub.a11
protein subunit in a cell also expressing a GHSR, exposing the cell
to a GHS, and detecting the response.
[0086] Ligands detected using assays described herein may be used
in the treatment of conditions which occur when there is a shortage
of growth hormone, such as observed in growth hormone deficient
children, elderly patients with musculoskeletal impairment and
recovering from hip fracture, and osteoporosis.
[0087] The GHSR and fragments are immunogenic. Thus, another aspect
of this invention is antibodies and antibody fragments which can
bind to GHSR or a GHSR fragment. These antibodies may be monoclonal
antibodies and produced using either hybridoma technology or
recombinant methods. They may be used as part of assay systems or
to deduce the function of a GHSR present on a cell membrane.
[0088] A further aspect of this invention are antisense
oligonucleotides nucleotides which can bind to GHSR nucleotides and
modulate receptor function or expression.
[0089] A further aspect of this invention is a method of increasing
the amount of GHSRs on a cell membrane comprising, introducing into
the cell a nucleic acid encoding a GHSR, and allowing expression of
the GHSR.
[0090] A GHS receptor, preferably imobilized on a solid support,
may be used diagnostically for the determination of the
concentration of growth hormone secretagogues, or metabolites
thereof, in physiological fluids, e.g., body fluids, including
serum, and tissue extracts, as for example in patients who are
undergoing therapy with a growth hormone secretagogue.
[0091] The administration of a GHS receptor to a patient may also
be employed for purposes of: amplifying the net effect of a growth
hormone secretagogue by providing increased downstream signal
following administration of the growth hormone secretagogue thereby
diminishing the required dosage of growth hormone secretagogue; or
diminishing the effect of an overdosage of a growth hormone
secretagogue during therapy.
[0092] The following, non-limiting Examples are presented to better
illustrate the invention.
EXAMPLE 1
[0093] Oocyte Preparation and Selection
[0094] Xenopus laevis oocytes were isolated and injected using
standard methods previously described by Arena, et al., 1991, Mol.
Pharmacol. 40, 368-374, which is hereby incorporated by reference.
Adult female Xenopus laevis frogs (purchased from Xenopus One, Ann
Arbor, Mich.) were anesthetized with 0.17% tricaine
methanesulfonate and the ovaries were surgically removed and placed
in a 60 mm culture dish (Falcon) containing OR-2 medium without
calcium (82.5 mM NaCl, 2 mM KCl, 2.5 mM sodium pyruvate, 1 mM
MgCl.sub.2, 100 m/ml penicillin, 1 mg/ml streptomycin, 5 mM HEPES,
pH=7.5; ND-96 from Specialty Media, N.J.). Ovarian lobes were
broken open, rinsed several times, and oocytes were released from
their sacs by collagenase A digestion (Boehringer-Mannheim; 0.2%
for 2-3 hours at 18.degree. C.) in calcium-free OR-2. When
approximately 50% of the follicular layers were removed, Stage V
and VI oocytes were selected and placed in ND-86 with calcium (86
mM NaCl, 2 mM KCl, 1 mM MgCl.sub.2, 1.8 mM CaCl.sub.2, 2.5 mM
sodium pyruvate, 0.5 mM theopylline, 0.1 mM gentamycin, 5 mM HEPES
[pH=7.5]). For each round of injection, typically 3-5 frogs were
pre-tested for their ability to express a control G-protein linked
receptor (human gonadotropin-releasing hormone receptor) and show a
robust phospholipase C intracellular signaling pathway (incubation
with 1% chicken serum which promotes calcium mobilization by
activation of phospholipase C). Based on these results, 1-2 frogs
were chosen for library pool injection (50 nl of cRNA at a
concentration of 25 ng (complex pools) to 0.5 ng (pure clone) per
oocyte usually 24 to 48 hours following oocyte isolation.
EXAMPLE 2
[0095] mRNA Isolation
[0096] Total RNA from swine (50-80 kg, Yorkshire strain)
pituitaries (snap-frozen in liquid nitrogen within 1-2 minutes of
animal sacrifice) was prepared by a modified phenol:guanidinium
thiocyanate procedure (Chomczynski, et al., 1987 Anal. Biochem.
162:156-159, using the TRI-Reagent LS as per the manufacturer's
instructions (Molecular Research Center, Cincinnati, Ohio).
