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J. Biol. Chem., Vol. 278, Issue 49, 48890-48897, December 5, 2003

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Direct Observation of G-protein Binding to the Human -Opioid Receptor Using Plasmon-Waveguide Resonance Spectroscopy^*

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

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

Using a recently developed method (Salamon, Z., Macleod, H. A., and Tollin, G. (1997) Biophys. J. 73, 2791–2797), plasmon-waveguide resonance spectroscopy, we have been able, for the first time, to directly measure the binding between the human brain -opioid receptor (hDOR) and its G-protein effectors in real-time. We have found that the affinity of the G-proteins toward the receptor is highly dependent on the nature of the ligand pre-bound to the receptor. The highest affinity was observed when the receptor was bound to an agonist (10 nM); the lowest when receptor was bound to an antagonist (500 nM); and no binding at all was observed when the receptor was bound to an inverse agonist. We also have found direct evidence for the existence of an additional G-protein binding conformational state that corresponds to the unliganded receptor, which has a G-protein binding affinity of 60 nM. Furthermore, GTP binding to the receptor·G-protein complex was only observed when the agonist was pre-bound. Similar studies were carried out using the individual G-protein subtypes for both the agonist and the unliganded receptor. Significant selectivity toward the different G-protein subtypes was observed. Thus, the unliganded receptor had highest affinity toward the G_o (K_d 20 nM) and lowest affinity toward the G_i2 (590 nM) subtypes, whereas the agonist-bound state had highest affinity for the G_o and G_i2 subtypes (K_d 9 nM and 7 nM, respectively). GTP binding was also highly selective, both with respect to ligand and G-protein subtype. We believe that this methodology provides a powerful new way of investigating transmembrane signaling.

Received for publication, June 27, 2003 , and in revised form, September 11, 2003.

^* This work was supported by a grant from the Vice President for Research, University of Arizona (to G. T. and V. J. H.), by National Institutes of Health (NIH) Grant GM59630 (to G. T. and Z. S.), and by United States Public Health Services and NIDA, NIH Grants DA-06284 and DA-13449 (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.

The on-line version of this article (available at http://www.jbc.org) contains the "Methods" section.

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

^** To whom correspondence should 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.

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