Dautzenberg F M, Wille S, Lohmann R, Spiess J
Max-Planck Institute for Experimental Medicine, Department of Molecular Neuroendocrinology, Hermann-Rein-Strasse 3, 37075 Goettingen, Germany.
Proc Natl Acad Sci U S A. 1998 Apr 28;95(9):4941-6. doi: 10.1073/pnas.95.9.4941.
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 Arg76, Asn81, Gly83, Leu88, and Ala89 in hCRF-R1 with the corresponding amino acids of xCRF-R1 (Gln76, Gly81, Val83, His88, and Leu89) resulted in a receptor that had approximately 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 (approximately 2.5-fold) effect on the ligand selectivity of the mutant receptor. Substitution of Arg76, Asn81, and Gly83 in hCRF-R1 with the corresponding sequence of xCRF-R1 (Gln76, Gly81, and Val83) resulted in a receptor with approximately 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.
对非选择性的人类促肾上腺皮质激素释放因子受体1（hCRF-R1）和配体选择性的非洲爪蟾促肾上腺皮质激素释放因子受体1（xCRF-R1）进行了比较。为了解铃蟾肽和绵羊促肾上腺皮质激素释放因子（CRF）的相互作用，二者都是hCRF-R1的高亲和力配体，但令人惊讶的是对xCRF-R1而言却是弱配体，构建了hCRF-R1和xCRF-R1的嵌合受体，随后进行双点或多点突变。结合研究和环磷酸腺苷（cAMP）分析表明，N端结构域表现出xCRF-R1完整的配体选择性。重要区域定位在氨基酸70至89之间；将hCRF-R1中的氨基酸精氨酸76、天冬酰胺81、甘氨酸83、亮氨酸88和丙氨酸89替换为xCRF-R1的相应氨基酸（谷氨酰胺76、甘氨酸81、缬氨酸83、组氨酸88和亮氨酸89），得到一种对人/大鼠CRF的亲和力比对铃蟾肽高约30倍的受体。将xCRF-R1中的这些氨基酸突变为人类序列完全消除了xCRF-R1的配体选择性。对hCRF-R1或xCRF-R1中的氨基酸88和89进行诱变，对突变受体的配体选择性只有轻微（约2.5倍）影响。将hCRF-R1中的精氨酸76、天冬酰胺81和甘氨酸83替换为xCRF-R1的相应序列（谷氨酰胺76、甘氨酸81和缬氨酸83），得到一种对人/大鼠CRF的亲和力比对绵羊CRF或铃蟾肽高约11倍的受体。当这三个氨基酸中只有两个发生突变时，未观察到对配体选择性有影响。基于这些数据，提示促肾上腺皮质激素释放因子受体1的氨基酸70 - 89对配体结合位点很重要。
