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J. Biol. Chem., Vol. 281, Issue 37, 27072-27080, September 15, 2006

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Fluorescent Indicators Distributed throughout the Pharmacophore of Cholecystokinin Provide Insights into Distinct Modes of Binding and Activation of Type A and B Cholecystokinin Receptors^*

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

Ligand probes with fluorescent indicators positioned throughout the pharmacophoric domain can provide important insights into the molecular basis of receptor binding and activation as reflected in the microenvironment of each indicator while docked at a receptor. We developed three cholecystokinin-like probes with Aladan situated at the N terminus, in the mid-region, and at the C terminus (positions 24, 29, and 33, respectively). These were studied in solution and docked at type A and B cholecystokinin receptors. This study demonstrated clear differences in mechanisms of cholecystokinin binding and activation of these structurally related receptors with distinct agonist structure-activity relationships. The fluorescence characteristics of Aladan are highly sensitive to the polarity of its microenvironment. The mid-region probe was least accessible to the aqueous milieu as determined by fluorescence emission spectra and iodide quenching, which was not altered by changes in conformation from active to inactive. Accessibility of the N- and C-terminal probes was affected by receptor conformation. The position 24 probe was more easily quenched in the active than in the G protein-uncoupled conformation for both receptors. However, the position 33 probe docked at the type A cholecystokinin receptor was more easily quenched in the active conformation, whereas the same probe docked at the type B cholecystokinin receptor was more easily quenched in the inactive conformation. Fluorescence anisotropy and red edge excitation shift determinations confirmed these observations and supported the proposed movements. Although both type A and B cholecystokinin receptors bind cholecystokinin with high affinity, resulting in fully efficacious biological responses, these receptors utilize distinct molecular modes of binding.

Received for publication, May 30, 2006 , and in revised form, July 18, 2006.

^* This work was supported by National Institutes of Health Grant DK32878 and 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 To whom correspondence should be addressed: Cancer Center, Mayo Clinic, 13400 East Shea Blvd., Scottsdale, AZ 85259. Tel.: 480-301-6650; Fax: 480-301-6969; E-mail: miller{at}mayo.edu.

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