Interaction with caveolin-1 modulates G protein coupling of the mouse beta3-adrenoceptor

Research output: Contribution to journalArticleResearchpeer-review

Abstract

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 beta(3)-adrenoceptor (AR) signaling is dependent on caveolin-1, however it is not known whether caveolin-1 is associated with the beta(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 beta(3a)- and beta(3b)-AR isoforms that diverge at the distal C-terminus. Only the beta(3b)-AR promotes PTX-sensitive cAMP accumulation. When cells expressing the beta(3a)-AR were treated with filipin III to disrupt membrane rafts, or transfected with caveolin-1 siRNA, the cyclic AMP response to the beta(3)-AR agonist CL316243 became PTX sensitive, suggesting Galphai/o coupling. The beta(3a)-AR C-terminus, SP(384)PLNRF(389)DGY(392)EGARPF(398)PT, resembles a caveolin interaction motif. Mutant beta(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 beta(3a)-AR but not the mutant receptors. In membrane preparations, the beta(3b)-AR activated Galphao and mediated PTX-sensitive cAMP responses, whereas the beta(3a)-AR did not activate Galphai/o proteins. The endogenous beta(3a)-AR displayed Galphai/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 beta(3a)-AR with caveolin inhibits coupling to Galphai/o proteins, and suggest that signaling is modulated by a raft-enriched complex containing the beta(3a)-AR, caveolin-1, Galphas and adenylyl cyclase.
Original languageEnglish
Pages (from-to)20674 - 20688
Number of pages15
JournalJournal of Biological Chemistry
Volume287
Issue number24
DOIs
Publication statusPublished - 2012

Cite this

@article{d17f795b2f9642cda9a685cad5f4ec0c,
title = "Interaction with caveolin-1 modulates G protein coupling of the mouse beta3-adrenoceptor",
abstract = "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 beta(3)-adrenoceptor (AR) signaling is dependent on caveolin-1, however it is not known whether caveolin-1 is associated with the beta(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 beta(3a)- and beta(3b)-AR isoforms that diverge at the distal C-terminus. Only the beta(3b)-AR promotes PTX-sensitive cAMP accumulation. When cells expressing the beta(3a)-AR were treated with filipin III to disrupt membrane rafts, or transfected with caveolin-1 siRNA, the cyclic AMP response to the beta(3)-AR agonist CL316243 became PTX sensitive, suggesting Galphai/o coupling. The beta(3a)-AR C-terminus, SP(384)PLNRF(389)DGY(392)EGARPF(398)PT, resembles a caveolin interaction motif. Mutant beta(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 beta(3a)-AR but not the mutant receptors. In membrane preparations, the beta(3b)-AR activated Galphao and mediated PTX-sensitive cAMP responses, whereas the beta(3a)-AR did not activate Galphai/o proteins. The endogenous beta(3a)-AR displayed Galphai/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 beta(3a)-AR with caveolin inhibits coupling to Galphai/o proteins, and suggest that signaling is modulated by a raft-enriched complex containing the beta(3a)-AR, caveolin-1, Galphas and adenylyl cyclase.",
author = "Masaaki Sato and Hutchinson, {Dana S} and Halls, {Michelle L} and Furness, {Sebastian G B} and Tore Bengtsson and Evans, {Bronwyn A} and Summers, {Roger J}",
year = "2012",
doi = "10.1074/jbc.M111.280651",
language = "English",
volume = "287",
pages = "20674 -- 20688",
journal = "Journal of Biological Chemistry",
issn = "1083-351X",
publisher = "American Society for Biochemistry and Molecular Biology",
number = "24",

}

Interaction with caveolin-1 modulates G protein coupling of the mouse beta3-adrenoceptor. / Sato, Masaaki; Hutchinson, Dana S; Halls, Michelle L; Furness, Sebastian G B; Bengtsson, Tore; Evans, Bronwyn A; Summers, Roger J.

In: Journal of Biological Chemistry, Vol. 287, No. 24, 2012, p. 20674 - 20688.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Interaction with caveolin-1 modulates G protein coupling of the mouse beta3-adrenoceptor

AU - Sato, Masaaki

AU - Hutchinson, Dana S

AU - Halls, Michelle L

AU - Furness, Sebastian G B

AU - Bengtsson, Tore

AU - Evans, Bronwyn A

AU - Summers, Roger J

PY - 2012

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N2 - 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 beta(3)-adrenoceptor (AR) signaling is dependent on caveolin-1, however it is not known whether caveolin-1 is associated with the beta(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 beta(3a)- and beta(3b)-AR isoforms that diverge at the distal C-terminus. Only the beta(3b)-AR promotes PTX-sensitive cAMP accumulation. When cells expressing the beta(3a)-AR were treated with filipin III to disrupt membrane rafts, or transfected with caveolin-1 siRNA, the cyclic AMP response to the beta(3)-AR agonist CL316243 became PTX sensitive, suggesting Galphai/o coupling. The beta(3a)-AR C-terminus, SP(384)PLNRF(389)DGY(392)EGARPF(398)PT, resembles a caveolin interaction motif. Mutant beta(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 beta(3a)-AR but not the mutant receptors. In membrane preparations, the beta(3b)-AR activated Galphao and mediated PTX-sensitive cAMP responses, whereas the beta(3a)-AR did not activate Galphai/o proteins. The endogenous beta(3a)-AR displayed Galphai/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 beta(3a)-AR with caveolin inhibits coupling to Galphai/o proteins, and suggest that signaling is modulated by a raft-enriched complex containing the beta(3a)-AR, caveolin-1, Galphas and adenylyl cyclase.

AB - 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 beta(3)-adrenoceptor (AR) signaling is dependent on caveolin-1, however it is not known whether caveolin-1 is associated with the beta(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 beta(3a)- and beta(3b)-AR isoforms that diverge at the distal C-terminus. Only the beta(3b)-AR promotes PTX-sensitive cAMP accumulation. When cells expressing the beta(3a)-AR were treated with filipin III to disrupt membrane rafts, or transfected with caveolin-1 siRNA, the cyclic AMP response to the beta(3)-AR agonist CL316243 became PTX sensitive, suggesting Galphai/o coupling. The beta(3a)-AR C-terminus, SP(384)PLNRF(389)DGY(392)EGARPF(398)PT, resembles a caveolin interaction motif. Mutant beta(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 beta(3a)-AR but not the mutant receptors. In membrane preparations, the beta(3b)-AR activated Galphao and mediated PTX-sensitive cAMP responses, whereas the beta(3a)-AR did not activate Galphai/o proteins. The endogenous beta(3a)-AR displayed Galphai/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 beta(3a)-AR with caveolin inhibits coupling to Galphai/o proteins, and suggest that signaling is modulated by a raft-enriched complex containing the beta(3a)-AR, caveolin-1, Galphas and adenylyl cyclase.

UR - http://www.jbc.org/content/287/24/20674.full.pdf

U2 - 10.1074/jbc.M111.280651

DO - 10.1074/jbc.M111.280651

M3 - Article

VL - 287

SP - 20674

EP - 20688

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 1083-351X

IS - 24

ER -