Cloning and functional pharmacology of two corticotropin-releasing factor receptors from a teleost fish

Although it is well established that fish possess corticotropin-releasing f actor (CRF) and a CRF-like peptide, urotensin I, comparatively little is kn own about the pharmacology of their cognate receptors. Here we report the i solation and functional expression of two complementary DNAs (cDNAs), from the chum salmon Oncorhynchus keta, which encode orthologues of the mammalia n and amphibian CRF type 1 (CRF) and type 2 (CRF2) receptors. Radioligand c ompetition binding experiments have revealed that the salmon CRF1 and CRF2 receptors bind urotensin I with similar to 8-fold higher affinity than rat/ human CRF. These two peptides. together with two related CRF-like peptides, namely, sauvagine and urocortin, were also tested in cAMP assays; for cell s expressing the salmon CRF1 receptor, EC50 values for the stimulation of c AMP production were between 4.5 +/- 1.8 and 15.3 +/- 3.1 nM. For the salmon CRF2 receptor, the corresponding values were: rat/human CRF, 9.4 +/- 0.4 n M; urotensin I, 21.2 +/- 2.1 nM; sauvagine, 0.7 +/- 0.1 nM; and urocortin, 2.2 +/- 0.7 nM. We have also functionally coupled the O. keta CRF1 receptor , in Xenopus laevis oocytes, to the endogenous Ca2+-activated chloride cond uctance by co-expression with the G-protein alpha subunit, G(alpha 16). The EC50 value for channel activation by rat/human CRF (11.2 +/- 2.6 nM) agree s well with that obtained in cAMP assays (15.3 +/- 3.1 nM). We conclude tha t although sauvagine is 13- and 30-fold more potent than rat/human CRF and urotensin I, respectively, in activating the salmon CRF2 receptor, neither receptor appears able to discriminate between the native ligands. CRF and u rotensin I. (C) 2001 Elsevier Science B.V. All rights reserved.