Role of Clathrin-mediated Endocytosis in Agonist-induced Down-regulation of the β2-Adrenergic Receptor*
- From the Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Abstract
Previous studies have demonstrated that non-visual arrestins function as adaptors in clathrin-mediated endocytosis to promote agonist-induced internalization of the β2-adrenergic receptor (β2AR). Here, we characterized the effects of arrestins and other modulators of clathrin-mediated endocytosis on down-regulation of the β2AR. In COS-1 and HeLa cells, non-visual arrestins promote agonist-induced internalization and down-regulation of the β2AR, whereas dynamin-K44A, a dominant-negative mutant of dynamin that inhibits clathrin-mediated endocytosis, attenuates β2AR internalization and down-regulation. In HEK293 cells, dynamin-K44A profoundly inhibits agonist-induced internalization and down-regulation of the β2AR, suggesting that receptor internalization is critical for down-regulation in these cells. Moreover, a dominant-negative mutant of β-arrestin, β-arrestin-(319–418), also inhibits both agonist-induced receptor internalization and down-regulation. Immunofluorescence microscopy analysis reveals that the β2AR is trafficked to lysosomes in HEK293 cells, where presumably degradation of the receptor occurs. These studies demonstrate that down-regulation of the β2AR is in part due to trafficking of the β2AR via the clathrin-coated pit endosomal pathway to lysosomes.
Footnotes
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↵* This research was supported in part by National Institutes of Health Grants GM44944 and GM47417 and by National Institutes of Health Training Grant 5-T32-CA09662 (to A. W. G).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ An established investigator of the American Heart Association. To whom correspondence should be addressed: Thomas Jefferson University, 233 S. 10th St., Philadelphia, PA 19107. Tel.: 215-503-4607; Fax: 215-923-1098; E-mail: benovic{at}lac.jci.tju.edu.
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↵1 The abbreviations used are: GPR, G protein-coupled receptor; β2AR, β2-adrenergic receptor; GFP, green fluorescent protein; PBS, phosphate-buffered saline.
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- Received December 3, 1997.
- Revision received January 20, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
