HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 282, Issue 40, 29089-29100, October 5, 2007

This Article Full Text Full Text (PDF) All Versions of this Article: 282/40/29089    most recent M700577200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hamdan, F. F. Articles by Bouvier, M. Search for Related Content PubMed PubMed Citation Articles by Hamdan, F. F. Articles by Bouvier, M. Social Bookmarking                      What's this?

Unraveling G Protein-coupled Receptor Endocytosis Pathways Using Real-time Monitoring of Agonist-promoted Interaction between -Arrestins and AP-2^*

Fadi F. Hamdan¹²³, Moulay Driss Rochdi¹³, Billy Breton, Delphine Fessart, Douce E. Michaud, Pascale G. Charest⁴, Stéphane A. Laporte⁵, and Michel Bouvier⁶

From the Department of Biochemistry and Groupe de Recherche Universitaire sur le Médicament, Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, Québec H3C 3J7 and the Department of Medicine, McGill University, Montréal, Québec H3A 1A1, Canada

The most widely studied pathway underlying agonist-promoted internalization of G protein-coupled receptors (GPCRs) involves -arrestin and clathrin-coated pits. However, both -arrestin- and clathrin-independent processes have also been reported. Classically, the endocytic routes are characterized using pharmacological inhibitors and various dominant negative mutants, resulting sometimes in conflicting results and interpretational difficulties. Here, taking advantage of the fact that -arrestin binding to the 2 subunit of the clathrin adaptor AP-2 (2-adaptin) is needed for the -arrestin-mediated targeting of GPCRs to clathrin-coated pits, we developed a bioluminescence resonance energy transfer-based approach directly assessing the molecular steps involved in the endocytosis of GPCRs in living cells. For 10 of the 12 receptors tested, including some that were previously suggested to internalize via clathrin-independent pathways, agonist stimulation promoted -arrestin 1 and 2 interaction with 2-adaptin, indicating a -arrestin- and clathrin-dependent endocytic process. Detailed analyses of -arrestin interactions with both the receptor and 2-adaptin also allowed us to demonstrate that recruitment of -arrestins to the receptor and the ensuing conformational changes are the leading events preceding AP-2 engagement and subsequent clathrin-mediated endocytosis. Among the receptors tested, only the endothelin A and B receptors failed to promote interaction between -arrestins and 2-adaptin. However, both receptors recruited -arrestins upon agonist stimulation, suggesting a -arrestin-dependent but clathrin-independent route of internalization for these two receptors. In addition to providing a new tool to dissect the molecular events involved in GPCR endocytosis, the bioluminescence resonance energy transfer-based -arrestin/2-adaptin interaction assay represents a novel biosensor to assess receptor activation.

Received for publication, January 19, 2007 , and in revised form, July 3, 2007.

^* This work was supported in part by research grants from the Canadian Institute of Health Research (CIHR) (to M. B. and S. A. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² Current address: Saint-Justine Hospital Research Center, Dept. of Genetics, Université de Montréal.

³ Recipients of CIHR postdoctoral fellowships.

⁴ Current address: Biological Sciences Dept., University of California, San Diego.

⁵ Holds a Canada research chair in molecular endocrinology.

⁶ Michel Bouvier holds a Canada research chair in signal transduction/molecular pharmacology. To whom correspondence should be addressed: Institute for Research in Immunology and Cancer, Université de Montréal, C.P. 6128, succursale Center-Ville, Montréal, Québec H3C 3J7, Canada. Tel.: 514-343-6319; Fax: 514-343-6843; E-mail: michel.bouvier{at}umontreal.ca.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				J. J. Linderman Modeling of G-protein-coupled Receptor Signaling Pathways J. Biol. Chem., February 27, 2009; 284(9): 5427 - 5431. [Full Text] [PDF]

				M. Scarselli and J. G. Donaldson Constitutive Internalization of G Protein-coupled Receptors and G Proteins via Clathrin-independent Endocytosis J. Biol. Chem., February 6, 2009; 284(6): 3577 - 3585. [Abstract] [Full Text] [PDF]

				P. A. Keyel, J. R. Thieman, R. Roth, E. Erkan, E. T. Everett, S. C. Watkins, J. E. Heuser, and L. M. Traub The AP-2 Adaptor {beta}2 Appendage Scaffolds Alternate Cargo Endocytosis Mol. Biol. Cell, December 1, 2008; 19(12): 5309 - 5326. [Abstract] [Full Text] [PDF]

				S. K. Mishra, A. Jha, A. L. Steinhauser, V. A. Kokoza, C. H. Washabaugh, A. S. Raikhel, W. A. Foster, and L. M. Traub Internalization of LDL-receptor superfamily yolk-protein receptors during mosquito oogenesis involves transcriptional regulation of PTB-domain adaptors J. Cell Sci., April 15, 2008; 121(8): 1264 - 1274. [Abstract] [Full Text] [PDF]