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J. Biol. Chem., Vol. 282, Issue 45, 32834-32843, November 9, 2007

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Fluorescence Resonance Energy Transfer Analysis of Secretin Docking to Its Receptor

MAPPING DISTANCES BETWEEN RESIDUES DISTRIBUTED THROUGHOUT THE LIGAND PHARMACOPHORE AND DISTINCT RECEPTOR RESIDUES^*

Kaleeckal G. Harikumar, Polo C.-H. Lam, Maoqing Dong, Patrick M. Sexton^¶1, Ruben Abagyan, and Laurence J. Miller²

From the Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona 85259, the Department of Molecular Biology, Scripps Research Institute and Molsoft LLC, La Jolla, California 92037, and the ^¶Department of Pharmacology, Monash University, Clayton, Victoria 3800, Australia

Full structural characterization of G protein-coupled receptors has been limited to rhodopsin, with its uniquely stable structure and ability to be crystallized. For other members of this important superfamily, direct structural insights have been limited to NMR structures of soluble domains. Two members of the Class II family have recently had the structures of their isolated amino-terminal regions solved by NMR, yet it remains unclear how that domain is aligned with the heptahelical transmembrane bundle domain of those receptors. Indeed, three distinct orientations have been suggested for different members of this family. In the current work, we have utilized fluorescence resonance energy transfer to establish the distances between four residues distributed throughout fully biologically active, high affinity analogues of secretin and distinct residues in each of four extracellular regions of the intact secretin receptor. These 16 distance constraints were utilized along with nine photoaffinity labeling spatial approximation constraints to study the three proposed orientations of the peptide-binding amino terminus and helical bundle domains of this receptor. In the best model, the carboxyl terminus of secretin was found to bind in a groove above the -hairpin region of the receptor amino terminus, with its amino-terminal end adjacent to the third extracellular loop and top of transmembrane segment VI. This refined model of the intact receptor was also fully consistent with the spatial approximation of the Trp⁴⁸-Asp⁴⁹-Asn⁵⁰ endogenous agonist segment with the third extracellular loop region that it has been shown to photolabel. This provides strong evidence for the orientation of peptide-binding and signaling domains of a prototypic Class II G protein-coupled receptor.

Received for publication, June 4, 2007 , and in revised form, August 21, 2007.

^* This work was supported by National Institutes of Health Grant DK46577 (to L. J. M.), National Health and Medical Research Council of Australia (NHMRC) Grant 436780 (to P. M. S.), and by the Fiterman Foundation. 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.

¹ An NHMRC Principal Research Fellow.

² To whom correspondence should be addressed: Mayo Clinic, 13400 E. Shea Blvd., Scottsdale, AZ 85259. Tel.: 480-301-6650; Fax: 480-301-6969; E-mail: miller{at}mayo.edu.

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