Residue 17 of Sauvagine Cross-links to the First Transmembrane Domain of Corticotropin-releasing Factor Receptor 1 (CRFR1)*
- ‡Endocrine Division, Department of Internal Medicine, Wayne State University School of Medicine, University Health Center, Detroit, Mighigan 48201 and the §Department of Chemistry, Dartmouth College, Hanover, New Hampshire 03755
- 1 To whom correspondence should be addressed: Chief Endocrine Division, Dept. of Internal Medicine, Wayne State University School of Medicine, 4H-UHC, 4201 St. Antoine, Detroit, MI 48201. Tel.: 313-745-4008; Fax: 313-993-0903; E-mail: asamra{at}med.wayne.edu.
Abstract
Corticotropin-releasing factor receptor 1 (CRFR1) mediates the physiological actions of corticotropin-releasing factor in the anterior pituitary gland and the central nervous system. Using chemical cross-linking we have previously reported that residue 16 of sauvagine (SVG) is in a close proximity to the second extracellular loop of CRFR1. Here we introduced p-benzoylphenylalanine (Bpa) at position 17 of a sauvagine analog, [Tyr0, Gln1, Bpa17]SVG, to covalently label CRFR1 and characterize the cross-linking site. Using a combination of receptor mutagenesis, peptide mapping, and N-terminal sequencing, we identified His117 within the first transmembrane domain (TM1) of CRFR1 as the cross-linking site for Bpa17 of 125I-[Tyr0, Gln1, Bpa17]SVG. These data indicate that, within the SVG-CRFR1 complex, residue 17 of the ligand lies withina9Å distance from residue 117 of the TM1 of CRFR1. The molecular proximity between residue 17 of the ligand and TM1 of CRFR1 described here and between residue 16 of the ligand and the CRFR1 second extracellular loop described previously provides useful molecular constraints for modeling ligand-receptor interaction in mammalian cells expressing CRFR1.
Footnotes
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↵2 The abbreviations used are: CRF, corticotropin-releasing factor; SVG, sauvagine; UCN, urocortin; CRFR1, CRF receptor type 1; Bpa, p-benzoylphenylalanine; MAPK, mitogen-activated protein kinase; J-domain, juxtamembrane domain; TM, transmembrane-spanning domain; HPLC, high pressure liquid chromatography; m, murine; PBS, phosphate-buffered saline; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PTH, parathyroid hormone; MM, molecular mass(es).
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↵* This work was supported, in whole or in part, by National Institutes of Health Grants 7RO1 DK63211 (to A. B. A.-S.) and GM54082 (to D. F. M.). 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.
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- Received August 18, 2008.
- Revision received October 3, 2008.
- The American Society for Biochemistry and Molecular Biology, Inc.
