Regulated Endocytosis of G-protein-coupled Receptors by a Biochemically and Functionally Distinct Subpopulation of Clathrin-coated Pits*
- From the ‡Program in Cell Biology and the Departments of §Biochemistry and Biophysics, ‖Cellular and Molecular Pharmacology, and **Psychiatry, University of California, San Francisco, California 94143-0984
Abstract
β-2 Adrenergic receptors (B2ARs) are endocytosed by clathrin-coated pits. This process serves specialized functions in signal transduction and receptor regulation, raising the question of whether B2ARs are associated with biochemically specialized membrane vesicles during their endocytic trafficking. Here we show that B2ARs are endocytosed by a distinct subpopulation of clathrin-coated pits, which represent a limited subset of coated pits present in the plasma membrane, even in cells overexpressing both B2ARs and β-arrestin. Coated pits mediating agonist-induced endocytosis of B2ARs differ from other coated pits mediating constitutive endocytosis of transferrin receptors in their temperature dependence for fission from the plasma membrane and in the association of their membrane coats with β-arrestin. Endocytosis of these coated pits generates endocytic vesicles selectively enriched in B2ARs, which fuse within ∼10 min after their formation with a common population of endosomes containing both B2ARs and transferrin receptors. These observations demonstrate, for the first time, the existence of a functionally and biochemically distinct subpopulation of clathrin-coated pits that mediate the agonist-regulated endocytosis of G-protein-coupled receptors, and they suggest a new model for the formation of compositionally specialized membrane vesicles at the earliest stage of the endocytic pathway.
Footnotes
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↵* These studies were supported by National Institutes of Health Grant DA00218, a grant-in-aid from the American Heart Association, and a National Alliance for Research on Schizophrenia and Depression Young Investigator award.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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↵¶ Supported by National Institutes of Health Institutional Training Grant T32GM08120.
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↵‡ Present address: Athersys Inc., 11000 Cedar Ave., Cleveland, OH 44106-3052.
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↵§§ To whom correspondence should be addressed: Nina Ireland Laboratory, Rm. LP-A104, University of California, San Francisco, 401 Parnassus Ave., San Francisco, CA 94143-0984. Tel.: 415- 476-7855; Fax: 415-476-7884; E-mail zastrow{at}itsa.ucsf.edu.
- Abbreviations:
- GPCR
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G-protein-coupled receptor
- B2AR
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β-2 adrenergic receptor
- TfnR
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transferrin receptor
- MAPK
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mitogen-activated protein kinase
- PBS
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phosphate-buffered saline
- GFP
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green fluorescent protein
- TBS
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Tris-buffered saline.
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- Received May 6, 1998.
- Revision received June 18, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