Typically, 5 mg of total RNA was obtained from 3.5 g wet weight of
pituitary tissue. Poly (A).sup.+ RNA was isolated from total RNA by
column chromatography (two passes) on oligo (dT) cellulose
(Pharmacia, Piscataway, N.J.). The yield of poly (A).sup.+ mRNA
from total RNA was usually 0.5%. RNA from other tissues was
isolated similarly.
EXAMPLE 3
[0097] cDNA Library Construction
[0098] First-strand cDNA was synthesized from poly (A).sup.+ mRNA
using M-MLV RNAse (-) reverse transcriptase (Superscript,
GIBCO-BRL, Gaithersberg, Md.) as per the manufacturer's
instructions with an oligo (dT)/Not I primer-adapter. Following
second-strand cDNA synthesis, double-stranded cDNA was subjected to
the following steps: 1) ligation to EcoR I adapters, 2) Not I
digestion, and 3) enrichment for large cDNAs and removal of excess
adapters by gel filtration chromatography on a Sephacryl S-500
column (Pharmacia). Fractions corresponding to high molecular
weight cDNA were ligated to EcoR I/Not I digested pSV-7, a
eucaryotic expression vector capable of expressing cloned cDNA in
mammalian cells by transfection (driven by SV-40 promoter) and in
oocytes using in vitro transcripts (initiated from the T7 RNA
polymerase promoter). pSV-7 was constructed by replacing the
multiple cloning site in pSG-5 (Stratagene, La Jolla, Calif.;
Green, S. et al., 1988 Nucleic Acids Res. 16:369), with an expanded
multiple cloning site. Ligated vector:cDNA was transformed into
E.coli strain DH10B (GIBCO-BRL) by electroporation with a
transformation efficiency of 1.times.10.sup.6 pfu/10 ng
double-stranded cDNA. The library contained approximately
3.times.10.sup.6 independent clones with greater than 95% having
inserts with an average size approximating 1.65 kb (range 0.8-2.8
kb). Unamplified library stocks were frozen in glycerol at
-70.degree. C. until needed. Aliquots of the library were amplified
once prior to screening by a modification of a solid-state method
(Kriegler, M. in Gene Transfer and Expression: A Laboratory Manual
Stockton Press, N.Y. 1990). Library stocks were titered on LB
plates and then the equivalent of 500-1000 colonies was added to 13
ml of 2.times.YT media containing 0.3% agarose and 100 mg/ml
carbenicillin in a 14 ml round-bottom polypropylene tube (Falcon).
The bacterial suspension was chilled in a wet ice bath for 1 hour
to solidify the suspension, and then grown upright at 37.degree. C.
for 24 hrs. The resultant bacterial colonies were harvested by
centrifugation at 2000.times.g at RT for 10 min, resuspended in 3
ml 2.times.YT/ carbenicillin. Aliquots were taken for frozen stocks
(5%) and plasmid DNA preparation.
EXAMPLE 4
[0099] Plasmid DNA Preparation and cRNA Transcription
[0100] Plasmid DNA was purified from pellets of solid-state grown
bacteria (1000 pools of 500 independent clones each) using the
Wizard Miniprep kit according to the manufacturer's instructions
(Promega Biotech, Madison, Wis.). The yield of plasmid DNA from a
14 ml solid-state amplification was 5-10 mg. In preparation for
cRNA synthesis, 4 mg of DNA was digested with Not I, and the
subsequent linearized DNA was made protein and RNase-free by
proteinase K treatment (10 mg for 1 hour at 37.degree. C.),
followed by two phenol, two chloroform/isoamyl alcohol extractions,
and two ethanol precipitations. The DNA was resuspended in
approximately 15 ml of RNase-free water and stored at -70.degree.
C. until needed. cRNA was synthesized using a kit from Promega
Biotech with modifications. Each 50 ml reaction contained: 5 ml of
linearized plasmid (approximately 1 mg), 40 mM Tris-HCl (pH=7.5), 6
mM MgCl.sub.2, 2 mM spermidine, 10 mM NaCl, 10 mM DTT, 0.05 mg/ml
bovine serum albumin, 2 units/ml RNasin, 800 mM each of ATP, CTP
and UTP, 200 mM GTP, 800 mM m7G(5')ppp(5')G, 80 units of T7 RNA
polymerase, and approximately 20,000 cpm of .sup.32P-CTP as a trace
for quantitation of synthesized RNA by TCA precipitation. The
reaction was incubated for 3 hrs. at 30.degree. C.; 20 units of
RNase-free DNase was added, and the incubation was allowed to
proceed for an additional 15 min. at 37.degree. C. cRNA was
purified by two phenol, chloroform/isoamyl alcohol extractions, two
ethanol precipitations, and resuspended at a concentration of 500
ng/ml in RNase-free water immediately before use.
