IDENTIFICATION OF 2 CORTICOTROPIN-RELEASING FACTOR RECEPTORS FROM XENOPUS-LAEVIS WITH HIGH LIGAND SELECTIVITY - UNUSUAL PHARMACOLOGY OF THETYPE-1 RECEPTOR

Two cDNA clones encoding distinct members of the corticotropin-releasi ng factor (CRF) receptor family have been isolated from Xenopus laevis with PCR-based approaches. The first full-length cDNA amplified from Xenopus brain encoded a 415-amino acid protein with similar to 80% ide ntity to mammalian CRF receptor type I (CRFR-1). The second full-lengt h cDNA isolated from Xenopus brain and heart encoded a 413-amino acid protein with similar to 81 % identity to the alpha-variant of mammalia n CRF receptor, type 2 (CRF-R2). No evidence could be obtained that th e beta-variant of CRF-R2 existed in Xenopus laevis. Binding studies us ing human embryonic kidney 293 (HEK 293) cells stably transfected with xenopus CRF-R2 showed that the CRF analogues urotensin I, urocortin, and sauvagine were bound with higher affinities than human/rat CRF, xe nopus CRF, and ovine CRF. In contrast to human CRF-R1, xenopus CRF-R1 (xCRF-R1) was very selective for different CRF ligands. Urotensin I, u rocortin, human/rat CRF, and xenopus CRF were bound with significantly (10-22-fold) higher affinities than ovine CRF (K-D = 31.7 nM) and sau vagine (K-D = 51.4 nM). In agreement with these binding data, EC50 val ues of 39.7 and 1.1 nM were found for sauvagine and for human/rat CRF or xenopus CRF, respectively, when the cyclic AMP production in HEK 29 3 cells stably transfected with xCRF-R1 was determined.