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Interaction with Caveolin-1 Modulates G Protein Coupling of Mouse β3-Adrenoceptor*

  • Masaaki Sato
    Affiliations
    Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and the Department of Pharmacology, Monash University, Parkville, Victoria 3052, Australia

    Department of Physiology, The Wenner-Gren Institute, Arrhenius Laboratories F3, Stockholm University, SE-106 91 Stockholm, Sweden
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  • Dana S. Hutchinson
    Footnotes
    Affiliations
    Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and the Department of Pharmacology, Monash University, Parkville, Victoria 3052, Australia
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  • Michelle L. Halls
    Footnotes
    Affiliations
    Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and the Department of Pharmacology, Monash University, Parkville, Victoria 3052, Australia
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  • Sebastian G.B. Furness
    Affiliations
    Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and the Department of Pharmacology, Monash University, Parkville, Victoria 3052, Australia
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  • Tore Bengtsson
    Affiliations
    Department of Physiology, The Wenner-Gren Institute, Arrhenius Laboratories F3, Stockholm University, SE-106 91 Stockholm, Sweden
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  • Bronwyn A. Evans
    Correspondence
    To whom correspondence should be addressed: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 399 Royal Parade, Parkville, Victoria 3052, Australia. Tel.: 61-3-9903-9086;
    Affiliations
    Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and the Department of Pharmacology, Monash University, Parkville, Victoria 3052, Australia
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  • Roger J. Summers
    Affiliations
    Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and the Department of Pharmacology, Monash University, Parkville, Victoria 3052, Australia
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  • Author Footnotes
    * This work was supported by National Health and Medical Research Council of Australia Program Grant 519461 (to P. M. Sexton, A. Christopoulos, and R. J.), National Health and Medical Research Council of Australia Career Development Award 545952 (to D. S. H.), National Health and Medical Research Council of Australia C. J. Martin Fellowship 519581 (to M. L. H.), and by Australian Research Council Linkage International Fellowship LX0989791 (to M. S.).
    This article contains supplemental Tables S1–S3.
    1 Both authors contributed equally to this work.
Open AccessPublished:April 25, 2012DOI:https://doi.org/10.1074/jbc.M111.280651
      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 β3-adrenoceptor (β3-AR) signaling is dependent on caveolin-1; however, it is not known whether caveolin-1 is associated with the β3-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 β3a- and β3b-AR isoforms that diverge at the distal C terminus. Only the β3b-AR promotes pertussis toxin (PTX)-sensitive cAMP accumulation. When cells expressing the β3a-AR were treated with filipin III to disrupt membrane rafts or transfected with caveolin-1 siRNA, the cyclic AMP response to the β3-AR agonist CL316243 became PTX-sensitive, suggesting Gαi/o coupling. The β3a-AR C terminus, SP384PLNRF389DGY392EGARPF398PT, resembles a caveolin interaction motif. Mutant β3a-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 β3a-AR but not the mutant receptors. In membrane preparations, the β3b-AR activated Gαo and mediated PTX-sensitive cAMP responses, whereas the β3a-AR did not activate Gαi/o proteins. The endogenous β3a-AR displayed Gαi/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 β3a-AR with caveolin inhibits coupling to Gαi/o proteins and suggest that signaling is modulated by a raft-enriched complex containing the β3a-AR, caveolin-1, Gαs, and adenylyl cyclase.

      Introduction

      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 (
      • Simons K.
      • Ikonen E.
      Functional rafts in cell membranes.
      ,
      • Sharma P.
      • Varma R.
      • Sarasij R.C.
      • Ira
      • Gousset K.
      • Krishnamoorthy G.
      • Rao M.
      • Mayor S.
      Nanoscale organization of multiple GPI-anchored proteins in living cell membranes.
      ). 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 multiprotein complexes, acting as signaling platforms that govern association between receptors and effector proteins (reviewed in Ref.
      • Patel H.H.
