Structural Requirements for the Activation of the Human Growth Hormone Secretagogue Receptor by Peptide and Nonpeptide Secretagogues
Scott D. Feighner1,
Andrew D. Howard1,
Kristine Prendergast1,
Oksana C. Palyha,
Donna L. Hreniuk,
Ravi Nargund,
Dennis Underwood,
James R. Tata,
Dennis C. Dean,
Carina P. Tan,
Karen Kulju McKee,
John W. Woods,
Arthur A. Patchett,
Roy G. Smith and
Lex H. T. Van der Ploeg
Departments of Biochemistry and Physiology, Medicinal Chemistry,
Molecular Systems, and Drug Metabolism Merck Research
Laboratories Rahway, New Jersey 07065
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ABSTRACT
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Antibodies raised against an intracellular and
extracellular domain of the GH secretagogue receptor (GHS-R)
confirmed that its topological orientation in the lipid bilayer is as
predicted for G protein-coupled receptors with seven transmembrane
domains. A strategy for mapping the agonist-binding site of the human
GHS-R was conceived based on our understanding of ligand binding in
biogenic amine and peptide hormone G protein-coupled receptors. Using
site-directed mutagenesis and molecular modeling, we classified GHS
peptide and nonpeptide agonist binding in the context of its receptor
environment. All peptide and nonpeptide ligand classes shared a common
binding domain in transmembrane (TM) region 3 of the GHS-R. This
finding was based on TM-3 mutation E124Q, which eliminated the
counter-ion to the shared basic N+ group of all
GHSs and resulted in a nonfunctional receptor. Restoration of function
for the E124Q mutant was achieved by a complementary change in the
MK-0677 ligand through modification of its amine side-chain to the
corresponding alcohol. Contacts in other TM domains [TM-2 (D99N), TM-5
(M213K, S117A), TM-6 (H280F), and extracellular loop 1 (C116A)] of the
receptor revealed specificity for the different peptide, benzolactam,
and spiroindolane GHSs. GHS-R agonism, therefore, does not require
identical disposition of all agonist classes at the ligand-binding
site. Our results support the hypothesis that the ligand-binding pocket
in the GHS-R is spatially disposed similarly to the well characterized
catechol-binding site in the
ß2-adrenergic receptor.
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INTRODUCTION
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GH release from pituitary somatotrophs is controlled through the
coordinated action of the agonist GH-releasing hormone and the
antagonist somatostatin, which activate distinct G protein-coupled
receptors (GPC-Rs). The synthetic hexapeptide GH-releasing peptide
(GHRP-6) and nonpeptide mimetics of GHRP-6 (L-692, 429, and MK-0677)
can also mediate GH release through interaction with a separate
receptor, the GH secretagogue receptor (GHS-R) (1, 2, 3, 4, 5). The finding of a
distinct GHS-R indicated that the GHSs may mimic an undiscovered
hormone involved in pulsatile GH release (5, 6). The potential clinical
benefit of GH replacement therapy is being evaluated in several
clinical trials (7).
The GHS-R is a member of the GPC-R family, sharing seven transmembrane
domains (TM). GHS-R activation leads to generation of inositol
trisphosphate (IP3) and Ca2+ release, through
activation of the G protein subunit G
11 (5, 6). The full-length
human and swine GHS-R complementary DNAs (cDNAs; type 1a) encode a
polypeptide of 366 amino acids, whereas the full-length rat gene
encodes a protein of 364 amino acids (5, 6). Each of these receptors
expressed in mammalian cells appears pharmacologically similar to the
native GHS-R. A second GHS-R cDNA (type 1b) encodes a truncated version
of the GHS-R (stop codon at the end of the third intracellular loop).
As a result, type 1b cDNAs expressed in eukaryotic cells fail to
respond to GHS addition. The nucleotide and amino acid sequence of the
GHS-R is unique; its closest relative is the neurotensin receptor, with
34% protein sequence identity (5, 9).
