THE FIRST EXTRACELLULAR DOMAIN OF CORTICOTROPIN-RELEASING FACTOR-R1 CONTAINS MAJOR BINDING DETERMINANTS FOR UROCORTIN AND ASTRESSIN

The CRF receptors are members of a Ti-transmembrane receptor family th at Includes GK-releasing hormone (GRF), calcitonin, vasoactive intesti nal peptide (VIP), secretin, and PTH receptors. To determine the struc tural features of the CRF receptor that may influence ligand recogniti on, a series of mutant receptors was analyzed for binding to astressin , a CRF antagonist, and to urocortin, a CRF agonist. Mutant receptors included chimeras between the CRF-R1 and GRF-R or Activin IIB-R, a sin gle membrane spanning receptor serine/threonine kinase. Binding to the mutant receptors was assessed using I-125-[DTyr(1)] astressin (Ast) and I-125-[Tyr(0)]-rat urocortin (Ucn). There was no binding to a chi meric receptor in which the first extracellular domain (E1(c)) (i.e. t he N-terminal region) of the CRF-RI was replaced by that of the GRF-R. The complementary chimera in which El domain of the GRF-R was replace d by that of the CRF-R1 bound astressin and urocortin with K-i values approximately 10 nM, compared with inhibitory binding dissociation con stant (K-i) values of approximately 2-4 nhl for the wild-type CRF-R1. The chimera in which El of the activin LIE: receptor was replaced by E l of the CRF-R1 bound astressin with a K-i approximately 4 nM. A chime ra in which both the first and fourth extracellular domains of the CRF -R1 replaced the corresponding domains of the GRF-R bound astressin wi th K-i approximately 4 nM and urocortin with a K-i approximately 2 avl . A chimera in which all four extracellular domains of the CRF recepto r replaced those of the GRF-R bound astressin and urocortin with K-i v alues approximately 4 nM and approximately I nM, respectively. In conc lusion? the major determinants for high affinity binding of CRF agonis ts and antagonists to CRF-RI are found in the first extracellular doma in of the receptor.