      • Murray F.
      • Insel P.A.
      G-protein-coupled receptor-signaling components in membrane raft and caveolae microdomains.
      ). Caveolae represent a subset of membrane rafts that have a distinctive membrane structure delineated by the presence of caveolin proteins as well as the protein cavin (
      • Parton R.G.
      • Simons K.
      The multiple faces of caveolae.
      ,
      • Hill M.M.
      • Bastiani M.
      • Luetterforst R.
      • Kirkham M.
      • Kirkham A.
      • Nixon S.J.
      • Walser P.
      • Abankwa D.
      • Oorschot V.M.
      • Martin S.
      • Hancock J.F.
      • Parton R.G.
      PDRF-Cavin, a conserved cytoplasmic protein required for caveola formation and function.
      ). Caveolin-1, -2, and -3 consist of a cytoplasmic N terminus, a 21-amino acid hairpin structure that inserts into the cell membrane, and a cytoplasmic C terminus with three palmitoylation sites. Caveolins interact with signaling proteins via a conserved scaffolding domain (for example, amino acids 82–101 of caveolin-1). Caveolae are thought to contain 100–200 caveolin molecules (
      • Parton R.G.
      • Hanzal-Bayer M.
      • Hancock J.F.
      Biogenesis of caveolae. A structural model for caveolin-induced domain formation.
      ); however, caveolins may form smaller noncaveolar oligomers of at least 15 molecules that have been termed caveolin scaffolds (
      • Lajoie P.
      • Goetz J.G.
      • Dennis J.W.
      • Nabi I.R.
      Lattices, rafts, and scaffolds. Domain regulation of receptor signaling at the plasma membrane.
      ). Noncaveolar caveolins may also modulate signaling (
      • Parton R.G.
      • Simons K.
      The multiple faces of caveolae.
      ), for example by growth factor receptors (
      • Lajoie P.
      • Partridge E.A.
      • Guay G.
      • Goetz J.G.
      • Pawling J.
      • Lagana A.
      • Joshi B.
      • Dennis J.W.
      • Nabi I.R.
      Plasma membrane domain organization regulates EGFR signaling in tumor cells.
      ) and G protein-coupled receptors (GPCRs)
      The abbreviations used are: GPCR
      G protein-coupled receptor
      AC
      adenylyl cyclase
      β-AR
      β-adrenoceptor
      β3-AR
      β3-adrenoceptor
      BAT
      brown adipose tissue
      cav-1
      caveolin-1
      s
      stimulatory guanine-nucleotide binding protein
      i/o
      inhibitory guanine nucleotide-binding protein
      PTX
      pertussis toxin
      GTPγS
      guanosine 5′-3-O-(thio)triphosphate
      IBMX
      3-isobutyl-1-methylxanthine
      ANOVA
      analysis of variance
      fsk
      forskolin.
      (
      • Head B.P.
      • Insel P.A.
      Do caveolins regulate cells by actions outside of caveolae?.
      ).
      The three β-adrenoreceptor subtypes (β-ARs) are highly conserved GPCRs that share common determinants for coupling to the α subunit of the stimulatory guanine nucleotide-binding protein (Gαs); however, functional diversity is generated by sequence-specific protein-protein interactions and by differential enrichment in membrane domains. For example, interaction of the β2-AR with inhibitory guanine nucleotide-binding proteins (Gαi/o) is dependent on the presence of a functional type 1 PSD-95/Drosophila Discs Large/ZO-1 (PDZ) docking site at the receptor C terminus (DSLL) (
      • Xiang Y.
      • Kobilka B.
      The PDZ-binding motif of the β2-adrenoceptor is essential for physiologic signaling and trafficking in cardiac myocytes.
      ), whereas the β1-AR C-terminal PDZ motif (ESKV) inhibits receptor internalization and Gαi coupling (
      • Xiang Y.
      • Devic E.
      • Kobilka B.