As described below, the availability of the cloned human GHS-R provides
a context in which to begin to understand the requirements of receptor
agonism and antagonism. Antibodies against the GHS-R validated the
membrane orientation of the receptor. As diverse chemical entities
exist that bind and activate the GHS-R, our objective was to determine
the interactions between various peptide and nonpeptide GHSs and
specific GHS-R amino acid residues. To guide the choice of receptor
mutagenesis, a preliminary three-dimensional receptor model of the
human GHS-R was constructed using two-dimensional sequence alignments
of highly conserved motifs of GPC-Rs and homology modeling, based on
bacteriorhodopsin.
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RESULTS AND DISCUSSION
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Many models of GPC-Rs have appeared in the literature since the
structure of bacteriorhodopsin was determined by electron
cryomicroscopy (8). Although bacteriorhodopsin is not a GPC-R, it
contains seven TM-spanning
-helixes and is functionally related to
rhodopsin, a GPC-R that also has been the subject of experimental
structure determinations (10). Spirited debate has centered on the
consequences of the subtle differences in helical position between
these two systems. Unfortunately, the low resolution of the rhodopsin
dataset compromises its utility in three-dimensional model building.
Models based on the bacteriorhodopsin footprint guided mutagenesis
experiments in diverse GPC-R systems, building confidence in the merits
of such an approach (see, for example, Refs. 11 and 12). In part, one
can attribute the utility of these models to the abundant sequence data
available for GPC-Rs (>800 identified; SWISS-PROT database release
35). Although the overall amino acid identity is low, the seven TM
helixes contain recognizable motifs that help to identify conserved
structural elements used in nucleating the two-dimensional sequence
alignments. Diversity in the TM region (especially the presence of a
charge in the hydrophobic helixes) can give clues to the locations of
sites specifically tailored to interact with ligands. The topology of
the receptors is partially constrained by a conserved disulfide bond
that restricts the conformational freedom of a small portion of the
extracellular loops. On the whole, the loops are of unknown structure
and are generally not considered in detail for model-building
exercises.
The location of essential amino acid residues of the GHS-R was
initially based on a functional two-dimensional sequence alignment of
highly conserved motifs in related GPC-Rs and homology modeling based
on the helical footprint of bacteriorhodopsin. In the two-dimensional
sequence alignment, a comparison was made with the angiotensin II,
ß2-adrenergic, neurotensin, and somatostatin-2 receptors
and human, swine, and rat GHS-Rs (Fig. 1
;
predicted essential amino acid residues are highlighted in red).

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Figure 1. Functional Alignment of Type 1a GHS-Rs to Related
GPC-Rs
The alignment was hand constructed by nucleating on several highly
conserved regions noted in blue. The TM helical
boundaries are not rigidly assigned. Sites that are known or postulated
to be important to ligand recognition are colored red.
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The starting conformation for binding of the biphenyl agonist,
L-692,585, follows from previous modeling work on a related structure,
L-692,429 (13). L-692,585 was docked into the receptor using the
relative orientation proposed for biphenyl derivatives in angiotensin
II AT1 (14). Docking of MK-0677 and GHRP-6 into the receptor model
followed from the superpositions among L-692,585, MK-0677, and GHRP-6
that were successfully employed in small molecule design and were also
consistent with nuclear magnetic resonance experiments (15). If the
docked conformation is relevant to the bioactive conformation of bound
agonist, the small molecule must reflect a character complimentary to
the receptor site. In our model, the basic amine of L-692,585 was
within contact distance of E124 in TM3. H280 in TM6 was within contact
distance of the tetrazole of L-692,585. The biphenyl group was situated
along a nonpolar face comprised of two highly conserved tryptophans in
TM4 and TM6. Similar complementarity can be seen with MK-0677 and
GHRP-6. Before mutagenesis, these data were combined to form a
preliminary three-dimensional receptor model of the GHS-R docked with
representative members of three classes of GHSs: the peptide, GHRP-6;
the biphenyl benzolactam L-692,585; and the spiroindolane MK-0677 (Fig. 2
).

