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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-affinity ligands for hCRF-R1 but surprisingly weak ligands for xCRF-R1, chimeric receptors of hCRF-R1 and xCRF-R1 followed by double or multiple 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 ∼30-fold higher affinity for human/rat CRF than for sauvagine. Mutagenesis of these amino acids in xCRF-R1 to the human sequence completely abolished the ligand selectivity of xCRF-R1. Mutagenesis of amino acids 88 and 89 in hCRF-R1 or xCRF-R1 had only a minor (∼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 ∼11-fold higher affinity for human/rat CRF compared with ovine CRF or sauvagine. When only 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.