      The PDZ-binding motif of the β1-adrenergic receptor modulates receptor trafficking and signaling in cardiac myocytes.
      ). The signaling properties of the β2-AR are clearly regulated by partitioning in membrane rafts or in caveolae (
      • Pontier S.M.
      • Percherancier Y.
      • Galandrin S.
      • Breit A.
      • Galés C.
      • Bouvier M.
      Cholesterol-dependent separation of the β2-adrenergic receptor from its partners determines signaling efficacy: insight into nanoscale organization of signal transduction.
      ). In cardiac myocytes, disruption of caveolae has no effect on the inotropic response to β1-AR stimulation, although it significantly enhances β2-AR-mediated Ca2+ transients and L-type Ca2+ channel currents (
      • Balijepalli R.C.
      • Foell J.D.
      • Hall D.D.
      • Hell J.W.
      • Kamp T.J.
      Localization of cardiac L-type Ca2+ channels to a caveolar macromolecular signaling complex is required for β2-adrenergic regulation.
      ,
      • Calaghan S.
      • White E.
      Caveolae modulate excitation-contraction coupling and β2-adrenergic signaling in adult rat ventricular myocytes.
      ).
      Although no studies to date have reported localization of the β3-AR in membrane rafts or caveolae, there is firm evidence that caveolin-1 regulates β3-AR signaling in adipocytes. In both white and brown adipocytes, β3-ARs stimulate the Gαs/adenylyl cyclase/protein kinase A (PKA) pathway, promoting breakdown of fat (lipolysis) via phosphorylation of perilipin and hormone-sensitive lipase. Brown adipocytes also display β3-AR-mediated thermogenesis via induction of the mitochondrial uncoupling protein UCP1. The role of caveolin-1 in both white and brown adipocytes has been examined using caveolin-1−/− mice. Stimulation of lipolysis by the β3-AR selective agonist CL316243 is reduced substantially in white adipocytes isolated from caveolin-1−/− mice compared with wild type mice, due to disruption of a signaling complex that normally includes caveolin-1, the catalytic subunit of PKA and perilipin (
      • Cohen A.W.
      • Razani B.
      • Schubert W.
      • Williams T.M.
      • Wang X.B.
      • Iyengar P.
      • Brasaemle D.L.
      • Scherer P.E.
      • Lisanti M.P.
      Role of caveolin-1 in the modulation of lipolysis and lipid droplet formation.
      ). A similar pattern is seen in differentiated 3T3-L1 adipocytes treated with caveolin-1 siRNA (
      • Ahmad F.
      • Lindh R.
      • Tang Y.
      • Ruishalme I.
      • Ost A.
      • Sahachartsiri B.
      • Strålfors P.
      • Degerman E.
      • Manganiello V.C.
      Differential regulation of adipocyte PDE3B in distinct membrane compartments by insulin and the β3-adrenergic receptor agonist CL316243. Effects of caveolin-1 knockdown on formation/maintenance of macromolecular signaling complexes.
      ). In control cells, CL316243 promotes phosphorylation of perilipin, hormone-sensitive lipase, and also the phosphorylation, activation, and recruitment of phosphodiesterase 3B into complexes that contain caveolin-1, β3-AR, and PKA regulatory subunit RII. Knockdown of caveolin-1 blocks the activation of PDE3B and its recruitment into plasma membrane signaling complexes. In brown adipose tissue from caveolin-1−/− mice, perilipin phosphorylation and the mobilization of triglycerides usually associated with fasting/cold exposure are substantially reduced (
      • Cohen A.W.
      • Schubert W.
      • Brasaemle D.L.
      • Scherer P.E.
      • Lisanti M.P.
      Caveolin-1 expression is essential for proper nonshivering thermogenesis in brown adipose tissue.
      ). Upstream cAMP responses are also reduced, in part due to decreased adenylyl cyclase activity and β3-AR abundance (
      • Mattsson C.L.
