Evidence for the abundant expression of arginine 185 containing human CRF2alpha receptors and the role of position 185 for receptor-ligand selectivity

The abundance of a histidine residue at position 185 (His(185)) of the huma n corticotropin-releasing factor (CRF) type 2 alpha receptor (hCRF(2 alpha) ) was investigated. His(185) has only been reported in hCRF(2); CRF2 protei ns from other species and all CRF1 receptors encode an arginine (Arg(185)) at the corresponding position. Cloning of partial and full-length hCRF(2) c DNAs from a variety of neuronal and peripheral tissues revealed the existen ce of receptor molecules encoding Arg(185) only. Sequence analysis of the h CRF2 gene verified the existence of Arg(185) also on genomic level. Full-le ngth cDNAs encoding either the His(185) (R2H(185)) or the Arg(185) (R2R(185 )) variants of hCRF(2 alpha) were stably expressed in HEK293 cells and test ed for ligand binding properties. In displacement studies R2H(185) and R2R( 185) displayed a similar substrate specificity, human and rat urocortin, an d the peptide antagonists astressin and alpha-helical CRF(9-41) were bound with high affinity whereas human and ovine CRF were low-affinity ligands. S ignificant differences were observed for sauvagine and urotensin I, which b ound with 3-fold (sauvagine) and 9-fold (urotensin I) higher affinity to R2 R(185). These data indicate that hCRF(2), like all vertebrate CRF1 and CRF2 proteins encodes an arginine residue at the junction between extracellular domain 2 and transmembrane domain 3 and that this amino acid plays a role fur the discrimination of some CRF peptide ligands. (C) 2000 Elsevier Scien ce Ltd. All rights reserved.