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J. Biol. Chem., Vol. 281, Issue 14, 9773-9780, April 7, 2006

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Agonist-stimulated -Adrenergic Receptor Internalization Requires Dynamic Cytoskeletal Actin Turnover^*

Zoya M. Volovyk, Matthew J. Wolf, Sathyamangla V. Naga Prasad, and Howard A. Rockman^¶1

From the Departments of Medicine, Cell Biology, and ^¶Molecular Genetics, Duke University Medical Center, Durham, North Carolina 27710

Stimulation of -adrenergic receptors (ARs) leads to sequential recruitment of -arrestin, AP-2 adaptor protein, clathrin, and dynamin to the receptor complex, resulting in endocytosis. Whether a dynamic actin cytoskeleton is required for AR endocytosis is not known. In this study, we have used ₁- and ₂ ARs, two ubiquitously expressed members of the AR family, to comprehensively evaluate the requirement of the actin cytoskeleton in receptor internalization. The integrity of the actin cytoskeleton was manipulated with the agent latrunculin B (LB) and mutants of cofilin to depolymerize actin filaments. Treatment of cells with LB resulted in dose-dependent depolymerization of the cortical actin cytoskeleton that was associated with significant attenuation in internalization of ₂ARs, ₁ARs, and mutants of ₁ARs that internalize via either clathrin- or caveolin-dependent pathways. Importantly, LB treatment did not inhibit -arrestin translocation or dynamin recruitment to the agonist-stimulated receptor. To unequivocally demonstrate the requirement of the actin cytoskeleton for ₂AR endocytosis, we used an actin-binding protein cofilin that biochemically depolymerizes and severs actin filaments. Isoproterenol-mediated internalization of ₂AR was completely blocked in the presence of wild type cofilin, which could be rescued by a mutant of cofilin that mimics a constitutive phosphorylated state and leads to normal agonist-stimulated ₂AR endocytosis. Finally, treatment with jasplakinolide, an inhibitor of actin turnover, resulted in dose-dependent inhibition of ₂AR internalization, suggesting that turnover of actin filaments at the receptor complex is required for endocytosis. Taken together, these data demonstrate that intact and functional dynamic actin cytoskeleton is required for normal AR internalization.

Received for publication, October 20, 2005 , and in revised form, January 18, 2006.

^* This work was supported by National Institutes of Health Grant HL61558 (to H. A. R.). 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: Duke University Medical Center, DUMC 3104, Durham, NC 27710. Tel.: 919-668-2521; Fax: 919-668-2524; E-mail: h.rockman{at}duke.edu.

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