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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha}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.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {alpha}-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. 1Go; predicted essential amino acid residues are highlighted in red).



View larger version (76K):
[in this window]
[in a new window]
 
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.

 
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. 2Go).



View larger version (48K):
[in this window]
[in a new window]
 
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).

 
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. 3Go. 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. 3Go, 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. 3Go, 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.



View larger version (29K):
[in this window]
[in a new window]
 
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.

 
A summary of GHS-R functional activity obtained for each of the mutant GHS-Rs is presented in the table accompanying Fig. 3Go. 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 4Go 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. 2Go. 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. 5Go, 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).



View larger version (20K):
[in this window]
[in a new window]
 
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).

 


View larger version (11K):
[in this window]
[in a new window]
 
Figure 5. Functional Activity of E124Q Mutant and Wild-Type Receptors in Response to L-168,740 and MK-0677

See Fig. 4Go for details. {blacksquare}, Wild-type receptor; {square}, E124Q mutant.

 
The binding of 0.2 nM (1 x Kd) [35S]MK-0677 to each of the different mutants is shown in Fig. 4Go (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. 4Go (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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 manufacturer’s 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 48–72 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 Freund’s 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 30–60 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).


    FOOTNOTES
 
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. Back

Received for publication August 22, 1997. Revision received October 7, 1997. Accepted for publication October 8, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Bowers CY, Sartor RA, Reynolds GA 1991 On the actions of the growth hormone-releasing hexapeptide GHRP. Endocrinology 128:2027–2035[Abstract]
  2. 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 260:1640–1643[Medline]
  3. 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 92:7001–7005[Abstract]
  4. 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 10:57–61[Abstract]
  5. 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 273:974–977[Abstract]
  6. 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 11:415–423[Abstract/Free Full Text]
  7. Laron Z 1995 Growth hormone secretagogues: clinical experience and therapeutic potential. Drugs 50:595–601[Medline]
  8. 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 213:899–929[Medline]
  9. Probst WC, Snyder LA, Schuster DI 1992 Sequence alignment of the G-protein coupled receptor superfamily. DNA Cell Biol 11:1–20[Medline]
  10. Schertler G, Villa C, Henderson R 1993 Projection structure of rhodopsin. Nature 362:770–772[CrossRef][Medline]
  11. Pittel Z, Wess J 1994 Intramolecular interactions in muscarinic acetylcholine-receptors studied with chimeric m2/m5 receptors. Mol Pharmacol 45:61–64[Abstract]
  12. 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 269:2728–2732[Abstract/Free Full Text]
  13. 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 37:897–906[Medline]
  14. 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 1:211–221
  15. 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
  16. 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 114:1531–1536[Abstract]
  17. 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 92:11165–11169[Abstract]
  18. Schwartz TW 1994 Locating ligand-binding sites in 7TM receptors by protein engineering. Curr Opin Biotechnol 5:434–444[Medline]
  19. Fong TM 1996 Mechanistic hypotheses for the activation of G protein-coupled receptors. Cell Signal 8:217–224[CrossRef][Medline]
  20. Schwartz TW, Rosenkilde MM 1996 Is there a lock for all agonist keys in 7TM receptors? Trends Pharmacol Sci 17:213–216[CrossRef][Medline]
  21. 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 264:16470–16477[Abstract/Free Full Text]
  22. 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 129:3337–3342[Abstract]
  23. 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 2:793–803
  24. 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 14:135–138[Abstract]
  25. Button D, Brownstein M 1993 Aequorin-expressing mammalian cell lines used to report Ca2+ mobilization. Cell Calcium 14:663–671[Medline]
  26. Hein L, Ishii K, Coughlin SR, Kobilka BK 1994 Intracellular targeting and trafficking of thrombin receptors. J Biol Chem 269:27719–27726[Abstract/Free Full Text]
  27. Kunkel TA 1985 Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA 82:488[Abstract]
  28. van Rhee AM, Jacobson KA 1996 Molecular architecture of G protein-coupled receptors. Drug Dev Res 37:1–38[CrossRef]
  29. 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 4:187–217