EXAMPLE 5
[0101] Aequorin Bioluminescence Assay (ABA) and Clone
Identification
[0102] The ABA requires injection of library pool cRNA (25 ng/egg
for pool sizes of 500 to 10,000) with aequorin cRNA (2 ng/egg)
supplemented with the G-protein alpha subunit Gal 1(2 ng/egg). To
facilitate stabilization of synthetic transcripts from aequorin and
G.sub.a11 plasmids, the expression vector pCDNA-3 was modified
(termed pcDNA-3v2) by insertion (in the Apa I restriction enzyme
site of the polylinker) of a cassette to append a poly (A) tract on
all cRNA's which initiate from the T7 RNA polymerase promoter. This
cassette includes (5' to 3'): a Bgl II site, pA (20) and a Sfi I
site which can be used for plasmid linearization. Polymerase chain
reaction (PCR) was utilized to generate a DNA fragment
corresponding to the open reading frame (ORF) of the aequorin cDNA
with an optimized Kosak translational initiation sequence (Inouye,
S. et. al., 1985, Proc. Natl. Acad. Sci. USA 82:3154-3158). This
DNA was ligated into pCDNA-3v2 linearized with EcoR I and Kpn I in
the EcoR I/Kpn I site of pCDNA-3v2. G.sub.a11 cDNA was excised as a
Cla I/Not I fragment from the pCMV-5 vector (Woon, C. et. al., 1989
J. Biol. Chem. 264: 5687-93), made blunt with Klenow DNA polymerase
and inserted into the EcoR V site of pcDNA-3v2. cRNA was injected
into oocytes using the motorized "Nanoject" injector (Drummond Sci.
Co., Broomall, Pa.) in a volume of 50 nl. Injection needles were
pulled in a single step using a Flaming/Brown micropipette puller,
Model P-87 (Sutter Instrument Co) and the tips were broken using
53.times.magnification such that an acute angle was generated with
the outside diameter of the needle being <3 mm. Following
injection, oocytes were incubated in ND-96 medium, with gentle
orbital shaking at 18.degree. C. in the dark. Oocytes were
incubated for 24 to 48 hours (depending on the experiment and the
time required for expression of the heterologous RNA) before
"charging" the expressed aequorin with the essential chromophore
coelenterazine. Oocytes were "charged" with coelenterazine by
transferring them into 35 mm dishes containing 3 ml charging medium
and incubating for 2-3 hours with gentle orbital shaking in the
dark at 18.degree. C. The charging medium contained 10 mM
coelenterazine (Molecular Probes, Inc., Eugene, Oreg.) and 30 mM
reduced glutathione in OR-2 media (no calcium). Oocytes were then
returned to ND-86 medium with calcium medium described above and
incubation continued in the dark with orbital shaking until
bioluminescence measurements were initiated. Measurement of GHSR
expression in oocytes was performed using a Berthold Luminometer
LB953 (Wallac Inc., Gaithersburg, Md.) connected to a PC running
the Autolumat-PC Control software (Wallac Inc., Gaithersburg, Md.).
Oocytes (singly or in pairs) were transferred to plastic tubes
(75.times.12 mm, Sarstedt) containing 2.9 ml Ca.sup.++-free OR-2
medium. Each cRNA pool was tested using a minimum of 3 tubes
containing oocytes. Bioluminescence measurements were triggered by
the injection of 0.1 ml of 30 mM MK-677 (1 mM final concentration)
and recordings were followed for 2 min. to observe kinetic
responses consistent with an IP.sub.3-mediated response.