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Figure 2. Three-Dimensional Docking Model of GHSs to the
Human GHS-R
Shown to the right of each model are the structures of
L-692,585 (top panel), MK-0677 (middle
panel), and GHRP-6 (lower panel).
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An important feature of GHS agonist bioactivity is the presence of a
basic amino group (2, 3, 16, 17). Based on conservation among human,
swine, and rat GHS-Rs and the growing body of evidence implicating TM
helixes 3, 5, and 6 and extracellular loops 2 and 3 in ligand binding
in other GPC-Rs (18, 19, 20), nine negatively charged amino acid residues
in the human GHS-R were identified as potential candidates for
stabilizing the positive charge. However, the three-dimensional
model of the human GHS-R suggested that E124 in helix 3 was disposed
similarly (but not identically) to D113 of the
ß2-adrenergic receptor (21). Given the well documented
role of this residue in ligand binding for adrenergic receptors, E124
was proposed to serve as the counter-ion to the basic N+
found in GHS agonists such as MK-0677, L-692,585, and the
amino-terminus of GHRP-6. In addition, residues on helixes 5 and 6
paralleling other sites of interaction (e.g. TM-5 may bind
the tetrazole moiety of the benzolactam L-692,585) were targeted for
mutation based on the three-dimensional model. Schematic representation
of the predicted topology (N-terminus extracellular, C-terminus
intracellular) of the GHS-R with mutated residues highlighted is shown
in Fig. 3
. To confirm the cell membrane orientation of the expressed
GHS-R, indirect immunofluorescence was performed with anti-GHS-R
antibodies. Antibodies raised against a predicted intracellular peptide
(Fig. 3
, peptide 7; amino acid sequence
in parentheses) failed to react with the receptor expressed
in COS-7 or HEK-293 cells unless the cells were permeabilized (Woods,
J. W., et al., unpublished observations). This result
indicates that the C-terminus of the protein is disposed
intracellularly. The predicted second extracellular loop peptide
antibodies (Fig. 3
, peptide 3a; amino acid sequence in
parentheses) reacted with the expressed GHS-R without prior
cell permeabilization, providing direct evidence that the membrane
topology of the GHS-R is similar to that of a typical GPC-R.

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Figure 3. Predicted TM Topology of the Human Type 1a GHS-R
and Summary of Mutants and Effect on Aequorin Functional Activity in
HEK-293 Cells and Specific [35S]MK-0677 Binding in COS-7
Cells
Left panel, Predicted TM topology of the human type 1a
GHS-R. TM helixes are numbered. Mutated residues are shown highlighted
in red. Gray amino acid residues indicate epitopes for antibody
generation. Right panel, Summary of mutants and effect
on aequorin functional activity in HEK-293 cells and specific
[35S]MK-0677 binding in COS-7 cells. In both assays, a
plus sign denotes little or no effect ( 2-fold)
relative to wild-type receptor binding (0.2 nM
[35S]MK-0677) and function (challenge by 100
nM MK-0677). A dash signifies that the
mutation conferred a significant reduction (>2-fold) relative to
wild-type binding and/or function.
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A summary of GHS-R functional activity obtained for each of the mutant
GHS-Rs is presented in the table accompanying Fig. 3
. Receptor function
was assessed using an assay that measures GHS-dependent,
IP3-coupled mobilization of intracellular calcium and
concomitant calcium-induced aequorin bioluminescence, a variant of an
assay previously used to expression clone the GHS-R (5). The original
rationale for this assay was based on earlier observations that showed
that GHRPs and nonpeptide mimetics induce a transient increase in the
concentration of intracellular calcium in somatotrophs, an increase in
the intracellular concentration of IP3, and an associated
increase in the activity of protein kinase C (22, 23, 24). Functional
activity was evaluated in response to 100 nM MK-0677
addition to HEK-293 cells that had been transiently transfected with
the GHS-R gene and stably transfected with the gene encoding the
jellyfish bioluminescent protein aequorin (293-AEQ17 cells) (25).
