MAPPING OF THE LIGAND-SELECTIVE DOMAIN OF THE XENOPUS-LAEVIS CORTICOTROPIN-RELEASING FACTOR-RECEPTOR-1 - IMPLICATIONS FOR THE LIGAND-BINDING SITE

The nonselective human corticotropin-releasing factor receptor 1 (hCRF -R1) and the ligand-selective Xenopus CRF-R1 (xCRF-R1) were compared. To understand the interactions of sauvagine and ovine CRF, both high-a ffinity ligands for hCRF-R1 but surprisingly weak ligands for xCRF-R1, chimeric receptors of hCRF-R1 and xCRF-R1 followed by double or multi ple point mutations were constructed. Binding studies and CAMP assays demonstrated that the N-terminal domain exhibited the complete ligand selectivity of xCRF-R1, The important region was mapped between amino acids 70 and 89; replacement of amino acids Arg(76), Asn(81), Gly(83), Leu(88), and Ala(89) in hCRF-R1 with the corresponding amino acids of xCRF-R1 (Gln(76), Gly(81), Val(83), His(88), and Leu(89)) resulted in a receptor that had similar to 30-fold higher affinity fbr human/rat CRF than for sauvagine, Mutagenesis of these amino acids in xCRF-R1 to the human sequence completely abolished the ligand selectivity of xCR F-R1, Mutagenesis of amino acids 88 and 89 in hCRF-R1 or xCRF-R1 had o nly a minor (similar to 2.5-fold) effect on the ligand selectivity of the mutant receptor. Substitution of Arg(76), Asn(81), and Gly(83) in hCRF-R1 with the corresponding sequence of xCRF-R1 (Gln(76), Gly(81), and Val(83)) resulted in a receptor with similar to 11-fold higher aff inity for human/rat CRF compared with ovine CRF or sauvagine, When onl y two of these three amino acids were mutated, no effect on the ligand selectivity was observed. On the basis of these data, it is suggested that amino acids 70-89 of CRF-R1 are important for the ligand binding site.