RESUMO
A series of small molecules derived from MK-0677, a potent synthetic GHS, mimicking the N-terminal Gly-Ser-O-(n-octanoyl)-L-Ser-Phe segment of ghrelin was synthesized and tested in a binding and in a functional assay measuring intracellular calcium elevation in HEK-293 cells expressing hGHSR1a. Replacement of Phe in this tetrapeptide with a spiro(indoline-3,4'-piperidine) group, Gly-Ser with 2-aminoisobutyric acid, and O-(n-octanoyl)-L-Ser with O-benzyl-D-Ser provided synthetic GHS agonists with similar functional potency as ghrelin.
Assuntos
Cálcio/metabolismo , Indóis/metabolismo , Hormônios Peptídicos , Peptídeos/metabolismo , Piperidinas/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G , Compostos de Espiro/metabolismo , Sítios de Ligação/fisiologia , Células Cultivadas , Grelina , Humanos , Indóis/química , Concentração Inibidora 50 , Luminescência , Mimetismo Molecular , Peptídeos/química , Piperidinas/síntese química , Ligação Proteica/fisiologia , Receptores de Grelina , Compostos de Espiro/químicaRESUMO
We have previously reported the cloning and characterization of a new orphan G-protein-coupled receptor (GPC-R), the growth hormone secretagogue receptor (GHS-R), and shown that this receptor mediates the activity of the growth hormone-releasing peptides (GHRPs) and nonpeptide ligands such as L-692,429 and MK-0677. Because the GHS-R obviously does not belong to any of the known GPC-R subfamilies, we searched for GHS-R family members by screening a human genomic library using low-stringency hybridization and screening a Pufferfish genomic library. The Pufferfish was selected because of its compact genome. From the human genomic library, a homolog, GPR38, with 52% identity to the GHS-R was isolated. From the Pufferfish library, three family members were isolated. The Pufferfish gene having 58% identity to the GHS-R, on expression in HEK293 cells, was activated with GHRP-6 and MK-0677. These results indicate that the GHS-R has been conserved for at least 400 million years and that the Pufferfish genome is appropriate for isolation of GHS-R family members. In our search for endogenous ligands for the orphan receptors GHS-R and GPR38, we showed that adenosine is a partial agonist of the GHS-R and that motilin is the endogenous ligand for GPR38. We also confirmed that the endogenous ligand ghrelin is a full agonist of the GHS-R.
Assuntos
Hormônios Peptídicos , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G , Adenosina/farmacologia , Animais , Benzazepinas/metabolismo , Linhagem Celular , Clonagem Molecular , Regulação da Expressão Gênica/efeitos dos fármacos , Biblioteca Genômica , Grelina , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Humanos , Indóis/metabolismo , Indóis/farmacologia , Estrutura Molecular , Motilina/farmacologia , Oligopeptídeos/farmacologia , Peptídeos/farmacologia , Receptores de Superfície Celular/agonistas , Receptores de Superfície Celular/metabolismo , Receptores de Grelina , Homologia de Sequência de Aminoácidos , Compostos de Espiro/metabolismo , Compostos de Espiro/farmacologia , Tetrazóis/metabolismo , Extratos de Tecidos/farmacologiaRESUMO
The recently discovered growth hormone secretagogue, ghrelin, is a potent agonist at the human growth hormone secretagogue receptor 1a (hGHSR1a). To elucidate structural features of this peptide necessary for efficient binding to and activation of the receptor, several analogues of ghrelin with various aliphatic or aromatic groups in the side chain of residue 3, and several short peptides derived from ghrelin, were prepared and tested in a binding assay and in an assay measuring intracellular calcium elevation in HEK-293 cells expressing hGHSR1a. Bulky hydrophobic groups in the side chain of residue 3 turned out to be essential for maximum agonist activity. Also, short peptides encompassing the first 4 or 5 residues of ghrelin were found to functionally activate hGHSR1a about as efficiently as the full-length ghrelin. Thus the entire sequence of ghrelin is not necessary for activity: the Gly-Ser-Ser(n-octanoyl)-Phe segment appears to constitute the "active core" required for agonist potency at hGHSR1a.