Specific [35S]MK-0677 binding (0.2 nM) was
evaluated in COS-7 and 293-AEQ17 cells, and similar results were
obtained. Note that most of the mutant receptors were still functional
and bound [35S]MK-0677 with high affinity. This indicates
that the effects of each mutation are related to a relatively specific
change in receptor function, rather than an overall nonspecific
disruption of productive receptor conformation. Based on these initial
results, a more limited set of mutants was chosen for further analysis:
TM-2, D99>N; extracellular loop 1, C116>A; TM-3, E124>D and E124>Q;
TM-5, M213>K and S217>A; and TM-6, F279>L. Figure 4
summarizes the relative functional
activities of the mutants in response to a 100 nM challenge
from representatives of the three different GHS classes: MK-0677,
GHRP-6, and L-692,585, whose structures are given in Fig. 2
. A
decreased or increased response to a given ligand in the aequorin
bioluminescence assay probably indicates a change in ligand-mediated
receptor activation. The requirement for E124 in helix 3 is
demonstrated by the effect of mutating E to Q. Consistent with their
basic side-chain, MK-0677, GHRP-6, and L-692,585 are no longer able to
signal in the E to Q mutant. To confirm the role of this residue in
receptor activation, an analog of MK-0677 (L-168,740) was synthesized
in which the primary amine side-chain was modified to the corresponding
alcohol, which could then serve as the counter-ion to E124. As shown in
Fig. 5
, L-168,740 did not activate the
wild-type receptor at concentrations as high as 10 µm. However,
functional rescue of the E124Q mutant was achieved by L-168,740, with
an EC50 of 1.3 µm. Shown for comparison from the same
experiment are dose-response curves for the activation of wild-type and
E124Q mutant receptor by MK-0677, which produced a more than 200-fold
decrease in potency against the E124Q mutant (EC50:
wild-type, 13.6 nM; E124Q, 3200 nM). The
conservative change, E124>D, is not disruptive, suggesting that the
basic amine on the agonist forms a salt bridge to the negative charge
on the GHS-R. Although the interaction with E124 is common to most
agonists, interaction with M213 in helix 5 is not general. M213 is
disposed similarly to the important S204 and S207 in
ß2-adrenergic receptor as well as the essential K199 in
the angiotensin II AT1 receptor. M213 appears to be critical to binding
of the biphenyl derivative L-692,585 and to a lesser extent the peptide
GHRP-6, without affecting receptor activation by MK-0677. Of the
residues in helix 6 targeted for mutagenesis, mutating H280>F
essentially normalized the responses to all three agonists. On the
other hand, mutating C116 to A at the top of helix 3 abolishes activity
for all agonists. This cysteine is believed to make a disulfide bond
that is conserved in all GPC-Rs (9).

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Figure 4. Functional Activities of Select Human GHS-R Mutants
with Different GHS Agonists, Immunoblot Analysis of Crude Membranes
from Transfected HEK-AEQ17 Cells, and Binding of
[35S]MK-0677 to Membranes from GHS-R-Transfected
HEK-AEQ17 Cells
Top panel, Functional activities of select human GHS-R
mutants with different GHS agonists. The HEK-293 cell line stably
expressing aequorin (293-AEQ17) (25) was transfected with a GHS-R
expression plasmid. Two days after transfection, cells were
"charged" with coelentrazine, scraped, transferred into ECB buffer,
and centrifuged in plastic tubes for luminometer measurements. Data for
each point on the dose-response curve represent the average of
triplicate measurements for each sample ( 3 x 105
cells/tube). Middle panel, Immunoblot analysis of crude
membranes from transfected HEK-AEQ17 cells. Membranes were prepared and
immunoblotted as described in Materials and Methods. The
antireceptor antibody (peptide 7) was used for all samples. Fifty
thousand cell equivalents ( 1 µg membrane protein) were loaded on
each lane of a 16% SDS-PAGE gel. The region of the gel shown
corresponds to the approximate size range of 40 kDa. Lower
panel, Binding of [35S]MK-0677 to membranes from
GHS-R-transfected HEK-AEQ17 cells. Two days after transfection with a
GHS-R cDNA expression construct, crude cell membranes were prepared and
assayed for specific [35S]MK-0677 binding (0.2
nM; 1 µg membrane protein).
