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J Biol Chem. 2008 Sep 5;283(36):24659-72. doi: 10.1074/jbc.M800778200. Epub 2008 Jun 19.

Cholesterol-dependent separation of the beta2-adrenergic receptor from its partners determines signaling efficacy: insight into nanoscale organization of signal transduction.

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  • 1Institut de Recherche en Immunologie et Cancérologie, Département de Biochimie, Université de Montréal, Montréal, Québec H3C 3J7, Canada.

Abstract

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 beta2 adrenergic receptor (beta2AR) signaling. The data revealed that whereas beta2AR 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 Galphas 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 beta2AR-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 beta2AR 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 beta2AR-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 Gs and AC allows their lateral separation from the receptor, thus restraining the basal activity and controlling responsiveness of beta2AR signaling machinery within larger signaling platforms.

PMID:
18566454
[PubMed - indexed for MEDLINE]
PMCID:
PMC3259828
Free PMC Article
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