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.
Institut de Recherche en Immunologie et Cancérologie, Département de Biochimie, Université de Montréal, Montréal, Québec H3C 3J7, Canada.
Stéphanie M
SM
Pontier
Yann
Y
Percherancier
Ségolène
S
Galandrin
Andreas
A
Breit
Céline
C
Galés
Michel
M
Bouvier
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