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The binding of 0.2 nM (1 x Kd)
[35S]MK-0677 to each of the different mutants is shown in
Fig. 4
(bottom panel). In mutants that showed little or no
binding with 0.2 nM [35S]MK-0677, raising the
radioligand concentration to
10 nM still failed to
produce measurable specific binding (a high background binding >10
nM precluded further increases in the radioligand
concentration). To document that differences in receptor function and
binding are not due to variable levels of mutant protein synthesis
and/or transport to the cell membrane, immunoblotting was performed
using antipeptide antibodies raised against the C-terminal
intracellular domain of the wild-type receptor (peptide 7) or an
amino-terminally placed FLAG epitope. The presence of the FLAG sequence
(eight amino acids) had no effect on the binding or function of the
receptor, as observed with other GPC-Rs (data not shown) (26). As shown
in Fig. 4
(middle panel), the expression level of each of
the mutant receptors was similar to that of the wild-type receptor (a
2-fold difference cannot be excluded). We conclude that the
decreased specific binding with [35S]MK-677 observed for
different mutant proteins resulted from a reduced binding affinity
rather than a drastic decrease in the binding capacity. Finally, for
some mutants a significant reduction in specific binding affinity is
not paralleled by an equal decrease in functional activity
(e.g. pFLAG-E124D; pFLAG-M213K). We attribute this to
altered association/dissociation rates at the receptor-binding pocket
as a result of the mutation, still allowing for receptor
activation.
In summary, a strategy was used for selecting mutants based on a
conserved binding orientation of biphenyl compounds in GPC-Rs. All of
the ligands studied in this work were affected by at least one mutation
in common with L-692,585, suggesting that the agonist-binding site is
partially overlapping. However, there are distinct regions of the
receptor that are selective for particular classes of agonists,
illustrating that receptor agonism does not require identical
disposition of all agonists in the ligand-binding site.
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MATERIALS AND METHODS
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Materials
All chemicals were reagent grade and of the highest purity
available. Peptide and nonpeptide GHSs were obtained from Peninsula
Laboratories (Belmont, CA) and the Department of Medicinal Chemistry,
Merck Research Laboratories (Drs. Ravi Nargund and Arthur A. Patchett).
[35S]MK-0677 (SA, 1000 Ci/mmol) was provided by Dr.
Dennis Dean (Department of Drug Metabolism, Merck Research
Laboratories). Coelenterazine was purchased from Molecular Probes
(Eugene, OR). Peptides for immunization were synthesized by Research
Genetics (Huntsville, AL). FLAG reagents were obtained from Kodak
Scientific Imaging Systems (Rochester, NY).
Mutagenesis
Point mutants (single or double nucleotide changes) were
constructed using a dut-ung- system (Bio-Rad, Richmond, CA) (27). Nucleotide
sequence analysis of all mutants verified that nucleotide sequence
errors had not occurred.
Transfection of COS-7 and 293-AEQ17 Cells
Transient transfections of the GHS-R (complete
1.1-kilobase
open reading frame without 5'- or 3'-untranslated sequence) in the
mammalian expression vectors pcDNA-3 (Invitrogen, San Diego, CA)
and pFLAG-CMV-2 (Kodak Scientific Imaging Systems) were conducted using
the Lipofectamine procedure (Life Technologies, Gaithersburg, MD)
according to the manufacturers instructions. Transfections were
performed in 60-mm dishes (80% confluent cells) with 30 µg
Lipofectamine and 2.5 µg GHS-R plasmid DNA. Receptor expression was
allowed to proceed for 4872 h.