Assuntos
Hormônios Peptídicos , Peptídeos/química , Receptores de Superfície Celular/agonistas , Receptores Acoplados a Proteínas G , Sequência de Aminoácidos , Sítios de Ligação , Cálcio/metabolismo , Linhagem Celular , Grelina , Humanos , Medições Luminescentes , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Peptídeos/metabolismo , Peptídeos/farmacologia , Receptores de Superfície Celular/metabolismo , Receptores de Grelina , Relação Estrutura-AtividadeRESUMO
Neuromedin U (NMU) is a neuropeptide with potent activity on smooth muscle which was isolated first from porcine spinal cord and later from other species. It is widely distributed in the gut and central nervous system. Peripheral activities of NMU include stimulation of smooth muscle, increase of blood pressure, alteration of ion transport in the gut, control of local blood flow and regulation of adrenocortical function. An NMU receptor has not been molecularly identified. Here we show that the previously described orphan G-protein-coupled receptor FM-3 (ref. 15) and a newly discovered one (FM-4) are cognate receptors for NMU. FM-3, designated NMU1R, is abundantly expressed in peripheral tissues whereas FM-4, designated NMU2R, is expressed in specific regions of the brain. NMU is expressed in the ventromedial hypothalamus in the rat brain, and its level is significantly reduced following fasting. Intracerebroventricular administration of NMU markedly suppresses food intake in rats. These findings provide a molecular basis for the biochemical activities of NMU and may indicate that NMU is involved in the central control of feeding.
Assuntos
Comportamento Alimentar/fisiologia , Proteínas de Membrana , Neuropeptídeos/metabolismo , Receptores de Superfície Celular/fisiologia , Receptores de Neurotransmissores/fisiologia , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Linhagem Celular , Clonagem Molecular , Jejum , Humanos , Ligantes , Camundongos , Dados de Sequência Molecular , Obesidade/metabolismo , RNA Mensageiro/metabolismo , Ensaio Radioligante , Ratos , Receptores de Superfície Celular/análise , Receptores de Neurotransmissores/análise , Alinhamento de Sequência , Distribuição TecidualRESUMO
Synthetic ligands have been identified that reset and amplify the cycle of pulsatile GH secretion by interacting with the orphan GH-secretagogue receptor (GHS-R). The GHS-R is rhodopsin like, but does not obviously belong to any of the established G protein-coupled receptor (GPCR) subfamilies. We recently characterized the closely related orphan family member, GPR38, as the motilin receptor. A common property of both receptors is that they amplify and sustain pulsatile biological responses in the continued presence of their respective ligands. To efficiently identify additional members of this new GPCR family, we explored a vertebrate species having a compact genome, that was evolutionary distant from human, but where functionally important genes were likely to be conserved. Accordingly, three distinct full-length clones, encoding proteins of significant identity to the human GHS-R, were isolated from the Pufferfish (Spheroides nephelus). Southern analyses showed that the three cloned Pufferfish genes are highly conserved across species. The gene with closest identity (58%) was activated by three synthetic ligands that were chosen for their very high selectivity on the GHS-R as illustrated by their specificity in activating the wild-type human GHS-R but not the E124Q mutant. These results indicate that the ligand activation domain of the GHS-R has been evolutionary conserved from Pufferfish to human (400 million years), supporting the notion that the GHS-R and its natural ligand play a fundamentally important role in biology. Furthermore, they illustrate the power of exploiting the compact Pufferfish genome for simplifying the isolation of endocrinologically important receptor families.
