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J. Biol. Chem., Vol. 283, Issue 36, 24659-24672, September 5, 2008

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Cholesterol-dependent Separation of the β₂-Adrenergic Receptor from Its Partners Determines Signaling Efficacy

INSIGHT INTO NANOSCALE ORGANIZATION OF SIGNAL TRANSDUCTION^*

From the Institut de Recherche en Immunologie et Cancérologie, Département de Biochimie, and Groupe de Recherche Universitaire sur le Médicament, Université de Montréal, Montréal, Québec H3C 3J7, Canada

Determining the role of lipid raft nanodomains in G protein-coupled receptor signaling remains fraught by the lack of assays directly monitoring rafts in native membranes. We thus combined extensive biochemical and pharmacological approaches to a nanoscale strategy based on bioluminescence resonance energy transfer (BRET) to assess the spatial and functional influence of cholesterol-rich liquid-ordered lipid nanodomains on β₂ adrenergic receptor (β₂AR) signaling. The data revealed that whereas β₂AR did not partition within liquid-ordered lipid phase, a pool of G protein and adenylyl cyclase (AC) were sequestered in these domains. Destabilization of the liquid-ordered phase by cholesterol depletion led to a lateral redistribution of G_s and AC that favored interactions between the receptor and its signaling partners as assessed by BRET. This resulted in an increased basal and agonist-promoted β₂AR-stimulated cAMP production that was partially dampened as a result of constitutive protein kinase A-dependent phosphorylation and desensitization of the receptor. This restraining influence of nanodomains on β₂AR signaling was further substantiated by showing that liquid-ordered lipid phase stabilization using caveolin overexpression or increasing membrane cholesterol amount led to an inhibition of β₂AR-associated signaling. Given the emerging concept that clustering of receptors and effectors into signaling platforms contributes to the efficacy and selectivity of signal transduction, our results support a model whereby cholesterol-promoted liquid-ordered lipid phase-embedding G_s and AC allows their lateral separation from the receptor, thus restraining the basal activity and controlling responsiveness of β₂AR signaling machinery within larger signaling platforms.

Received for publication, January 30, 2008 , and in revised form, June 10, 2008.

^* This work was supported by grants from the Canadian Institute for Health Research (CIHR) and the Heart and Stroke Foundation of Canada (to M. B.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ Both authors contributed equally to this work.

² Current address: Molecular Oncology Group, McGill University, Royal Victoria Hospital, Room H5.21, 687 Pine Ave. West, Montreal, Quebec H3A 1A1, Canada.

³ Recipient of a fellowship from the Institut National de la Santé etdela Recherche Médicale (INSERM, France) and a postdoctoral fellowship from the CIHR. Current address: CNRS FRE 2944, Institut Lwoff, 7 rue Guy Moquet, 94801 Villejuif, France.

⁴ To whom correspondence should be addressed: Institut de Recherche en Immunologie et Cancérologie, Université de Montréal, C.P. 6128 Succursale Centre-ville, Montréal, Québec H3C 3J7, Canada. Tel.: 514-343-6319; Fax: 514-343-6843; E-mail: michel.bouvier{at}umontreal.ca.

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