[35S]MK-0677 Binding Assay
Binding of [35S]MK-0677 (SA, 1200 Ci/mmol) to
crude membranes prepared from COS-7 cell transfectants was performed as
previously described (4, 5).
Twenty-five micrograms of membrane
protein were used. Specific binding (>90% of the total) is defined as
the difference between total binding and nonspecific binding conducted
in the presence of 50 nM unlabeled MK-0677.
Aequorin Bioluminescence Assay
Measurement of GHS-R expression in the aequorin-expressing
stable reporter cell line 293-AEQ17 was performed as previously
described (6, 25). Luminescence recordings were made for 2 min to
observe responses consistent with IP3-mediated
kinetics.
Production and Characterization of Antipeptide Antibodies
Antipeptide antibodies were raised in female New England White
rabbits (six rabbits per peptide) using eight different peptides
directed against various regions of the human GHS-R type 1a (Covance,
Denver, PA). The peptides were conjugated to keyhole limpet hemocyanin
as a carrier. The following protocol was used: priming ip injection of
B. pertussis antigen (250 µg) on day -3, primary
injection of peptide conjugate (100 µg) emulsified with Freunds
complete adjuvant given intranodally on day 1, and booster injections
given sc on day 21 (500 µg) and days 45 and 66 (250 µg), and at
3-week intervals maintenance boost injections given (125 µg
peptide conjugate). End-point enzyme-linked immunosorbent assay titers
gave a positive response to the injected peptide (
1:100,000
dilution) for all peptides in the majority of rabbits. Both peptide 3a
(amino acid sequence, HENGTDPWDTNECR; second extracellular loop) and
peptide 7 (amino acid sequence, RAWTESSINT; C-terminal intracellular
domain) detected the receptor expressed in mammalian cells by indirect
immunofluorescence (Woods, J. W., Baker, J., Feighner, S. D.,
Hreniuk, D. L., Howard, A. D., Palyha, O. C., and Smith,
R. G., unpublished observations). However, antibodies against
peptide 7 were the only antibodies with utility for immunoblotting
techniques. IgG fractions were isolated from crude serum using the
Immunopure IgG purification kit (Pierce Chemical Co., Rockford, IL).
Immunoblotting of crude cell membranes from transfected cells was
carried out using
50,000 cell equivalents (
1 µg protein).
SDS-denatured proteins (heated at 65 C for 2 min) were run on an
SDS-PAGE gel (16% acrylamide, Novex, San Diego, CA) and
electrotransferred to polyvinylidene difluoride membranes (Millipore,
Bedford, MA). Membranes were treated with 5% nonfat dry milk in
Tris-buffered saline (TBS; pH 7.5) at 37 C for 1 h, washed twice
in TBS for 2 min each time, and incubated with protein A-purified
peptide 7 antibodies (10 µg/ml) for 3060 min at room temperature.
The blots were washed three times for 2 min each time and incubated
with donkey antirabbit horseradish peroxidase conjugate (1:1,000
dilution) for 30 min. The Western blots were then washed three times
for 15 min each time in TBS, and proteins were detected by
chemiluminescence (Amersham ECL kit, Arlington Heights, IL) using
Hyperfilm (2-sec exposure).
Molecular Modeling
A three-dimensional receptor model of the GHS-R was constructed
using two-dimensional sequence alignment on highly conserved motifs in
the TM domains of many guanine nucleotide protein-coupled receptors
(28). Sequence alignments were performed by hand using the tools
available in QUANTA (version 4.0, Molecular Simulations, Boston, MA).
Homology modeling was based on the helical footprint of
bacteriorhodopsin, using the published angiotensin II AT1a receptor
model as a template to position and orient both the helixes and the
common side-chains (14). Side-chains not common with AT1a were built
using a standard rotamer library available in the protein design module
of QUANTA. The resultant model was energy minimized with the CHARMm
force field (29).