      • Andersson E.R.
      • Nedergaard J.
      Differential involvement of caveolin-1 in brown adipocyte signaling. Impaired β3-adrenergic, but unaffected LPA, PDGF, and EGF receptor signaling.
      ,
      • Mattsson C.L.
      • Csikasz R.I.
      • Shabalina I.G.
      • Nedergaard J.
      • Cannon B.
      Caveolin-1-ablated mice survive in cold by nonshivering thermogenesis despite desensitized adrenergic responsiveness.
      ). It cannot be determined from these studies, however, whether caveolin-1 is associated functionally with the β3-AR itself or whether the diminished responses in knock-out mice are due solely to effects on downstream signaling, for example via PKA and perilipin.
      We have been able to address this question by taking advantage of the distinct signaling properties of two mouse β3-AR isoforms generated by alternative splicing (
      • Evans B.A.
      • Papaioannou M.
      • Hamilton S.
      • Summers R.J.
      Alternative splicing generates two isoforms of the β3-adrenoceptor which are differentially expressed in mouse tissues.
      ,
      • Hutchinson D.S.
      • Bengtsson T.
      • Evans B.A.
      • Summers R.J.
      Mouse β3a- and β3b-adrenoceptors expressed in Chinese hamster ovary cells display identical pharmacology but utilize distinct signaling pathways.
      ). The β3a- and β3b-AR isoforms differ only in their distal C-terminal tail, yet cAMP accumulation mediated by the β3b-AR is increased following pretreatment of cells with pertussis toxin (PTX), whereas the β3a-AR response is PTX-insensitive. Use of cell-permeable peptides corresponding to the unique β3a- and β3b-AR C termini demonstrated that the β3a-AR C-terminal tail interacts with a distinct protein or signaling complex (
      • Sato M.
      • Hutchinson D.S.
      • Bengtsson T.
      • Floren A.
      • Langel U.
      • Horinouchi T.
      • Evans B.A.
      • Summers R.J.
      Functional domains of the mouse β3-adrenoceptor associated with differential G protein coupling.
      ). We proposed that binding of proteins such as caveolin or other scaffolding proteins to the β3a-AR C terminus may localize the receptor to membrane microdomains or intracellular compartments where it cannot couple to Gαi/o.
      We demonstrate here that when CHO-K1 cells expressing the β3a-AR are treated with filipin III to disrupt membrane rafts, the cyclic AMP response to CL316243 becomes PTX-sensitive. In contrast, there is no change in the PTX sensitivity of the β3b-AR response. This suggests that residues present in the β3a-AR C-terminal tail may direct localization of the receptor to membrane rafts, and this in turn may govern its capacity to couple to Gαi/o proteins. The β3a-AR C terminus, SP384PLNRF389DGY392EGARPF398PT, contains a motif that is similar to the caveolin interaction motif of many proteins (φXφXXXXφ or φXXXXφXXφ, where φ is an aromatic residue (
      • Couet J.
      • Li S.
      • Okamoto T.
      • Ikezu T.
      • Lisanti M.P.
      Identification of peptide and protein ligands for the caveolin-scaffolding domain. Implications for the interaction of caveolin with caveolae-associated proteins.
      )). We show that cAMP accumulation is PTX-sensitive in cells expressing β3a-ARs carrying mutations in the putative caveolin-binding site. Knockdown of caveolin-1 in CHO-K1 cells expressing the wild type β3a-AR or in mouse brown adipocytes expressing endogenous β3a-ARs also alters the PTX sensitivity of cAMP accumulation and glucose uptake. We demonstrate that caveolin-1 interacts with the wild type β3a-AR but not with mutant β3a-ARs lacking key residues within the interaction motif. Our findings also indicate that PTX treatment increases cAMP responses in membranes derived from cells expressing the β3b-AR via inhibition of receptor-Gαo coupling.