Assuntos
Peixes/genética , Receptores de Superfície Celular/química , Receptores Acoplados a Proteínas G , Sequência de Aminoácidos , Animais , Southern Blotting , Linhagem Celular , Clonagem Molecular , Sequência Conservada , Biblioteca Genômica , Humanos , Ligantes , Modelos Genéticos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Receptores de Superfície Celular/genética , Receptores de Grelina , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
A series of structurally diverse growth hormone (GH) releasing substances have been synthesized that are distinct from the naturally occurring GH releasing hormone (GHRH). These synthetic molecules range from the family of GH releasing peptides and mimetics such as MK-0677. The physiological importance of these molecules and their receptor is exemplified by studies in the elderly. For example, when MK-0677 was administered chronically to 70- to 90-year-old subjects, once daily, the age-related reduced amplitude of GH pulses was reversed to that of the physiological profile typical of young adults. In 1996, the synthesis of (35)S-MK-0677 was reported and used as a ligand to characterize a common receptor (GH secretagogue receptor [GHS-R]) for the GH releasing substances. The GHS-R is distinct from the GHRH receptor. Subsequently, the GHS-R gene was cloned and shown to encode a unique G-protein coupled receptor with a deduced protein sequence that was 96% identical in human and rat. Because of the physiological importance of the GHS-R, a search for family members (FMs) was initiated and its molecular evolution investigated. Three FMs GPR38, GPR39 and FM3 were isolated from human genomic libraries. To accelerate the identification of other FMs, a vertebrate organism with a compact genome distant in evolutionary terms from humans was exploited. The pufferfish (Spheroides nephelus) genome provides an ideal model for the discovery of human genes. Three distinct full-length clones encoding proteins of significant sequence identity to the human GHS-R were cloned from the pufferfish. Remarkably, the pufferfish gene with highest sequence homology to the human receptor was activated by the hexapeptide and non-peptide ligands. These intriguing results show that the structure and function of the ligand binding pocket of the human GHS-R has been highly conserved in evolution ( approximately 400 million years) and strongly suggests that an endogenous natural ligand has been conserved. This new information is consistent with a natural ligand for the GHS-R playing a fundamentally important and conserved role in physiology.
Assuntos
Hormônio Liberador de Hormônio do Crescimento , Receptores de Superfície Celular/fisiologia , Receptores Acoplados a Proteínas G , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Animais , Sítios de Ligação , Hormônio do Crescimento Humano/metabolismo , Humanos , Indóis/farmacologia , Dados de Sequência Molecular , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Receptores de Grelina , Receptores de Neuropeptídeos , Receptores de Hormônios Reguladores de Hormônio Hipofisário , Compostos de Espiro/farmacologiaRESUMO
Motilin is a 22-amino acid peptide hormone expressed throughout the gastrointestinal (GI) tract of humans and other species. It affects gastric motility by stimulating interdigestive antrum and duodenal contractions. A heterotrimeric guanosine triphosphate-binding protein (G protein)-coupled receptor for motilin was isolated from human stomach, and its amino acid sequence was found to be 52 percent identical to the human receptor for growth hormone secretagogues. The macrolide antibiotic erythromycin also interacted with the cloned motilin receptor, providing a molecular basis for its effects on the human GI tract. The motilin receptor is expressed in enteric neurons of the human duodenum and colon. Development of motilin receptor agonists and antagonists may be useful in the treatment of multiple disorders of GI motility.
Assuntos
Colo/metabolismo , Mucosa Gástrica/metabolismo , Intestino Delgado/metabolismo , Motilina/metabolismo , Receptores dos Hormônios Gastrointestinais/química , Receptores dos Hormônios Gastrointestinais/genética , Receptores de Neuropeptídeos/química , Receptores de Neuropeptídeos/genética , Processamento Alternativo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Cálcio/metabolismo , Linhagem Celular , Mapeamento Cromossômico , Cromossomos Humanos Par 13 , Clonagem Molecular , Eritromicina/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Humanos , Hibridização In Situ , Ligantes , Dados de Sequência Molecular , Motilina/análogos & derivados , Receptores dos Hormônios Gastrointestinais/metabolismo , Receptores de Neuropeptídeos/metabolismo , Glândula Tireoide/metabolismo , TransfecçãoRESUMO
The primary hormonal regulator of pigmentation is melanocyte stimulating hormone derived from proopiomelanocortin by proteolytic processing. The melanocortin-1 receptor serves a key role in the regulation of pigmentation. We describe the identification of the first intron within a melanocortin receptor. A new melanocortin-1 receptor isoform, generated by alternative mRNA splicing, encodes an additional 65 amino acids at the predicted intracellular, C-terminal tail of the melanocortin-1 receptor. When expressed in heterologous cells, the new spliced form of the melanocortin-1 receptor (melanocortin-1 receptor B) appears pharmacologically similar to the non-spliced melanocortin-1 receptor. Melanocortin-1 receptor B is expressed in testis, fetal heart and melanomas.
