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Sheng Wu, Graeme J Roch, Laura A Cervini, Jean E Rivier, and Nancy M Sherwood

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 (EC50=1.8×10−9 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 (EC50=7.4×10−9 M) supports our suggestion that the VPAC2R and PHIR shared a common ancestral receptor.

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P J Lowry, S C Koerber, R J Woods, S Baigent, S Sutton, D P Behan, W Vale, and J Rivier


As the association of corticotrophin-releasing factor (CRF) with its binding protein (BP) to form a dimer complex (CRF2/BP2) appears to be dependent on the nature of the ligand we have compared the circular dichroism difference spectra after association of the BP with ovine (o) CRF, human (h) CRF and the α-helical CRF(9–41) antagonist. All three ligands caused a negative change in molar ellipticity above 210 nm, with oCRF having the least and hCRF the greatest effect. Below 210 nm there was a marked divergence of difference spectra, with the reaction with the natural peptides, hCRF and oCRF, resulting in a positive change in ellipticity, whilst that with the antagonist produced a negative change. In view of the BP spectrum indicating predominantly β-sheet and the peptides showing mainly α-helix these results were interpreted as the changes above 210 nm being due to dimerization and below 210 nm to a change in the conformation of ligand on binding. The opposite change in α-helicity of the antagonist observed on binding compared with the two natural CRF peptides could have fundamental pharmacological implications.