      DISCUSSION

      We show here that the β3b-AR is able to couple to both Gαs and Gαo, in agreement with the PTX sensitivity of CL316243-stimulated cAMP accumulation in whole cells and in crude membranes. In contrast, the β3a-AR is unable to couple to Gαi/o in either native mouse brown adipocytes or recombinant CHO-K1 cells. Our previous study indicated that residues present in the unique β3a-AR C-terminal tail may interfere with Gαi/o coupling due to interaction with other proteins such as caveolin (
      • Sato M.
      • Hutchinson D.S.
      • Bengtsson T.
      • Floren A.
      • Langel U.
      • Horinouchi T.
      • Evans B.A.
      • Summers R.J.
      Functional domains of the mouse β3-adrenoceptor associated with differential G protein coupling.
      ). This notion was reinforced by the observation that the β3a-AR C-terminal tail contains a motif that is similar to the caveolin interaction motif of many proteins (φXφXXXXφ or φXXXXφXXφ (
      • Couet J.
      • Li S.
      • Okamoto T.
      • Ikezu T.
      • Lisanti M.P.
      Identification of peptide and protein ligands for the caveolin-scaffolding domain. Implications for the interaction of caveolin with caveolae-associated proteins.
      )). We investigated this idea by treating CHO-K1 cells expressing the β3-AR isoforms with filipin III to disrupt membrane rafts or with a caveolin-1 siRNA and by examining the coupling of mutant β3a-ARs. We have also demonstrated a direct association between the β3a-AR and caveolin-1 using a proximity ligation assay, and we show that our findings in recombinant CHO-K1 cells are recapitulated in brown adipocytes derived from wild type or caveolin-1 knock-out mice.
      CHO-K1 cells express caveolin-1 and exhibit caveolar structures (
      • Abrami L.
      • Fivaz M.
      • Kobayashi T.
      • Kinoshita T.
      • Parton R.G.
      • van der Goot F.G.
      Cross-talk between caveolae and glycosylphosphatidylinositol-rich domains.
      ,
      • Zeng Y.
      • Tao N.
      • Chung K.N.
      • Heuser J.E.
      • Lublin D.M.
      Endocytosis of oxidized low density lipoprotein through scavenger receptor CD36 utilizes a lipid raft pathway that does not require caveolin-1.
      ,
      • Cheng Z.J.
      • Singh R.D.
      • Holicky E.L.
      • Wheatley C.L.
      • Marks D.L.
      • Pagano R.E.
      Co-regulation of caveolar and Cdc42-dependent fluid phase endocytosis by phosphocaveolin-1.
      ). As knockdown of caveolin-1 in CHO-K1 cells promoted coupling of the β3a-AR to Gαi/o, we sought further evidence that caveolin-1 interacts with the receptor C-terminal tail. Our first step was to make a series of β3a-ARs with mutations in single or multiple amino acids that might contribute to the caveolin-1 interaction. Single mutations of Phe-389 or Tyr-392 to alanine did not promote Gαi/o coupling of the β3a-AR, whereas cAMP responses mediated by the combined mutants F389A,Y392A or F389A,Y392A, F398A became PTX-sensitive (Fig. 4). We also mutated Pro-384 to serine to mimic the rat β3-AR sequence, as this receptor does couple to Gαi/o (
      • Lenard N.R.
      • Prpic V.
      • Adamson A.W.
      • Rogers R.C.
      • Gettys T.W.
      Differential coupling of β3A- and β3B-adrenergic receptors to endogenous and chimeric Gαs and Gαi.
      ). Interestingly, both the P384S single mutant and a P384S,F389A double mutant showed PTX sensitivity, albeit slightly less than the other composite mutants. This study indicated that the motif PXXXXFXXY is dominant in preventing Gαi/o coupling of the β3a-AR, and it gave us the opportunity to test directly whether the mutants differed from the wild type receptor in their capacity to interact with caveolin-1. We examined this interaction using the Duolink in situ proximity ligation assay, which is based on close juxtaposition of antibodies directed against the two interacting partners (
      • Söderberg O.
