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J. Biol. Chem., Vol. 280, Issue 2, 1044-1050, January 14, 2005

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Distinct Molecular Mechanisms for Agonist Peptide Binding to Types A and B Cholecystokinin Receptors Demonstrated Using Fluorescence Spectroscopy^*

Kaleeckal G. Harikumar, Jeremy Clain, Delia I. Pinon, Maoqing Dong, and Laurence J. Miller

From the Cancer Center and Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic Scottsdale, Scottsdale, Arizona 85259

Fluorescence spectroscopy provides a direct method for evaluating the environment of a fluorescent ligand bound to its receptor. We utilized this methodology to determine the environment of Alexa within a cholecystokinin (CCK)-like probe (Alexa⁴⁸⁸-Gly-[(Nle^28,31)CCK-26–33]; CCK-8 probe) bound to the type A CCK receptor (Harikumar, K. G., Pinon, D. L., Wessels, W. S., Prendergast, F. G., and Miller, L. J. (2002) J. Biol. Chem. 277, 18552–18560). Here, we study this probe at the type B CCK receptor and develop another probe with its fluorophore closer to the carboxyl-terminal pharmacophore of type B receptor ligands (Alexa⁴⁸⁸-Trp-Nle-Asp-Phe-NH₂; CCK-4 probe). Both probes bound to type B CCK receptors in a saturable and specific manner and represented full agonists. Similar to the type A receptor, at the type B receptor these probes exhibited shorter lifetimes and lower anisotropy when the receptor was in the active conformation than when it was shifted to its inactive, G protein-uncoupled state using guanosine 5'-[,-imido]-triphosphate trisodium salt. Absolute values for lifetime and anisotropy were lower for the CCK-8 probe bound to the type B receptor than for this probe bound to the type A receptor, and Alexa fluorescence was more easily quenched by iodide at the type B receptor. This represents the first direct evidence that, despite having identical affinities for binding and potencies for activating type A and B receptors, CCK is docked via distinct mechanisms, with the amino terminus more exposed to the aqueous milieu when bound to the type B CCK receptor than to the type A CCK receptor. Of interest, despite this difference in binding, activation of both receptors results in analogous direction of movement of the fluorescent indicator probes.

Received for publication, August 18, 2004 , and in revised form, October 12, 2004.

^* This work was supported by Grant DK32878 from the National Institutes of Health and a grant from 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.

To whom correspondence should be addressed: Cancer Center, Mayo Clinic Scottsdale, 13400 East Shea Blvd., Scottsdale, AZ 85259. Tel.: 480-301-4160; Fax: 480-301-4596; E-mail: miller{at}mayo.edu.

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This article has been cited by other articles:

				K. G. Harikumar, D. I. Pinon, and L. J. Miller Fluorescent Indicators Distributed throughout the Pharmacophore of Cholecystokinin Provide Insights into Distinct Modes of Binding and Activation of Type A and B Cholecystokinin Receptors J. Biol. Chem., September 15, 2006; 281(37): 27072 - 27080. [Abstract] [Full Text] [PDF]

				K. G. Harikumar, K. Hosohata, D. I. Pinon, and L. J. Miller Use of Probes with Fluorescence Indicator Distributed throughout the Pharmacophore to Examine the Peptide Agonist-binding Environment of the Family B G Protein-coupled Secretin Receptor J. Biol. Chem., February 3, 2006; 281(5): 2543 - 2550. [Abstract] [Full Text] [PDF]