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J. Biol. Chem., Vol. 279, Issue 43, 44673-44682, October 22, 2004

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Selectivity, Cooperativity, and Reciprocity in the Interactions between the -Opioid Receptor, Its Ligands, and G-proteins^*

Isabel D. Alves, Kathy A. Ciano¶, Valentina Boguslavski||, Eva Varga**, Zdzislaw Salamon, Henry I. Yamamura**, Victor J. Hruby||, and Gordon Tollin||

From the Department of Biochemistry and Molecular Biophysics, the ^¶Department of Molecular and Cellular Biology, the ^**Department of Pharmacology, and the ^||Department of Chemistry, the University of Arizona, Tucson, Arizona 85721

A better understanding of signal transduction mechanisms is of critical importance. Methodologies that allow studies to be done while receptors are incorporated into lipid bilayers are advantageous. One such technique is plasmon-waveguide resonance (PWR) spectroscopy, which can follow changes in conformation accompanying protein-ligand, protein-protein, and protein-lipid interactions occurring in G-protein-coupled receptors in real time with high sensitivity and without the need for molecular labeling. Here we investigated several aspects of human -opioid receptor (hDOR)-G-protein interactions: 1) the effect of different types of agonists on the interaction with individual G-protein subtypes; 2) the affinities of the separate G-protein and subunits to different ligand-occupied states of the receptor; and 3) the effect of the presence of the G-protein on the interactions of the ligand with the receptor. To accomplish this we have incorporated the receptor into a solid supported lipid bilayer in the presence of ligand or G-protein and monitored the PWR spectral changes induced by the reciprocal G-protein or ligand interactions. We found a high degree of selectivity in the interactions of different agonist-bound states of the receptor with the different G-protein subtypes. This has important implications for agonist-directed trafficking and selective drug design. Studies with the separated and subunits show that cooperativity exists in these interactions. The high affinities of the separated subunits to the receptor point to the possibility of independent promotion of specific signaling events. The presence of G-proteins increased the affinity of agonists to the hDOR, and caused faster binding kinetics and different ligand-induced conformational changes. Because ligand also influences G-protein binding, reciprocity exists between these two binding processes.

Received for publication, April 28, 2004 , and in revised form, August 16, 2004.

^* This work was supported in part by grants from Vice-President for Research, University of Arizona (to G. T. and V. J. H.), National Institutes of Health Grant GM59630 (to G. T. and Z. S.), United States Public Health Service Grant DA-06284, and National Institute on Drug Abuse Grant DA13449 (to V. J. H.). 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.

Recipient of a fellowship from the Institute for Biomedical Research and Biotechnology.

To whom correspondence may be addressed: 1306 E. University Blvd., Old Chemistry Bldg., University of Arizona, Tucson, AZ 85721. Tel.: 520-621-6332; Fax: 520-621-8407; E-mail: hruby{at}u.arizona.edu. To whom correspondence may be addressed: 1041 E. Lowell St., Dept. of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, AZ 85721. Tel.: 520-621-3447; Fax: 520-621-9288; E-mail: gtollin{at}u.arizona.edu.

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