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.
Highlights
The concept that signaling through membrane receptors occurs within large signaling complexes has been recently proposed [1,2,3,4,5,6,7,8,9,10]
The canonical signaling pathway associated with the seventransmembrane 2-adrenergic receptor (2AR)5 leads to the regulation of cyclic AMP production through the activation of adenylyl cyclase (AC) by the stimulatory G protein, Gs [21]
In agreement with what was previously observed in cardiomyocytes, COS, A6, Madin-Darby canine kidney, and Chinese hamster ovary cells [22,23,24,25,26, 28], heterologously expressed (Fig. 1A) as well as endogenous 2AR, G␣s, and AC V/VI were found to co-distribute with two raft molecular markers, flotillin and GM1, in light density membrane obtained from cell fractionation based on a detergent-free sodium carbonate preparation
Summary
Constructs—FLAG-2ARWT cloned into pcDNA3 was kindly provided by Dr Stefano Marullo (Cochin Institute, Paris). Membrane proteins were incubated in a total volume of 0.5 ml in a buffer containing 75 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 2 mM EDTA, protease inhibitors, and varying concentrations of 2AR ligands depending on the type of experiment. On the day of the experiment, living cells expressing similar amount of RLuc were fluorescently labeled with increasing concentrations of DiIC16 or FastDiI (Molecular Probes) at 4 °C for 5 min as described previously [42] in order to mark liquid-ordered or disordered membrane regions, respectively. BRET measurements were monitored in PBS following the addition of Coelenterazine h (5 M, NanoLight Technology) using a multi-well plate reader, Mithras LB 940 (Berthold), which allowed the sequential integration of signals detected in the 480 Ϯ 20 and 530 Ϯ 20 nm windows for donor (Rluc-fused proteins) and acceptor (DiIC16 or FastDiI) light emission, respectively. Statistical significance of the differences between the different conditions were calculated using one-way analysis of variance with a Bonferroni post-test for p values less than 0.05
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