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Threonine 180 Is Required for G-protein-coupled Receptor Kinase 3- and β-Arrestin 2-mediated Desensitization of the µ-Opioid Receptor in Xenopus Oocytes

Celvert, Jeremy P. and Lowe, Janet and Kovoor, Abraham and Gurevich, Vsevolod V. and Chavkin, Charles (2001) Threonine 180 Is Required for G-protein-coupled Receptor Kinase 3- and β-Arrestin 2-mediated Desensitization of the µ-Opioid Receptor in Xenopus Oocytes. Journal of Biological Chemistry, 276 (7). pp. 4894-4900. ISSN 0021-9258. http://resolver.caltech.edu/CaltechAUTHORS:CELjbc01

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Abstract

To determine the sites in the µ-opioid receptor (MOR) critical for agonist-dependent desensitization, we constructed and coexpressed MORs lacking potential phosphorylation sites along with G-protein activated inwardly rectifying potassium channels composed of Kir3.1 and Kir3.4 subunits in Xenopus oocytes. Activation of MOR by the stable enkephalin analogue, [D-Ala2,MePhe4,Glyol5]enkephalin, led to homologous MOR desensitization in oocytes coexpressing both G-protein-coupled receptor kinase 3 (GRK3) and beta -arrestin 2 (arr3). Coexpression with either GRK3 or arr3 individually did not significantly enhance desensitization of responses evoked by wild type MOR activation. Mutation of serine or threonine residues to alanines in the putative third cytoplasmic loop and truncation of the C-terminal tail did not block GRK/arr3-mediated desensitization of MOR. Instead, alanine substitution of a single threonine in the second cytoplasmic loop to produce MOR(T180A) was sufficient to block homologous desensitization. The insensitivity of MOR(T180A) might have resulted either from a block of arrestin activation or arrestin binding to MOR. To distinguish between these alternatives, we expressed a dominant positive arrestin, arr2(R169E), that desensitizes G protein-coupled receptors in an agonist-dependent but phosphorylation-independent manner. arr2(R169E) produced robust desensitization of MOR and MOR(T180A) in the absence of GRK3 coexpression. These results demonstrate that the T180A mutation probably blocks GRK3- and arr3-mediated desensitization of MOR by preventing a critical agonist-dependent receptor phosphorylation and suggest a novel GRK3 site of regulation not yet described for other G-protein-coupled receptors.


Item Type:Article
Additional Information:Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Received for publication, August 15, 2000, and in revised form, October 20, 2000. Originally published In Press as doi:10.1074/jbc.M007437200 on November 1, 2000 This work was supported by United States Public Health Service Grant DA11672 from the National Institute on Drug Abuse. 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.
Record Number:CaltechAUTHORS:CELjbc01
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:CELjbc01
Alternative URL:http://dx.doi.org/10.1074/jbc.M007437200
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:7002
Collection:CaltechAUTHORS
Deposited By: Archive Administrator
Deposited On:04 Jan 2007
Last Modified:26 Dec 2012 09:27

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