Agonist-stimulated β-Adrenergic Receptor Internalization Requires Dynamic Cytoskeletal Actin Turnover

• Published in: The Journal of biological chemistry Vol. 281; no. 14; pp. 9773 - 9780
• Main Authors: Volovyk, Zoya M., Wolf, Matthew J., Prasad, Sathyamangla V. Naga, Rockman, Howard A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 07.04.2006
• Subjects: Actin Cytoskeleton - metabolism; Actin Depolymerizing Factors - genetics; Actin Depolymerizing Factors - physiology; Actins - metabolism; Adrenergic beta-Agonists - pharmacology; Bridged Bicyclo Compounds, Heterocyclic - pharmacology; Cell Culture Techniques; Endocytosis - physiology; Humans; Isoproterenol - pharmacology; Kidney - cytology; Receptors, Adrenergic, beta-1 - metabolism; Receptors, Adrenergic, beta-2 - metabolism; Thiazoles - pharmacology; Thiazolidines
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M511435200

Stimulation of β-adrenergic receptors (βARs) leads to sequential recruitment of β-arrestin, AP-2 adaptor protein, clathrin, and dynamin to the receptor complex, resulting in endocytosis. Whether a dynamic actin cytoskeleton is required for βAR endocytosis is not known. In this study, we have used β1- and β2 ARs, two ubiquitously expressed members of the βAR 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 β2ARs, β1ARs, and mutants of β1ARs that internalize via either clathrin- or caveolin-dependent pathways. Importantly, LB treatment did not inhibit β-arrestin translocation or dynamin recruitment to the agonist-stimulated receptor. To unequivocally demonstrate the requirement of the actin cytoskeleton for β2AR endocytosis, we used an actin-binding protein cofilin that biochemically depolymerizes and severs actin filaments. Isoproterenol-mediated internalization of β2AR 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 β2AR endocytosis. Finally, treatment with jasplakinolide, an inhibitor of actin turnover, resulted in dose-dependent inhibition of β2AR 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 βAR internalization.