[0103] Pool S 10-20 was prepared from the unfractionated swine
pituitary cDNA library and was composed of 10 pools each of 1000
clones. S10-20 gave a positive signal on two luminometer
instruments and the component pools were then individually tested
for activity. From the 10 pools of 1000 clones, only pool S271 gave
a positive response. This pool was made from two pools of 500
clones designated P541 and P542. Again, only one of the pools,
P541, gave a positive bioluminescent signal in the presence of 1 mM
Compound A. At this point, the bacterial titer was determined in
the glycerol stock of P541 such that dilutions could be plated onto
LB agar plates containing 100 mg/ml carbenicillin to yield
approximately 50 colonies per plate. A total of 1527 colonies were
picked and replicated from 34 plates. The colonies on the original
plates were then washed off, plasmids isolated, cRNA synthesized
and injected into oocytes. cRNA prepared from 8 of the 34 plates
gave positive signals in oocytes. Two plates were selected and the
individual colonies from these plates were grown up, plasmid
isolated, cRNA prepared and injected into oocytes. A single clonal
isolate from each plate (designated as clones 7-3 and 28-18) gave a
positive bioluminescence response to 1 mM Compound A. Clone 7-3 was
further characterized.
EXAMPLE 6
[0104] Receptor Characterization
[0105] DNA sequencing was performed on both strands using an
automated Applied Biosystems instrument (ABI model 373) and
manually by the dideoxy chain termination method using Sequenase II
(US Biochemical, Cleveland, Ohio). Database searches (Genbank 88,
EMBL 42, Swiss-Prot 31, PIR 40, dEST, Prosite, dbGPCR ), sequence
alignments and analysis of the GHSR nucleotide and protein
sequences were carried out using the GCG Sequence Analysis Software
Package (Madison, Wis.; pileup, peptide structure and motif
programs), FASTA and BLAST search programs, and the PC/Gene
software suite from Intelligenetics (San Francisco, Calif.; protein
analysis programs). Northern blot analysis was conducted using
total (20 mg/lane) or poly (A)+ mRNA (5-10 mg/lane) prepared as
described above. RNA was fractionated on a 1% agarose gel
containing 2.2 M formaldehyde and blotted to a nitrocellulose
membrane. Southern blots were hybridized with a PCR generated probe
encompassing the majority of the ORF predicted by clone 7-3 (nt 291
to 1132). The probe was radiolabeled by random-priming with
[a].sup.32P-dCTP to a specific activity of greater than 10.sup.9
dpm/mg. Southern blots were pre-hybridized at 42.degree. C. for 4
hrs. in 5.times.SSC, 5.times.Denhardt's solution, 250 mg/ml tRNA,
1% glycine, 0.075% SDS, 50 mM NaPO.sub.4 (pH 6) and 50% formamide.
Hybridizations were carried out at 42.degree. C. for 20 hrs. in
5.times.SSC, 1.times.Denhardt's solution, 0.1% SDS, 50 mM
NaPO.sub.4, and 50% formamide. RNA blots were washed in
2.times.SSC, 0.2% SDS at 42.degree. C. and at -70.degree. C. RNA
size markers were 28S and 18S rRNA and in vitro transcribed RNA
markers (Novagen). Nylon membranes containing EcoR I and Hind III
digested genomic DNA from several species (Clontech; 10 mg/lane)
were hybridized for 24 hrs. at 30.degree. C. in 6.times.SSPE,
10.times.Denhardt's, 1% SDS, and 50% formamide. Genomic blots were
washed twice with room temperature 6.times.SSPE, twice with
55.degree. C. 6.times.SSPE, and twice with 55.degree. C.
4.times.SSPE. Additional swine GHSR clones from the swine cDNA
library (described above) were identified by hybridization to
plasmid DNA (in pools of 500 clones each) immobilized to nylon
membranes in a slot-blot apparatus (Scheicher and Schuell). Pure
clonal isolates were subsequently identified by colony
hybridization. Swine GHSR clones that extend further in a 5'
direction were identified using 5' RACE procedures (Frohman, M. A.,
1993 Methods Enzyniol. 218:340-358, which is incorporated by
reference) using swine pituitary poly (A).sup.+ mRNA as
template.
EXAMPLE 7
[0106] Human GHSR
[0107] Human pituitary homologues of the swine GHSR were obtained
by screening a commercially available cDNA library constructed in
the vector lambda ZAP II (Stratagene) as per the manufacturer's
instructions. Approximately 1.86.times.10.sup.6 phages were
initially plated and screened using a random-primer labeled portion
of swine clone 7-3 (described above) as hybridization probe. Twenty
one positive clones were plaque purified. The inserts from these
clones were excised from the bacteriophage into the phagemid
pBluescript II SK- by co-infection with helper phage as described
by the manufacturer (Stratagene). Human clones were characterized
as has been described above for the swine clone.
