Cholesterol-dependent separation of the beta2-adrenergic receptor from its partners determines signaling efficacy: insight into nanoscale organization of signal transduction.
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.
Pontier
Stéphanie M
SM
Institut de Recherche en Immunologie et Cancérologie, Département de Biochimie, Université de Montréal, Montréal, Québec H3C 3J7, Canada.
Percherancier
Yann
Y
Galandrin
Ségolène
S
Breit
Andreas
A
Galés
Céline
C
Bouvier
Michel
M
D000262:Adenylyl Cyclases / Q000378:metabolism; D000322:Adrenergic Agonists / Q000494:pharmacology; D022461:Caveolins / Q000096:biosynthesis; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D044385:GTP-Binding Protein alpha Subunits / Q000378:metabolism; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism*; D008954:Models, Biological; D010766:Phosphorylation / Q000187:drug effects; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*
D000322:Adrenergic Agonists; D022461:Caveolins; D044385:GTP-Binding Protein alpha Subunits; D018343:Receptors, Adrenergic, beta-2; D002784:Cholesterol; D017868:Cyclic AMP-Dependent Protein Kinases; D000262:Adenylyl Cyclases