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

• Published in: The Journal of biological chemistry 2008-09, Vol.283 (36), p.24659
• Main Authors: Pontier, Stéphanie M, Percherancier, Yann, Galandrin, Ségolène, Breit, Andreas, Galés, Céline, Bouvier, Michel
• Format: Article
• Language: English
• Subjects: Adenylyl Cyclases - metabolism; Adrenergic Agonists - pharmacology; Caveolins - biosynthesis; Cell Line; Cholesterol - metabolism; Cyclic AMP-Dependent Protein Kinases - metabolism; GTP-Binding Protein alpha Subunits - metabolism; Humans; Membrane Microdomains - metabolism; Models, Biological; Phosphorylation - drug effects; Receptors, Adrenergic, beta-2 - metabolism; Signal Transduction - drug effects; Signal Transduction - physiology
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M800778200

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.