Interaction between ! 3 -adrenoceptors and caveolin-1 1 Interaction with Caveolin-1 Modulates G Protein Coupling of the Mouse ! 3 -Adrenoceptor* Masaaki Sato 1,3 , Dana S. Hutchinson 1,2† , Michelle L. Halls 1,2† , Sebastian G.B. Furness 1,2 , Tore Bengtsson 3 , Bronwyn A. Evans 1,2 and Roger J. Summers 1,2 From 1 Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, 399 Royal Parade, Parkville, Victoria 3052 and 2 Department of Pharmacology, Monash University, Clayton, Victoria 3800, AUSTRALIA and 3 Department of Physiology, The Wenner-Gren Institute, Arrhenius Laboratories F3, Stockholm University, SE-106 91 Stockholm, SWEDEN *Running title: Interaction between ! 3 -adrenoceptors and caveolin-1 To whom correspondence should be addressed: Dr Bronwyn Evans, Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 399 Royal Parade, Parkville, Victoria 3052, AUSTRALIA. Tel: +61 3 9903 9086; Email: bronwyn.evans@monash.edu Keywords: adenylyl cyclase, adipocyte, adrenergic receptor, caveolin, G proteins Background: Caveolins affect signaling by G protein-coupled receptors (GPCRs). Results: Interaction between ! 3a -adrenoceptors and caveolin-1 facilitates Gs-mediated responses but prevents the receptor from coupling to inhibitory Gi/o proteins. Conclusion: Association of the ! 3a -adrenoceptor with caveolin-1 is important in determining the selectivity and efficiency of G protein coupling and signaling. Significance: We demonstrate the functional impact of a GPCR-caveolin association. SUMMARY Caveolins act as scaffold proteins in multiprotein complexes and have been implicated in signaling by G protein-coupled receptors. Studies using knockout mice suggest that ! 3 -AR signaling is dependent on caveolin-1, however it is not known whether caveolin-1 is associated with the ! 3 -AR or solely with downstream signaling proteins. We have addressed this question by examining the impact of membrane rafts and caveolin-1 on the differential signaling of mouse ! 3a - and ! 3b -AR isoforms that diverge at the distal C-terminus. Only the ! 3b -AR promotes PTX-sensitive cAMP accumulation. When cells expressing the ! 3a -AR were treated with filipin III to disrupt membrane rafts, or transfected with caveolin-1 siRNA, the cyclic AMP response to the ! 3 -AR agonist CL316243 became PTX sensitive, suggesting G" i/o coupling. The ! 3a -AR C-terminus, SP (384)PLNRF (389)DGY (392)EGARPF (398) PT, resembles a caveolin interaction motif. Mutant ! 3a -ARs (F389A/Y392A/F398A or P384S/F389A) promoted PTX-sensitive cAMP responses, and in situ proximity assays demonstrated an association between caveolin-1 and the wild type ! 3a -AR but not the mutant receptors. In membrane preparations, the ! 3b -AR activated G" o and mediated PTX-sensitive cAMP responses, whereas the ! 3a -AR did not activate G" i/o proteins. The endogenous ! 3a -AR displayed G" i/o coupling in brown adipocytes from caveolin-1 knockout mice, or in wild type adipocytes treated with filipin III. Our studies indicate that interaction of the ! 3a -AR with caveolin inhibits coupling to G" i/o proteins, and suggest that signaling is modulated by a raft-enriched complex containing the ! 3a -AR, caveolin-1, G" s and adenylyl cyclase. The plasma membrane is not a random or uniform array of lipids and proteins, but instead has physical heterogeneity as well as higher order structures that are critical to the functioning of receptors, ion channels and signaling proteins. Membrane rafts, or lipid rafts, are liquid-ordered lipid domains of 5-10 nm that are enriched in cholesterol and sphingolipids (1,2). Rafts display reduced lateral diffusion relative to the liquid-disordered phase, providing nucleation sites for further membrane organization to produce larger structures of 50- 150 nm. These higher order structures are enriched in multi-protein complexes, acting as signaling platforms that govern association between receptors and effector proteins (reviewed in (3)). Caveolae represent a subset of http://www.jbc.org/cgi/doi/10.1074/jbc.M111.280651 The latest version is at JBC Papers in Press. Published on April 25, 2012 as Manuscript M111.280651 Copyright 2012 by The American Society for Biochemistry and Molecular Biology, Inc. at Monash University (CAUL), on May 7, 2012 www.jbc.org Downloaded from