Interaction of Arrestins with Intracellular Domains of Muscarinic and α2-Adrenergic Receptors*
- From the ¶Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 and the ‡Department of Pharmacology, Medical University of South Carolina, Charleston, South Carolina 29425
Abstract
The intracellular domains of G-protein-coupled receptors provide sites for interaction with key proteins involved in signal initiation and termination. As an initial approach to identify proteins interacting with these receptors and the receptor motifs required for such interactions, we used intracellular subdomains of G-protein-coupled receptors as probes to screen brain cytosol proteins. Peptides from the third intracellular loop (i3) of the M2-muscarinic receptor (MR) (His208–Arg387), M3-MR (Gly308–Leu497), or α2A/D-adrenergic receptor (AR) (Lys224–Phe374) were generated in bacteria as glutathione S-transferase (GST) fusion proteins, bound to glutathione-Sepharose and used as affinity matrices to detect interacting proteins in fractionated bovine brain cytosol. Bound proteins were identified by immunoblotting following SDS-polyacrylamide gel electrophoresis. Brain arrestins bound to the GST-M3fusion protein, but not to the control GST peptide or i3 peptides derived from the α2A/D-AR and M2-MR. However, each of the receptor subdomains bound purified β-arrestin and arrestin-3. The interaction of the M3-MR and M2-MR i3 peptides with arrestins was further investigated. The M3-MR i3 peptide bound in vitro translated [3H]β-arrestin and [3H]arrestin-3, but did not interact with in vitro translated or purified visual arrestin. The properties and specificity of the interaction ofin vitro translated [3H]β-arrestin, [3H]visual arrestin, and [3H]β-arrestin/visual arrestin chimeras with the M2-MR i3 peptide were similar to those observed with the intact purified M2-MR that was phosphorylated and/or activated by agonist. Subsequent binding site localization studies indicated that the interaction of β-arrestin with the M3-MR peptide required both the amino (Gly308–Leu368) and carboxyl portions (Lys425–Leu497) of the receptor subdomain. In contrast, the carboxyl region of the M3-MR i3 peptide was sufficient for its interaction with arrestin-3.
Footnotes
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↵* This work was supported in part by Grants NS24821 (to S. M. L.) and GM47419 (to J. L. B.) from the National Institutes of Health and Grant 2235 (to S. M. L.) from the Council for Tobacco Research.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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↵§ Visiting scientist from the Chinese Academy of Medical Sciences and Peking Union Medical College.
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↵‖ Established Investigator of the American Heart Association.
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↵** To whom correspondence should be addressed: Dept. of Pharmacology, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29425. Tel.: 803-792-2574; Fax: 803-792-2475; E-mail:laniersm{at}musc.edu.
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↵1 The abbreviations used are: i3, third intracellular loop; MR, muscarinic receptor; AR, adrenergic receptor; PAGE, polyacrylamide gel electrophoresis.
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↵2 Due to differences in the degree of resin substitution for various fusion proteins, variable membrane transfer efficiencies, and relative signal intensities, it is difficult to accurately determine the affinity of arrestin binding to the resins. However, data obtained thus far indicate no dramatic differences in the affinity of the purified arrestins for the three i3 peptides.
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↵3 Based on the relative migration of β-arrestin and arrestin-3 in 10% denaturing polyacrylamide gels and the comigration of the partially purified brain arrestin with β-arrestin (Fig. 5), the arrestin identified in the 100 mm NaCl elution of the 90% ammonium sulfate precipitate is predominantly β-arrestin (G. Wu and S. M. Lanier, unpublished data).
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- Received December 24, 1996.
- Revision received April 23, 1997.