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FOOTNOTES
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Address requests for reprints to: Dr. Andrew D. Howard, Departments of Biochemistry and Physiology, Merck Research Laboratories, Building RY-80Y-265, P.O. Box 2000, Rahway, New Jersey 07065.
1 Co-first authors. 
Received for publication August 22, 1997.
Revision received October 7, 1997.
Accepted for publication October 8, 1997.
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REFERENCES
|
---|
-
Bowers CY, Sartor RA, Reynolds GA 1991 On the actions of
the growth hormone-releasing hexapeptide GHRP. Endocrinology 128:20272035[Abstract]
-
Smith RA, Cheng K, Schoen WR, Pong S-S, Hickey G, Jacks T,
Butler B, Chan WW, Chaung L-YP, Judith F, Taylor J, Wyvratt MJ, Fisher
M-H 1993 A nonpeptidyl growth hormone secretagogue. Science 260:16401643[Medline]
-
Patchett AA, Nargund RA, Tata JR, Chen M-M, Barakat KJ,
Johnston DBR, Cheng K, Chan WW-S, Butler B, Hickey G, Jacks T, Scheim
K, Pong S-S, Chaung L-YP, Chen HY, Frazer E, Leung KH, Chiu S-HL, Smith
RG 1995 Design and biological activities of L-163, 191 (MK-0677): a
potent and orally active growth hormone secretagogue. Proc Natl Acad
Sci USA 92:70017005[Abstract]
-
Pong S-S, Chaung L-YP, Dean DC, Nargund RP, Patchett AA,
Smith RG 1996 Identification of a new G protein-linked receptor for
growth hormone secretagogues. Mol Endocrinol 10:5761[Abstract]
-
Howard AD, Feighner SD, Cully DF, Arena JP, Liberator
PL, Rosenblum CI, Hamelin M, Hreniuk DL, Palyha OC, Anderson J, Paress
PS, Diaz C, Chou M, Liu KK, McKee K-K, Pong S-S, Chaung L-Y, Elbrecht
A, Dashkevicz M, Heavens R, Rigby M, Sirinathsinghji D, Dean DC,
Melillo DG, Patchett AA, Nargund R, Griffin PR, DeMartino JA, Gupta SK,
Schaeffer JA, Smith RG, Van der Ploeg LHT 1996 A receptor in pituitary
and hypothalamus that functions in growth hormone release. Science 273:974977[Abstract]
-
McKee K-K, Palyha OC, Feighner SD, Hreniuk DL, Tan C,
Phillips M, Smith RG, Van der Ploeg LHT, Howard AD 1997 Molecular
analysis of rat pituitary and hypothalamic growth hormone secretagogue
receptors. Mol Endocrinol 11:415423[Abstract/Free Full Text]
-
Laron Z 1995 Growth hormone secretagogues: clinical
experience and therapeutic potential. Drugs 50:595601[Medline]
-
Henderson R, Baldwin J, Ceska T, Zemlin F, Beckmann E,
Downing K 1990 Model for the structure of bacteriorhodopsin based on
high-resolution electron cryo-microscopy. J Mol Biol 213:899929[Medline]
-
Probst WC, Snyder LA, Schuster DI 1992 Sequence alignment of
the G-protein coupled receptor superfamily. DNA Cell Biol 11:120[Medline]
-
Schertler G, Villa C, Henderson R 1993 Projection structure of
rhodopsin. Nature 362:770772[CrossRef][Medline]
-
Pittel Z, Wess J 1994 Intramolecular interactions in
muscarinic acetylcholine-receptors studied with chimeric m2/m5
receptors. Mol Pharmacol 45:6164[Abstract]
-
Fong TM, Yu H, Cascieri MA, Underwood D, Swain CJ, Strader CD 1994 The role of histidine-265 in antagonist binding to the
neurokinin-1 receptor. J Biol Chem 269:27282732[Abstract/Free Full Text]
-
Schoen WR, Pisano JM, Prendergast K, Wyvratt MJ, Fisher MH,
Cheng K, Chan WW-S, Butler B, Smith RG, Ball RG 1994 A novel
3-substituted benzazepinone growth hormone secretagogue (L-692, 429).