      • Gullberg M.
      • Jarvius M.
      • Ridderstråle K.
      • Leuchowius K.J.
      • Jarvius J.
      • Wester K.
      • Hydbring P.
      • Bahram F.
      • Larsson L.G.
      • Landegren U.
      Direct observation of individual endogenous protein complexes in situ by proximity ligation.
      ). We were unable to perform this experiment with the β3b-AR because the β3-AR antibody is directed toward the β3a-AR C-terminal tail. We did show, however, that the antibody detected the F389A,Y392A,F398A and the P384S,F389A mutants as well as the wild type β3a-AR (Fig. 5E). The wild type receptor displayed robust interaction with caveolin-1 in this assay, whereas there was no signal in nontransfected CHO-K1 cells or in cells expressing the F389A,Y392A,F398A mutant. There was a very low signal in cells expressing the P384S,F389A β3a-AR, suggesting that this receptor retained weak association with caveolin-1. In combination with the functional properties of mutant receptors, the Duolink data indicate that an interaction with caveolin-1 does modulate the G protein coupling of the β3a-AR.
      The prototypical caveolin-binding motif consists of the sequences, φXφXXXXφ, φXXXXφXXφ, or a combination of the two (
      • Couet J.
      • Li S.
      • Okamoto T.
      • Ikezu T.
      • Lisanti M.P.
      Identification of peptide and protein ligands for the caveolin-scaffolding domain. Implications for the interaction of caveolin with caveolae-associated proteins.
      ). These consensus sequences were elucidated by screening phage display libraries using the caveolin scaffolding domain to select random peptide fragments. The most commonly occurring peptides were found to conform to one of the motifs above; however, there were many less abundant peptides in which the spacing or number of aromatic residues varied from the consensus (
      • Couet J.
      • Li S.
      • Okamoto T.
      • Ikezu T.
      • Lisanti M.P.
      Identification of peptide and protein ligands for the caveolin-scaffolding domain. Implications for the interaction of caveolin with caveolae-associated proteins.
      ). Among proteins known to interact with caveolin, there are exceptions to the strict requirement for a consensus caveolin-binding motif with 3 or 4 aromatic residues. For example, the motif φXφXXXXφXXφ is present in Gαi and Gαo proteins, but the sequence of other G proteins known to interact with caveolin varies at one or more key positions; for example, Gαs has the sequence TKFQVDKVNFHMFDA, and Gαq has YFDLQSVIFRMVDA (
      • Couet J.
      • Li S.
      • Okamoto T.
      • Ikezu T.
      • Lisanti M.P.
      Identification of peptide and protein ligands for the caveolin-scaffolding domain. Implications for the interaction of caveolin with caveolae-associated proteins.
      ). Our data indicate that the β3a-AR C terminus interacts with caveolin-1 despite lacking one of the consensus aromatic residues. More broadly, there may be many GPCRs that interact with the caveolin scaffolding domain of caveolin-1 despite lacking any cytoplasmic sequences that conform strictly to the φXφXXXXφ or φXXXXφXXφ consensus sites.
      It has been pointed out that there is no single sorting signal that directs localization of GPCRs to membrane rafts (
      • Bethani I.
      • Skånland S.S.
      • Dikic I.
      • Acker-Palmer A.
      Spatial organization of transmembrane receptor signaling.
      ). In addition, this localization may be unaffected, increased, or decreased by agonist treatment (
      • Patel H.H.
      • Murray F.
      • Insel P.A.
      G-protein-coupled receptor-signaling components in membrane raft and caveolae microdomains.
      ). For example, the δ-opioid receptor redistributes into raft domains upon agonist activation (
      • Alves I.D.
      • Salamon Z.
      • Hruby V.J.