Assuntos
Processamento Alternativo , Receptores da Corticotropina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Clonagem Molecular , Cricetinae , Etiquetas de Sequências Expressas , Humanos , Concentração Inibidora 50 , Modelos Genéticos , Dados de Sequência Molecular , Polimorfismo Genético , Ligação Proteica , Receptores da Corticotropina/metabolismo , Receptores de MelanocortinaRESUMO
Growth hormone secretagogues (GHS) are a group of synthetic peptide and nonpeptide molecules that potently stimulate the release of GH from the anterior pituitary gland through the activation of a novel G-protein-coupled receptor (GPC-R), the GHS-R. In our search for GHS-R family members, we recently described the cloning of two related GPC-Rs, GPR38 and 39. In the present report, we detail the isolation of a new GPC-R (FM-3) from human and mouse with moderate sequence identity to both the GHS-R and neurotensin-R. FM-3 is expressed in a diverse set of tissues.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Proteínas de Membrana , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G , Receptores de Neurotensina/genética , Receptores de Neurotransmissores , Linfócitos T/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , DNA/química , DNA/genética , DNA Complementar/química , DNA Complementar/genética , Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores de Grelina , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Linfócitos T/química , Distribuição Tecidual , TransfecçãoRESUMO
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 beta2-adrenergic receptor.
Assuntos
Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Hormônio do Crescimento Humano/metabolismo , Peptídeos/metabolismo , Peptídeos/fisiologia , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/fisiologia , Hormônio do Crescimento Humano/química , Hormônio do Crescimento Humano/genética , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Peptídeos/química , Peptídeos/genética , Ratos , Receptores de Superfície Celular/genética , Receptores de Grelina , SuínosRESUMO
Growth hormone release is under tight control by two hypothalamic hormones: growth hormone-releasing hormone and somatostatin. In addition, synthetic growth hormone secretagogues have also been shown to regulate growth hormone release through the growth hormone secretagogue receptor (GHS-R), suggesting the existence of an additional physiological regulator for growth hormone release. To understand the physiological role of the GHS-R in more detail, we mapped the expression of mRNA for the receptor by in situ hybridization and RNase protection assays using rat and human tissues. In the rat brain, the major signals were detected in multiple hypothalamic nuclei as well as in the pituitary gland. Intense signals were also observed in the dentate gyrus of the hippocampal formation. Other brain areas that displayed localized and discrete signals for the receptor include the CA2 and CA3 regions of the hippocampus, the substantia nigra, ventral tegmental area, and dorsal and median raphe nuclei. In resemblance to the results from rat brain, RNase protection assays using human tissues revealed specific signals in pituitary, hypothalamus and hippocampus. Moreover, a weak signal was noted in the pancreas. The demonstration of hypothalamic and pituitary localization of the GHS-R is consistent with its role in regulating growth hormone release. The expression of the receptor in other central and peripheral regions may implicate its involvement in additional as yet undefined physiological functions.
Assuntos
Encéfalo/metabolismo , Hipófise/metabolismo , Receptores de Superfície Celular/biossíntese , Receptores Acoplados a Proteínas G , Transcrição Gênica , Animais , Autorradiografia , Sequência de Bases , Giro Denteado/metabolismo , Éxons , Humanos , Hipotálamo/metabolismo , Hibridização In Situ , Íntrons , Masculino , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Especificidade de Órgãos , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Receptores de Superfície Celular/genética , Receptores de GrelinaRESUMO
Galanin (GAL) is a widely distributed neuropeptide with diverse biological effects including modulation of hormone release, antinociception and modification of feeding behavior. Its effects are mediated through G-protein-coupled receptors (GPCR) for which only a single type has been cloned, GAL receptor 1 (GALR1). We describe the cloning of a second galanin receptor type, GALR2, from rat hypothalamus. The GALR2 amino acid sequence is 38% identical to GALR1 and is pharmacologically similar to GALR1 when expressed in COS-7 cells. GALR2 is encoded by a single gene containing at least one intron and expressed in a diverse range of tissues.