J Med Chem 37:897906[Medline]
-
Underwood DJ, Strader CD, Rivero R, Patchett AA, Greenlee WJ,
Prendergast K 1994 Structural model of antagonist and agonist binding
to the angiotensin II, AT1 subtype, G protein-coupled
receptor. Curr Biol 1:211221
-
Bednarek MA, Arison B, Baum MW, Cheng K, Butler B, Chan WW-S,
Ren EN, Wu T-J, Prendergast K 1996 NMR and Structure-Function Studies
on Growth Hormone Releasing Peptides, GHRP-6 and GHRP-2.
Springer-Verlag, Edinburgh
-
Momany FA, Bowers CY, Reynolds GA, Hong A, Newlander K 1984 Conformational energy studies and in vitro and in vivo activity data on
growth hormone-releasing peptides. Endocrinology 114:15311536[Abstract]
-
McDowell RS, Elias KA, Stanley MS, Burdick DJ, Burnier JP,
Chan KS, Fairbrother WJ, Hammonds RG, Ingle GS, Jacobsen NE 1995 Growth
hormone secretagogues: characterization, efficacy, and minimal
bioactive conformation. Proc Natl Acad Sci USA 92:1116511169[Abstract]
-
Schwartz TW 1994 Locating ligand-binding sites in 7TM
receptors by protein engineering. Curr Opin Biotechnol 5:434444[Medline]
-
Fong TM 1996 Mechanistic hypotheses for the activation of G
protein-coupled receptors. Cell Signal 8:217224[CrossRef][Medline]
-
Schwartz TW, Rosenkilde MM 1996 Is there a lock for all
agonist keys in 7TM receptors? Trends Pharmacol Sci 17:213216[CrossRef][Medline]
-
Strader CD, Candelore MR, Hill WS, Dixon RAF, Sigal IS 1989 A
single amino acid subsitution in the beta-adrenergic receptor promotes
partial agonist activity from antagonists. J Biol Chem 264:1647016477[Abstract/Free Full Text]
-
Cheng K, Chan WWS, Butler B, Barrato A, Smith RA 1991 Evidence
for a role of protein kinase-C in
His-D-Trp-Ala-D-Phe-Lys-amide induced growth
hormone release from rat primary pituitary cells. Endocrinology 129:33373342[Abstract]
-
Bresson-Bepoldun L, Odessa MF, Dufy-Barbe L 1994 GHRP-6
stimulates calcium increase and growth hormone release in human
somatotrophs in vitro. Endocr J 2:793803
-
Lei T, Buchfelder M, Fahlbusch R, Adams EF 1995 Growth hormone
releasing peptide (GHRP-6) stimulates phosphatidylinositol (PI)
turnover in human pituitary somatotroph cells. J Mol Endocrinol 14:135138[Abstract]
-
Button D, Brownstein M 1993 Aequorin-expressing mammalian cell
lines used to report Ca2+ mobilization. Cell Calcium 14:663671[Medline]
-
Hein L, Ishii K, Coughlin SR, Kobilka BK 1994 Intracellular
targeting and trafficking of thrombin receptors. J Biol Chem 269:2771927726[Abstract/Free Full Text]
-
Kunkel TA 1985 Rapid and efficient site-specific mutagenesis
without phenotypic selection. Proc Natl Acad Sci USA 82:488[Abstract]
-
van Rhee AM, Jacobson KA 1996 Molecular architecture of G
protein-coupled receptors. Drug Dev Res 37:138[CrossRef]
-
Brooks BB, Bruccoleri RE, Olafson BD, States DJ, Swaminathan
S, Karplus M 1983 CHARMm: a program for macromolecular energy,
minimization, and dynamics calculations. J Comput Chem 4:187217