      • Tollin G.
      Ligand modulation of lateral segregation of a G-protein-coupled receptor into lipid microdomains in sphingomyelin/phosphatidylcholine solid-supported bilayers.
      ), possibly because the activated receptor adopts a longer conformation that has higher affinity for areas of the membrane bilayer such as rafts that are thickened due to enrichment with sphingomyelin (
      • Bethani I.
      • Skånland S.S.
      • Dikic I.
      • Acker-Palmer A.
      Spatial organization of transmembrane receptor signaling.
      ). The α1A-AR, on the other hand, colocalizes with raft markers both before and immediately after agonist stimulation, but it moves from membrane rafts within 3–10 min (
      • Morris D.P.
      • Lei B.
      • Wu Y.X.
      • Michelotti G.A.
      • Schwinn D.A.
      The α1a-adrenergic receptor occupies membrane rafts with its G protein effectors but internalizes via clathrin-coated pits.
      ). Another study has shown that signaling by the β2-AR is constrained by exclusion from cholesterol-rich raft nanodomains that are enriched in other components of the signaling machinery, including Gαs and AC (
      • Pontier S.M.
      • Percherancier Y.
      • Galandrin S.
      • Breit A.
      • Galés C.
      • Bouvier M.
      Cholesterol-dependent separation of the β2-adrenergic receptor from its partners determines signaling efficacy: insight into nanoscale organization of signal transduction.
      ). Increasing the abundance of liquid-ordered raft domains by increasing cholesterol content or overexpressing caveolin-3 inhibits β2-AR-mediated cAMP responses, whereas disruption of rafts by cholesterol extraction with methyl-β-cyclodextrin increases both basal and maximal agonist-stimulated cAMP accumulation. Similar effects have been demonstrated in C6 glioma cells that express endogenous β2-ARs, where disruption of membrane rafts with methyl-β-cyclodextrin or knockdown of caveolin-1/caveolin-2 by siRNA led to increased cAMP accumulation (
      • Allen J.A.
      • Yu J.Z.
      • Dave R.H.
      • Bhatnagar A.
      • Roth B.L.
      • Rasenick M.M.
      Caveolin-1 and lipid microdomains regulate Gs trafficking and attenuate Gs/adenylyl cyclase signaling.
      ). Our Duolink proximity assay suggests that the β3a-AR interacts with caveolin-1 in the absence of agonist, and in contrast to the β2-AR studies, our functional data with filipin III indicate that signaling is more efficient in the presence of membrane rafts, both in CHO-K1 cells expressing the β3a-AR and in brown adipocytes with endogenous receptors.
      Chimeric Gαq/Gαs and Gαq/Gαi chimeras have been used as an alternative tool to test the coupling of mouse β3a-AR and β3b-AR isoforms (
      • Lenard N.R.
      • Prpic V.
      • Adamson A.W.
      • Rogers R.C.
      • Gettys T.W.
      Differential coupling of β3A- and β3B-adrenergic receptors to endogenous and chimeric Gαs and Gαi.
      ). The Gα constructs consisted of Gαq with the C-terminal five amino acids that determine receptor coupling replaced by those from Gαs or Gαi (
      • Conklin B.R.
      • Herzmark P.
      • Ishida S.
      • Voyno-Yasenetskaya T.A.
      • Sun Y.
      • Farfel Z.
      • Bourne H.R.
      C-terminal mutations of Gqα and Gsα that alter the fidelity of receptor activation.
      ,
      • Coward P.
      • Chan S.D.
      • Wada H.G.
      • Humphries G.M.
      • Conklin B.R.
      Chimeric G proteins allow a high throughput signaling assay of Gi-coupled receptors.
