title: The first extracellular domain of corticotropin releasing factor-R1 contains major binding determinants for urocortin and astressin.
abstract: 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 (E1c) (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 Ki values approximately 10 nM, compared with inhibitory binding dissociation constant (Ki) 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 Ki 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 Ki approximately 4 nM and urocortin with a Ki 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 Ki 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.
authors.affiliation: The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California 92037-1099, USA. perrin@salk.edu
authors.forename: M H
authors.initials: MH
authors.lastname: Perrin
authors.forename: S
authors.initials: S
authors.lastname: Sutton
authors.forename: D L
authors.initials: DL
authors.lastname: Bain
authors.forename: W T
authors.initials: WT
authors.lastname: Berggren
authors.forename: W W
authors.initials: WW
authors.lastname: Vale
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D002678:Chimera; D003346:Corticotropin-Releasing Hormone / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D051381:Rats; D018019:Receptors, Corticotropin-Releasing Hormone / Q000235:genetics* / Q000378:metabolism*; D054832:Urocortins
chemical_list: D010446:Peptide Fragments; D018019:Receptors, Corticotropin-Releasing Hormone; D054832:Urocortins; C104416:astressin; D003346:Corticotropin-Releasing Hormone