title: Interaction with caveolin-1 modulates G protein coupling of mouse β3-adrenoceptor.
abstract: Caveolins act as scaffold proteins in multiprotein complexes and have been implicated in signaling by G protein-coupled receptors. Studies using knock-out mice suggest that β(3)-adrenoceptor (β(3)-AR) signaling is dependent on caveolin-1; however, it is not known whether caveolin-1 is associated with the β(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 β(3a)- and β(3b)-AR isoforms that diverge at the distal C terminus. Only the β(3b)-AR promotes pertussis toxin (PTX)-sensitive cAMP accumulation. When cells expressing the β(3a)-AR were treated with filipin III to disrupt membrane rafts or transfected with caveolin-1 siRNA, the cyclic AMP response to the β(3)-AR agonist CL316243 became PTX-sensitive, suggesting Gα(i/o) coupling. The β(3a)-AR C terminus, SP(384)PLNRF(389)DGY(392)EGARPF(398)PT, resembles a caveolin interaction motif. Mutant β(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 β(3a)-AR but not the mutant receptors. In membrane preparations, the β(3b)-AR activated Gα(o) and mediated PTX-sensitive cAMP responses, whereas the β(3a)-AR did not activate Gα(i/o) proteins. The endogenous β(3a)-AR displayed Gα(i/o) coupling in brown adipocytes from caveolin-1 knock-out mice or in wild type adipocytes treated with filipin III. Our studies indicate that interaction of the β(3a)-AR with caveolin inhibits coupling to Gα(i/o) proteins and suggest that signaling is modulated by a raft-enriched complex containing the β(3a)-AR, caveolin-1, Gα(s), and adenylyl cyclase.
authors.affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and the Department of Pharmacology, Monash University, Parkville, Victoria 3052, Australia.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Sato
authors.forename: Dana S
authors.initials: DS
authors.lastname: Hutchinson
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Halls
authors.forename: Sebastian G B
authors.initials: SG
authors.lastname: Furness
authors.forename: Tore
authors.initials: T
authors.lastname: Bengtsson
authors.forename: Bronwyn A
authors.initials: BA
authors.lastname: Evans
authors.forename: Roger J
authors.initials: RJ
authors.lastname: Summers
mesh_terms: D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D051242:Caveolin 1 / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D004149:Dioxoles / Q000494:pharmacology; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics / Q000378:metabolism*; D021962:Membrane Microdomains / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D020125:Mutation, Missense; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D022702:Receptors, Adrenergic, beta-3 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*
chemical_list: D000318:Adrenergic beta-Agonists; D051242:Caveolin 1; D004149:Dioxoles; D020033:Protein Isoforms; D022702:Receptors, Adrenergic, beta-3; C076126:disodium (R,R)-5-(2-((2-(3-chlorophenyl)-2-hydroxyethyl)-amino)propyl)-1,3-benzodioxole-2,3-dicarboxylate; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D000262:Adenylyl Cyclases