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J. Biol. Chem., Vol. 283, Issue 5, 2973-2985, February 1, 2008

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The _1a-Adrenergic Receptor Occupies Membrane Rafts with Its G Protein Effectors but Internalizes via Clathrin-coated Pits^*

Daniel P. Morris¹, Beilei Lei, Yue-Xuan Wu, Gregory A. Michelotti, and Debra A. Schwinn

From the Department of Anesthesiology, Duke University Medical Center, Durham, North Carolina 27710 and the Department of Anesthesiology, Pharmacology, and Genome Sciences, University of Washington, Seattle, Washington 98195

The _1a-adrenergic receptor (_1aAR) occupies intracellular and plasma membranes in both native and heterologous expression systems. Based on multiple independent lines of evidence, we demonstrate the _1aAR at the cell surface occupies membrane rafts but exits from rafts following stimulation. In non-detergent raft preparations, basal _1aAR is present in low density membrane rafts and colocalizes with its G protein effectors on density gradients. Raft disruption by cholesterol depletion with methyl-β-cyclodextrin eliminates these light rafts. To confirm the presence of the _1aAR in plasma membrane rafts, fluorescence resonance energy transfer measurements were used to demonstrate colocalization of surface receptor and the raft marker, cholera toxin B. This colocalization was largely lost following _1aAR stimulation with phenylephrine. Similarly, receptor stimulation causes exit of the _1aAR from light rafts within 3-10 min in contrast to the G proteins, which largely remain in light rafts. Importantly, this delayed exit of the _1aAR suggests acute receptor signaling and desensitization occur entirely within rafts. Interestingly, both confocal analysis and measurement of surface _1aAR levels indicate modest receptor internalization during the 10 min following stimulation, suggesting most of the receptor has entered non-raft plasma membrane. Nevertheless, activation does increase the rate of receptor internalization as does disruption of rafts with methyl-β-cyclodextrin, suggesting raft exit enables internalization. Confocal analysis of surface-labeled hemagglutinin-_1aAR reveals that basal and stimulated receptor occupies clathrin pits in fixed cells consistent with previous indirect evidence. The evidence presented here strongly suggests the _1aAR is a lipid raft protein under basal conditions and implies agonist-mediated signaling occurs from rafts.

Received for publication, July 16, 2007 , and in revised form, November 29, 2007.

^* This work was supported in part by National Institutes of Health Grant HL49103 (to D. A. S.) and American Heart Association Grant 0530175N (to D. P. M.). 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 Figs. s1-s6 and a movie.

¹ To whom correspondence should be addressed: 595 LaSalle St., Ste. 1027, Durham, NC 27710. Tel.: 919-681-4780; Fax: 919-681-4776; E-mail: morri082{at}mc.duke.edu.

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