RESUMO
A group of ten hormones in humans are structurally related and known as the secretin superfamily. These hormones bind to G-protein-coupled receptors that activate the cAMP pathway and are clustered as the secretin or B family. We used an evolutionary approach with zebrafish as a model to understand why some of these hormones, such as peptide histidine-methionine (PHM) and pituitary adenylate cyclase-activating polypeptide (PACAP)-related peptide (PRP) in humans lack a receptor. We used molecular techniques to clone two full-length receptor cDNAs in zebrafish, which were analyzed for amino acid sequence and ligand-binding motifs, phylogenetic position, synteny, tissue expression, functional response, and signaling pathway. Evidence is provided that the two cDNAs encoded the peptide histidine-isoleucine (PHI) receptor and PRP receptor, which is known as GHRH-like peptide (GHRH-LP) receptor in non-mammals. Further, we cloned a zebrafish cDNA encoding the peptides PHI and vasoactive intestinal peptide (VIP). The PHIR had been previously labeled as one type of a VIP-PACAP (VPAC2R) shared receptor based only on sequence data. The PHIR cDNA, transfected into COS7 cells, responded to zebrafish PHI in a sensitive and dose-dependent manner (EC(50)=1.8x10(-9) M) but not to PACAP and VIP. The GHRH-LP receptor responded to both zebrafish GHRH-LP1 and GHRH with a 3.5-fold greater response to the former. For comparison, two zebrafish receptors (PAC1R and VPAC1R) and two human receptors (VPAC2R and GHRHR) were tested with human and/or zebrafish peptides. Unexpectedly, zebrafish VIP activated its PAC1R suggesting that in evolution, PAC1R is not always a specific receptor for PACAP. We conclude that zebrafish, like goldfish, have a specific receptor for PHI and GHRH-LP. Our evidence that zebrafish PHI is more potent than human PHM in activating the human VPAC2R (EC(50)=7.4x10(-9) M) supports our suggestion that the VPAC2R and PHIR shared a common ancestral receptor.
Assuntos
Hormônio Liberador de Hormônio do Crescimento/metabolismo , Peptídeo PHI/metabolismo , Receptores de Superfície Celular/metabolismo , Secretina/metabolismo , Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Hormônio Liberador de Hormônio do Crescimento/genética , Humanos , Dados de Sequência Molecular , Peptídeo PHI/genética , Peptídeos/genética , Peptídeos/metabolismo , Filogenia , Receptores de Superfície Celular/classificação , Receptores de Superfície Celular/genética , Secretina/classificação , Secretina/genética , Alinhamento de Sequência , Distribuição Tecidual , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/classificação , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
GnRH agonists or antagonists are currently utilized as therapeutic agents in a number of diseases. A side-effect of prolonged treatment with GnRH analogues is hypoestrogenism. In this study, we tested the in vitro potency of different GnRH analogues originally found to be partial agonists (i.e. analogues with decreased efficacy for activating or stimulating their cognate receptor) as well as novel analogues, to identify compounds that might potentially be useful for partial blockade of gonadotrophin release. Cultured COS-7 cells transiently expressing the rat or human GnRH receptor (GnRHR) were exposed to increasing concentrations (10(-8) to 10(-5) M) of GnRH analogues (c(4-10)[Asp4,DNal6,Dpr10]-GnRH; c(4-10) [Dpr4,DNal6,Asp10]-GnRH; c(4-10)[Cys(4,10),DNal6]-GnRH; c[Eaca1,DNal6]-GnRH; c[Gly1,DNal6]-GnRH; c[betaAla1,DTrp6]-GnRH; c[Dava1,DNal6]-GnRH; c[Gaba1, DNal6]-GnRH), and the ability of these analogues to provoke or antagonize GnRH-stimulated inositol phosphate production was assessed. With both human and rat GnRHRs, c[Eaca1,DNal6]-GnRH, c[Gly1,DNal6]-GnRH, c[betaAla1,DTrp6]-GnRH and c[Dava1,DNal6]-GnRH exhibited partial agonist activity (35-87% of the maximal efficacy shown by 10(-6) M GnRH), whereas c[Gaba1,DNal6]-GnRH behaved as a partial agonist with the human GnRHR and as full agonist with the rat GnRHR. c(4-10)[Asp4, DNal6,Dpr10]-GnRH and c(4-10)[Dpr4,DNal6,Asp10]-GnRH exhibited full antagonist activity with both GnRHRs, and c(4-10) [Cys(4,10),DNal6]-GnRH was a weak, partial agonist with the human GnRHR and a full antagonist with the rat GnRHR. With the exception of c[Gaba1,DNal6]-GnRH stimulation of the human GnRHR, and c[Dava1,DNal6]-GnRH and c[Gaba1, DNal6]-GnRH stimulation of the rat GnRHR, all partial agonists also exhibited antagonist activity in the presence of the exogenous full agonist. The results demonstrate that structurally similar analogues display variable potencies and efficacies in vitro for a specific GnRHR as well as for the human versus the rat GnRHR. Their ultimate in vivo usefulness to treat clinical conditions in which complete suppression of gonadotroph activity is not required remains to be investigated.