EXAMPLE 8
[0108] Assays
[0109] Mammalian cells (COS-7) were transfected with GHSR
expression plasmids using Lipofectamine (GIBCO-BRL; Hawley-Nelson,
P. 1993, Focus 15:73). Transfections were performed in 60 mm dishes
on 80% confluent cells (approximately 4.times.10.sup.5 cells) with
8 mg of Lipofectamine and 32 mg of GHSR plasmid DNA.
[0110] Binding of .sup.35S-Compound A to swine pituitary membranes
and crude membranes prepared from COS-7 cells transfected with GHSR
expression plasmids was conducted. Crude cell membranes from COS-7
transfectants were prepared on ice, 48 hrs. post-transfection. Each
60 mm dish was washed twice with 3 ml of PBS, once with 1 ml
homogenization buffer (50 mM Tris-HCl [pH 7.4], 5 mM MgCl.sub.2,
2.5 mM EDTA, 30 mg/ml bacitracin). 0.5 ml of homogenization buffer
was added to each dish, cells were removed by scraping and then
homogenized using a Polytron device (Brinkmann, Syosset, N.Y.; 3
bursts of 10 sec. at setting 4). The homogenate was then
centrifuged for 20 min. at 11,000.times.g at 0.degree. C. and the
resulting crude membrane pellet (chiefly containing cell membranes
and nuclei) was resuspended in homogenization buffer supplemented
with 0.06% BSA (0.1 ml/60 mm dish) and kept on ice. Binding
reactions were performed at 20.degree. C. for 1 hr. in a total
volume of 0.5 ml containing: 0.1 ml of membrane suspension, 10 ml
of .sup.35S-Compound A (0.05 to 1 nM; specific activity
approximately 900 Ci/mmol), 10 ml of competing drug and 380-390 ml
of homogenization buffer. Bound radioligand was separated by rapid
vacuum filtration (Brandel 48-well cell harvester) through GF/C
filters pretreated for 1 hr. with 0.5% polyethylenimine. After
application of the membrane suspension to the filter, the filters
were washed 3 times with 3 ml each of ice cold 50 mM Tris-HCl [pH
7.4], 10 mM MgCl.sub.2, 2.5 mM EDTA and 0.015% Triton X-100, and
the bound radioactivity on the filers was quantitated by
scintillation counting. Specific binding (>90% of total) is
defined as the difference between total binding and non-specific
binding conducted in the presence of 50 nM unlabeled Compound
A.
EXAMPLE 9
[0111] Preparation of High Specific Activity Radioligand
[.sup.35S]-Compound A
[0112] [.sup.35S]-Compound A was prepared from an appropriate
precursor,
N-[1(R)-[(1,2-dihydrospiro[3H-indole-3,4'-piperidin]-1'-yl)-carbonyl]-2-(-
phenyl-methyloxy)ethyl]-2-amino-t-butoxycarbonyl-2-methylpropan-amide,
using methane [.sup.35S]sulfonyl chloride as described in Dean DC,
et al., 1995, In: Allen J, Voges R (eds) Synthesis and Applications
of Isotopically Labelled Compounds, John Wiley & Sons, New
York, pp. 795-801. Purification by semi-preparative HPLC (Zorbax
SB-phenyl column, 68% MeOH/water, 0.1% TFA, 5 ml/min) was followed
by N-t-BOC cleavage using 15% trifluro-acetic acid in
dichloromethane (25.degree. C., 3 hr) to give
[methylsulfonyl-.sup.35S]Compound A in near quantitative yield.
HPLC purification (Hamilton PRP-1 4.6.times.250 mm column, linear
gradient of 50-75% methanol-water with 1 mM HCl over 30 min, 1.3
ml/min) provided the ligand in >99% radiochemical purity. The
structure was established by HPLC coelution with unlabeled Compound
A and by mass spectral analysis. The latter method also indicated a
specific activity of .about.1000 Ci/mmol.
