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Measurement of Intermolecular Distances for the Natural Agonist Peptide Docked at the Cholecystokinin Receptor Expressed in Situ Using Fluorescence Resonance Energy Transfer

Mayo Clinic Scottsdale, Cancer Center and Department of Molecular Pharmacology and Experimental Therapeutics, Scottsdale, Arizona (K.G.H., D.I.P., L.J.M.); Mayo Clinic, Department of Molecular Pharmacology and Experimental Therapeutics, Rochester, Minnesota (W.S.W., F.G.P.), and Vanderbilt University, Department of Chemistry and Center for Structural Biology, Nashville, Tennessee (E.S.D., T.P.L.)

Fluorescence resonance energy transfer is a powerful biophysical technique used to analyze the structure of membrane proteins. Here, we used this tool to determine the distances between a distinct position within a docked agonist and a series of distinct sites within the intramembranous confluence of helices and extracellular loops of the cholecystokinin (CCK) receptor. Pseudo-wild-type CCK receptor constructs having single reactive cysteine residues inserted into each of these sites were developed. The experimental strategy included the use of the full agonist, Alexa⁴⁸⁸-CCK, bound to these receptors as donor, with Alexa⁵⁶⁸ covalently bound to the specific sites within the CCK receptor as acceptor. Site-labeling was achieved by derivatization of intact cells with a novel fluorescent methanethiosulfonate reagent. A high degree of spectral overlap was observed between receptor-bound donor and receptor-derivatized acceptors, with no transfer observed for a series of controls representing saturation of the receptor binding site with nonfluorescent ligand and use of a null-reactive CCK receptor construct. The measured distances between the fluorophore within the docked agonist and the sites within the first (residue 102) and third (residue 341) extracellular loops of the receptor were shorter than those directed to the second loop (residue 204) or to intramembranous helix two (residue 94). These distances were accommodated well within a refined molecular model of the CCK-occupied receptor that is fully consistent with all existing structure-activity and photoaffinity-labeling studies. This approach provides the initial insights into the conformation of extracellular loop regions of this receptor and establishes clear differences from analogous loops in the rhodopsin crystal structure.

Received May 19, 2003; accepted September 26, 2003.

Address correspondence to: Dr. Laurence J. Miller, Director, Cancer Center, Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic Scottsdale, 13400 East Shea Boulevard, Scottsdale, AZ 85259. E-mail: miller{at}mayo.edu

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