title: Cloning and functional pharmacology of two corticotropin-releasing factor receptors from a teleost fish.
abstract: Although it is well established that fish possess corticotropin-releasing factor (CRF) and a CRF-like peptide, urotensin I, comparatively little is known about the pharmacology of their cognate receptors. Here we report the isolation and functional expression of two complementary DNAs (cDNAs), from the chum salmon Oncorhynchus keta, which encode orthologues of the mammalian and amphibian CRF type 1 (CRF(1)) and type 2 (CRF(2)) receptors. Radioligand competition binding experiments have revealed that the salmon CRF(1) and CRF(2) receptors bind urotensin I with approximately 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 cells expressing the salmon CRF(1) receptor, EC(50) values for the stimulation of cAMP production were between 4.5+/-1.8 and 15.3+/-3.1 nM. For the salmon CRF(2) receptor, the corresponding values were: rat/human CRF, 9.4+/-0.4 nM; 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 CRF(1) receptor, in Xenopus laevis oocytes, to the endogenous Ca(2+)-activated chloride conductance by co-expression with the G-protein alpha subunit, G(alpha16). The EC(50) value for channel activation by rat/human CRF (11.2+/-2.6 nM) agrees well with that obtained in cAMP assays (15.3+/-3.1 nM). We conclude that although sauvagine is 13- and 30-fold more potent than rat/human CRF and urotensin I, respectively, in activating the salmon CRF(2) receptor, neither receptor appears able to discriminate between the native ligands CRF and urotensin I.
authors.affiliation: Institut für Zellbiochemie und Klinische Neurobiologie, Universitätsklinikum Hamburg-Eppendorf, Universität Hamburg, Martinistrasse 52, 20246, Hamburg, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Pohl
authors.forename: M G
authors.initials: MG
authors.lastname: Darlison
authors.forename: W C
authors.initials: WC
authors.lastname: Clarke
authors.forename: K
authors.initials: K
authors.lastname: Lederis
authors.forename: D
authors.initials: D
authors.lastname: Richter
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D002460:Cell Line; D003001:Cloning, Molecular; D003346:Corticotropin-Releasing Hormone / Q000378:metabolism / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D005260:Female; D015870:Gene Expression; D006801:Humans; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D008969:Molecular Sequence Data; D017685:Oncorhynchus keta / Q000235:genetics*; D009865:Oocytes; D010802:Phylogeny; D012313:RNA / Q000235:genetics / Q000378:metabolism; D051381:Rats; D018019:Receptors, Corticotropin-Releasing Hormone / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D014579:Urotensins / Q000378:metabolism / Q000494:pharmacology; D014982:Xenopus laevis; D000099052:CRF Receptor, Type 1
chemical_list: C101531:CRF receptor type 2; D018076:DNA, Complementary; D018019:Receptors, Corticotropin-Releasing Hormone; D014579:Urotensins; D000099052:CRF Receptor, Type 1; D012313:RNA; D003346:Corticotropin-Releasing Hormone; C029676:urotensin I; D000242:Cyclic AMP