title: Residues of corticotropin releasing factor-binding protein (CRF-BP) that selectively abrogate binding to CRF but not to urocortin 1.
abstract: Corticotropin releasing factor-binding protein (CRF-BP) binds CRF and urocortin 1 (Ucn 1) with high affinity, thus preventing CRF receptor (CRFR) activation. Despite recent progress on the molecular details that govern interactions between CRF family neuropeptides and their cognate receptors, little is known concerning the mechanisms that allow CRF-BP to bind CRF and Ucn 1 with picomolar affinity. We conducted a comprehensive alanine scan of 76 evolutionarily conserved residues of CRF-BP and identified several residues that differentially affected the affinity for CRF over Ucn 1. We determined that both neuropeptides derive their similarly high affinity from distinct binding surfaces on CRF-BP. Alanine substitutions of arginine 56 (R56A) and aspartic acid 62 (D62A) reduce the affinity for CRF by approximately 100-fold, while only marginally affecting the affinity for Ucn 1. The selective reduction in affinity for CRF depends on glutamic acid 25 in the CRF peptide, as substitution of Glu(25) reduces the affinity for CRF-BP by approximately 2 orders of magnitude, but only in the presence of both Arg(56) and Asp(62) in human CRF-BP. We show that CRF-BP(R56A) and CRF-BP(D62A) have lost the ability to inhibit CRFR1-mediated responses to CRF that activate luciferase induction in HEK293T cells and ACTH release from cultured rat anterior pituitary cells. In contrast, both CRF-BP mutants retain the ability to inhibit Ucn 1-induced CRFR1 activation. Collectively our findings demonstrate that CRF-BP has distinct and separable binding surfaces for CRF and Ucn 1, opening new avenues for the design of ligand-specific antagonists based on CRF-BP.
authors.affiliation: Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA. huising@salk.edu
authors.forename: Mark O
authors.initials: MO
authors.lastname: Huising
authors.forename: Joan M
authors.initials: JM
authors.lastname: Vaughan
authors.forename: Shaili H
authors.initials: SH
authors.lastname: Shah
authors.forename: Katherine L
authors.initials: KL
authors.lastname: Grillot
authors.forename: Cynthia J
authors.initials: CJ
authors.lastname: Donaldson
authors.forename: Jean
authors.initials: J
authors.lastname: Rivier
authors.forename: Gert
authors.initials: G
authors.lastname: Flik
authors.forename: Wylie W
authors.initials: WW
authors.lastname: Vale
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites / Q000502:physiology; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D003346:Corticotropin-Releasing Hormone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020125:Mutation, Missense; D010449:Peptide Mapping / Q000379:methods; D011485:Protein Binding / Q000502:physiology; D054832:Urocortins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D054832:Urocortins; C067133:corticotropin releasing factor-binding protein; D003346:Corticotropin-Releasing Hormone