Assuntos
Receptores dos Hormônios Gastrointestinais/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Ligação Competitiva , Clonagem Molecular , Expressão Gênica , Dados de Sequência Molecular , Família Multigênica , Ratos , Receptores de Galanina , Receptores dos Hormônios Gastrointestinais/efeitos dos fármacos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
GH release is thought to occur under the reciprocal regulation of two hypothalamic peptides, GH releasing hormone (GHRH) and somatostatin, via their engagement with specific cell surface receptors on the anterior pituitary somatotroph. In addition, GH-releasing peptides, such as GHRP-6 and the nonpeptide mimetics, L-692,429 and MK-0677, stimulate GH release through their activation of a distinct receptor, the GH secretagogue receptor (GHS-R). The recent cloning of the GHS-R from human and swine pituitary gland identifies yet a third G protein-coupled receptor (GPC-R) involved in the control of GH release and further supports the existence of an undiscovered hormone that may activate this receptor. Using the human GHS-R as a probe, we report the isolation of a rat pituitary GHS-R cDNA derived from an unspliced, precursor mRNA. The rat cDNA encodes a protein of 364 amino acids containing seven transmembrane domains (7-TM) with >90% sequence identity to both the human and swine GHS-Rs. A single intron of approximately 2 kb divides the open reading frame into two exons encoding TM 1-5 and TM 6-7, thus placing the GHS-R into the intron-containing class of GPC-Rs. The intron maps to the site of sequence divergence between the human and swine type 1a and 1b GHS-R mRNAs. In addition, determination of the nucleotide sequence for the human GHS-R gene confirmed the position of an intron in the human GHS-R gene at this position. A full-length contiguous cDNA from rat hypothalamus was isolated and shown to be identical in its nucleotide and deduced amino acid sequence to the rat pituitary GHS-R. The cloned rat GHS-R binds [35S]MK-0677 with high affinity [dissociation constant (K(D)) = 0.7 nM] and is functionally active when expressed in HEK-293 cells. Expression of the rat GHS-R was observed specifically in the pituitary and hypothalamus when compared with control tissues.
Assuntos
Hipotálamo/metabolismo , Hipófise/metabolismo , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G , Receptores da Somatotropina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Sequência Conservada , DNA Complementar , Humanos , Hibridização In Situ , Indóis/metabolismo , Íntrons , Cinética , Dados de Sequência Molecular , Fases de Leitura Aberta , Precursores de RNA , Ratos , Receptores de Grelina , Receptores da Somatotropina/biossíntese , Alinhamento de Sequência , Compostos de Espiro/metabolismo , Suínos , TransfecçãoRESUMO
The recent cloning of a growth hormone secretagogue receptor (GHS-R) from human pituitary gland and brain identified a third G protein-coupled receptor (GPC-R) involved in the control of growth hormone release. The nucleotide sequence of the GHS-R is most closely related to the neurotensin receptor-1 (NT-R1) (35% overall protein identity). Two human GPC-Rs related to both the type 1a GHS-R and NT-Rs were cloned and characterized. Hybridization at low posthybridizational stringency with restriction enzyme-digested human genomic DNA resulted in the identification of a genomic clone encoding a first GHS-R/NT-R family member (GPR38). A cDNA clone was identified encoding a second GHS-R-related gene (GPR39). GPR38 and GPR39 share significant amino acid sequence identity with the GHS-R and NT-Rs 1 and 2. An acidic residue (E124) in TM-3, essential for the binding and activation of the GHS-R by structurally dissimilar GHSs, was conserved in GPR38 and GPR39. GPR38 is encoded by a single gene expressed in thyroid gland, stomach, and bone marrow. GPR39 is encoded by a highly conserved single-copy gene, expressed in brain and other peripheral tissues. Fluorescence in situ hybridization localized the genes for GPR38 and GPR39 to separate chromosomes, distinct from the gene encoding the GHS-R and NT-R type 1. The ligand-binding and functional properties of GPR38 and GPR39 remain to be determined.
Assuntos
Cromossomos Humanos Par 13 , Cromossomos Humanos Par 2 , Proteínas de Ligação ao GTP/metabolismo , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G , Receptores de Neurotensina/genética , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , Clonagem Molecular , Sequência Conservada , Evolução Molecular , Expressão Gênica , Humanos , Dados de Sequência Molecular , Receptores de Grelina , Homologia de Sequência de AminoácidosRESUMO
Small synthetic molecules termed growth hormone secretagogues (GHSs) act on the pituitary gland and the hypothalamus to stimulate and amplify pulsatile growth hormone (GH) release. A heterotrimeric GTP-binding protein (G protein)-coupled receptor (GPC-R) of the pituitary and arcuate ventro-medial and infundibular hypothalamus of swine and humans was cloned and was shown to be the target of the GHSs. On the basis of its pharmacological and molecular characterization, this GPC-R defines a neuroendocrine pathway for the control of pulsatile GH release and supports the notion that the GHSs mimic an undiscovered hormone.