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J. Biol. Chem., Vol. 283, Issue 14, 8902-8912, April 4, 2008

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Residues of Corticotropin Releasing Factor-binding Protein (CRF-BP) That Selectively Abrogate Binding to CRF but Not to Urocortin 1^*

Mark O. Huising¹, Joan M. Vaughan, Shaili H. Shah, Katherine L. Grillot, Cynthia J. Donaldson, Jean Rivier, Gert Flik, and Wylie W. Vale²

From the Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, La Jolla, California 92037 and the Department of Animal Physiology, Radboud University Nijmegen, 6525 ED Nijmegen, The Netherlands

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 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 Glu²⁵ reduces the affinity for CRF-BP by approximately 2 orders of magnitude, but only in the presence of both Arg⁵⁶ and Asp⁶² 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.

Received for publication, December 4, 2007 , and in revised form, January 9, 2008.

^* This work was supported in part by the Adler Foundation, the Clayton Medical Research Foundation, Inc., and NIDDK, Grant P01 DK26741 from the National Institutes of Health. The following have been licensed by The Salk Institute for Biological Studies and/or The Clayton Foundation: CRF to Ferring Pharmaceuticals, CRF1 receptor and Ucn 2 to Neurocrin Biosciences, and Ucn 3 to Johnson & Johnson. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3 and Table S1.

² CMRF, Inc., Senior Investigator and cofounder, consultant, equity holder, and member of the Board of Directors of Neurocrine Biosciences and Acceleron Pharma.

¹ To whom correspondence should be addressed: 10010 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-453-4100 (ext. 1510); Fax: 858-552-1546; E-mail: huising{at}salk.edu.

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