EXAMPLE 10
[0113] DNA Encoding a Rat Growth Hormone Secretagogue Receptor
(GHSR) Type Ia
[0114] Cross-hybridization under reduced stringency was the
strategy utilized to isolate the rat GHSR type Ia. Approximately
10.sup.6phage plaques of a once-amplified rat pituitary cDNA
library in lambda gt11 (RL105 lb; Clontech, Palo Alto, Calif.) were
plated on E. coli strain Y1090r.sup.-. The plaques were transferred
to maximum-strength Nytran (Schleicher & Schuell, Keene, N.H.)
denatured, neutralized and screened with a 1.6 kb EcoRI/NotI
fragment containing the entire coding and untranslated regions of
the swine GHSR, clone 7-3. The membranes were incubated at
30.degree. C. in prehybridization solution (50% formamide,
2.times.Denhardts, 5.times.SSPE, 0.1% SDS, 100 mg/ml salmon sperm
DNA) for 3 hours followed by overnight incubation in hybridization
solution (50% formamide, 2.times.Denhardts, 5.times.SSPE, 0.1% SDS,
10% dextran sulfate, 100 mg/ml salmon sperm DNA) with
1.times.10.sup.6 cpm/ml of [.sup.32P]-labeled probe. The probe was
labeled with [.sup.32P]dCTP using a random priming kit (Gibco BRL,
Gaithersburg, N. Dak.). After hybridization the blots were washed
two times each with 2.times.SSC, 0.1% SDS (at 24.degree. C., then
37.degree. C., and finally 55.degree. C.). A single positive clone
was isolated following three rounds of plaque purification. Phage
containing the GHSR was eluted from plate plaques with
1.times.lambda buffer (0.1 M NaCl, 0.01M MgSO.sub.4-7H.sub.2O, 35mM
Tris-HCl, pH 7.5) following overnight growth of approximately 200
pfu/150 mm dish. After a ten minute centrifugation at
10,000.times.g to remove debris, the phage solution was treated
with 1 mg/ml RNAse A and DNAse I for thirty minutes at 24.degree.
C., followed by precipitation with 20% PEG (8000)/2M NaCl for two
hours on ice, and collection by centrifugation at 10,000.times.g
for twenty minutes. Phage DNA was isolated by incubation in 0.1%
SDS, 30 mM EDTA, 50 mg/ml proteinase K for one hour at 68.degree.
C., with subsequent phenol (three times) and chloroform (twice)
extraction before isopropanol precipitation overnight. The GHSR DNA
insert (.about.6.4 kb) was sub-cloned from lambda gt11 into the
plasmid vector Litmus 28 (New England Biolabs, Beverly, Mass.). 2
mg of phage DNA was heated to 65.degree. C. for ten minutes, then
digested with 100 units BsiWI (New England Biolab, Bevely, Mass.)
at 37.degree. C. overnight. A 6.5 kb fragment was gel purified,
electroeluted and phenol/chloroform extracted prior to ligation to
BsiWI-digested Litmus 28 vector.
[0115] Double-stranded DNA was sequenced on both strands on a ABI
373 automated sequencer using the ABI PRISM dye termination cycle
sequencing ready reaction kit (Perkin Elmer; Foster City,
Calif.).
[0116] Comparison of the complete ORF encoding the rat GHSR type Ia
protein sequence to human and swine GHSR homologs reveals a high
degree of sequence identity (rat vs. human, 95.1%; rat vs. swine
93.4%).
[0117] For sequence comparisons and functional expression studies,
a contiguous DNA fragment encoding the complete ORF (devoid of
intervening sequence) for the rat GHSR type Ia was generated. The
PCR was utilized to synthesize a amino-terminal fragment from Met-1
to Val-260 with EcoRI (5') and Hpal (3') restriction sites
appended, while a carboxyl-terminal fragment was generated from
Lys-261 to Thr-364 with Dra I (5') and Not I (3') restriction sites
appended. The ORF construct was assembled into the mammalian
expression vector pSV7 via a three-way ligation with EcoRI/Not
I-digested pSV7, EcoRI/Hpa I-digested NH.sub.2-terminal fragment,
and Dra I/Not I-digested C-terminal fragment.
[0118] Functional activity of the ORF construct was assessed by
transfecting (using lipofectamine; GIBCOJBRL) 5 mg of plasmid DNA
into the aequorin expressing reporter cell line (293-AEQ17)
cultured in 60 mm dishes. Following approximately 40 hours of
expression the aequorin in the cells was charged for 2 hours with
coelenterazine, the cells were harvested, washed and pelleted by
low speed centrifugation into luminometer tubes. Functional
activity was determined by measuring Compound A dependent
mobilization of intracellular calcium and concomitant calcium
induced aequorin bioluminescence. Shown in FIG. 26 are three
replicate samples exhibiting Compound A-induced luminescent
responses.
* * * * *