Arrestin/Clathrin Interaction
LOCALIZATION OF THE CLATHRIN BINDING DOMAIN OF NONVISUAL ARRESTINS TO THE CARBOXYL TERMINUS*
- From the Departments of Biochemistry and Molecular Pharmacology and Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Abstract
We have recently demonstrated that the nonvisual arrestins, β-arrestin and arrestin3, interact with high affinity and stoichiometrically with clathrin, and we postulated that this interaction mediates internalization of G protein-coupled receptors (Goodman, O. B., Jr., Krupnick, J. G., Santini, F., Gurevich, V. V., Penn, R. B., Gagnon, A. W., Keen, J. H., and Benovic, J. L. (1996)Nature 383, 447–450). In this study, we localized the clathrin binding domain of arrestin3 using a variety of approaches. Truncation mutagenesis demonstrated that the COOH-terminal half of arrestin3 is required for clathrin interaction. Assessment of the clathrin binding properties of various glutathione S-transferase-arrestin3 fusion proteins indicated that the predominant clathrin binding domain is contained within residues 367–385. Alanine scanning mutagenesis further localized this domain to residues 371–379, and site-directed mutagenesis demonstrated the critical importance of both hydrophobic (Leu-373, Ile-374, and Phe-376) and acidic (Glu-375 and Glu-377) residues in the arrestin3/clathrin interaction. These results are complementary to the observation that hydrophobic and basic residues in clathrin are critical for its interaction with nonvisual arrestins (Goodman, O. B., Jr., Krupnick, J. G., Gurevich, V. V., Benovic, J. L., and Keen, J. H. (1997)J. Biol. Chem. 272, 15017–15022). Lastly, an arrestin3 mutant in which Leu-373, Ile-374, and Phe-376 were mutated to Ala was significantly defective in its ability to promote β2-adrenergic receptor internalization in COS-1 cells when compared with wild-type arrestin3. Taken together, these results implicate a discrete region of arrestin3 in high affinity binding to clathrin, an interaction critical for agonist-promoted internalization of the β2-adrenergic receptor.
Footnotes
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↵* This work was supported in part by National Institutes of Health Grants GM44944 and GM47417 (to J. L. B.), GM28526 (to J. H. K.), and National Institutes of Health Training Grant 5-T32-DK07705-03 (to J. G. K. and O. B. G., Jr.).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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↵‡ Established investigator of the American Heart Association. To whom correspondence should be addressed: Kimmel Cancer Institute, Thomas Jefferson University, 233 S. 10th St., Philadelphia, PA 19107. Tel.: 215-503-4607; Fax: 215-923-1098.
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↵1 The abbreviations used are: GPR, G protein-coupled receptor; G protein, guanine nucleotide-binding protein; GRK, G protein-coupled receptor kinase; PCR, polymerase chain reaction; bp, base pair(s); GST, glutathione S-transferase; PBS, phosphate-buffered saline; MES, 2-(N-morpholino)ethanesulfonic acid; ROS, rod outer segment.
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- Received February 12, 1997.
