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J. Biol. Chem., Vol. 283, Issue 7, 4387-4394, February 15, 2008

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Efficient Coupling of Transducin to Monomeric Rhodopsin in a Phospholipid Bilayer^*

Matthew R. Whorton, Beata Jastrzebska, Paul S.-H. Park, Dimitrios Fotiadis^¶, Andreas Engel^¶, Krzysztof Palczewski, and Roger K. Sunahara¹

From the Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0632, the Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, and ^¶M. E. Müller Institute for Microscopy, University of Basel, CH-4056 Basel, Switzerland

G protein-coupled receptors (GPCRs) are seven transmembrane domain proteins that transduce extracellular signals across the plasma membrane and couple to the heterotrimeric family of G proteins. Like most intrinsic membrane proteins, GPCRs are capable of oligomerization, the function of which has only been established for a few different receptor systems. One challenge in understanding the function of oligomers relates to the inability to separate monomeric and oligomeric receptor complexes in membrane environments. Here we report the reconstitution of bovine rhodopsin, a GPCR expressed in the retina, into an apolipoprotein A-I phospholipid particle, derived from high density lipoprotein (HDL). We demonstrate that rhodopsin, when incorporated into these 10 nm reconstituted HDL (rHDL) particles, is monomeric and functional. Rhodopsin·rHDL maintains the appropriate spectral properties with respect to photoactivation and formation of the active form, metarhodopsin II. Additionally, the kinetics of metarhodopsin II decay is similar between rhodopsin in native membranes and rhodopsin in rHDL particles. Photoactivation of monomeric rhodopsin·rHDL also results in the rapid activation of transducin, at a rate that is comparable with that found in native rod outer segments and 20-fold faster than rhodopsin in detergent micelles. These data suggest that monomeric rhodopsin is the minimal functional unit in G protein activation and that oligomerization is not absolutely required for this process.

Received for publication, April 23, 2007 , and in revised form, October 25, 2007.

^* This work was supported in part by United States Public Health Service Grant NEI EY08061 from the National Institutes of Health (to K. P.), NIGMS Grants GM079191 (to K. P.) and GM068603 (to R. K. S.) from the National Institutes of Health, the Michigan Diabetes Research and Training Center NIDDK Grant P60DK-20572 (to R. K. S.) from the National Institutes of Health, and the University of Michigan Biological Sciences Scholars Program (to R. K. S.). 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 supplemental Fig. 7.

¹ To whom correspondence should be addressed: 1301 Medical Sciences Research Bldg. III, Ann Arbor, MI 48109. Tel.: 734-647-6277; Fax: 734-763-4450; E-mail: sunahara{at}umich.edu.

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