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The First Extracellular Domain of Corticotropin Releasing Factor-R1 Contains Major Binding Determinants for Urocortin and Astressin¹

The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California 92037-1099

Address all correspondence and requests for reprints to: Marilyn Perrin, Ph.D., The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, California 92037-1099. E-mail: perrin{at}salk.edu

The CRF receptors are members of a 7-transmembrane receptor family that includes GH-releasing hormone (GRF), calcitonin, vasoactive intestinal peptide (VIP), secretin, and PTH receptors. To determine the structural features of the CRF receptor that may influence ligand recognition, 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 single membrane spanning receptor serine/threonine kinase. Binding to the mutant receptors was assessed using ¹²⁵I-[DTyr¹] astressin (Ast*) and ¹²⁵I-[Tyr⁰]-rat urocortin (Ucn*). There was no binding to a chimeric receptor in which the first extracellular domain (E1_c) (i.e. the N-terminal region) of the CRF-R1 was replaced by that of the GRF-R. The complementary chimera in which E1 domain of the GRF-R was replaced by that of the CRF-R1 bound astressin and urocortin with K_i values approximately 10 nM, compared with inhibitory binding dissociation constant (K_i) values of approximately 2–4 nM for the wild-type CRF-R1. The chimera in which E1 of the activin IIB receptor was replaced by E1 of the CRF-R1 bound astressin with a K_i approximately 4 nM. A chimera in which both the first and fourth extracellular domains of the CRF-R1 replaced the corresponding domains of the GRF-R bound astressin with K_i approximately 4 nM and urocortin with a K_i approximately 2 nM. A chimera in which all four extracellular domains of the CRF receptor replaced those of the GRF-R bound astressin and urocortin with K_i values approximately 4 nM and approximately 1 nM, respectively. In conclusion, the major determinants for high affinity binding of CRF agonists and antagonists to CRF-R1 are found in the first extracellular domain of the receptor.

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