      ). This provides a single readout (increased intracellular Ca2+) to measure the relative efficiency of coupling to Gαs and Gαi subunits. The β3a-AR and β3b-AR both coupled less efficiently to Gαi than to Gαs, but there was no difference in the relative coupling of each isoform to Gαi. This result is entirely consistent with our data, as we have also shown that there is no inherent difference in the capacity of the β3a-AR and β3b-AR to couple to Gαi/o (
      • Sato M.
      • Hutchinson D.S.
      • Bengtsson T.
      • Floren A.
      • Langel U.
      • Horinouchi T.
      • Evans B.A.
      • Summers R.J.
      Functional domains of the mouse β3-adrenoceptor associated with differential G protein coupling.
      ). Instead, the difference between the two isoforms resides in their differential interaction with caveolin-1 and localization in membrane rafts/caveolae. In the study by Lenard et al. (
      • Lenard N.R.
      • Prpic V.
      • Adamson A.W.
      • Rogers R.C.
      • Gettys T.W.
      Differential coupling of β3A- and β3B-adrenergic receptors to endogenous and chimeric Gαs and Gαi.
      ), membrane localization of the chimeric Gα subunits would be determined by the common Gαq component. It has been shown previously that Gαq interacts with caveolin-1 (
      • de Weerd W.F.
      • Leeb-Lundberg L.M.
      Bradykinin sequesters B2 bradykinin receptors and the receptor-coupled Gα subunits Gαq and Gαi in caveolae in DDT1 MF-2 smooth muscle cells.
      ,
      • Bhatnagar A.
      • Sheffler D.J.
      • Kroeze W.K.
      • Compton-Toth B.
      • Roth B.L.
      Caveolin-1 interacts with 5-HT2A serotonin receptors and profoundly modulates the signaling of selected Gαq-coupled protein receptors.
      ) and is enriched in membrane raft fractions (
      • Sugawara Y.
      • Nishii H.
      • Takahashi T.
      • Yamauchi J.
      • Mizuno N.
      • Tago K.
      • Itoh H.
      The lipid raft proteins flotillins/reggies interact with Gαq and are involved in Gq-mediated p38 mitogen-activated protein kinase activation through tyrosine kinase.
      ), although others have shown that the raft localization of Gαq is dependent on the extraction procedure used (
      • Morris D.P.
      • Lei B.
      • Wu Y.X.
      • Michelotti G.A.
      • Schwinn D.A.
      The α1a-adrenergic receptor occupies membrane rafts with its G protein effectors but internalizes via clathrin-coated pits.
      ). Even if the Gαq chimeras were enriched in membrane rafts relative to bulk membrane, the high abundance of these proteins (
      • Lenard N.R.
      • Prpic V.
      • Adamson A.W.
      • Rogers R.C.
      • Gettys T.W.
      Differential coupling of β3A- and β3B-adrenergic receptors to endogenous and chimeric Gαs and Gαi.
      ) would likely mask any differences in the Gαq/i coupling of the β3a-AR and β3b-AR isoforms.
      In CHO-K1 cells, membrane rafts are enriched in Gαi/o and Gαs relative to the bulk membrane (
      • Ostrom R.S.
      • Post S.R.
      • Insel P.A.
      Stoichiometry and compartmentation in G protein-coupled receptor signaling: implications for therapeutic interventions involving G(s).
      ,
      • Razani B.
      • Woodman S.E.
      • Lisanti M.P.
      Caveolae. From cell biology to animal physiology.
      ,
      • Head B.P.
      • Patel H.H.
      • Roth D.M.
      • Murray F.
      • Swaney J.S.
      • Niesman I.R.
      • Farquhar M.G.
      • Insel P.A.
      Microtubules and actin microfilaments regulate lipid raft/caveolae localization of adenylyl cyclase signaling components.
      ). The two predominant adenylyl cyclase isoforms expressed are AC6 and AC7 (
      • Varga E.V.
      • Stropova D.
      • Rubenzik M.
      • Wang M.
      • Landsman R.S.
      • Roeske W.R.
      • Yamamura H.I.
      Identification of adenylyl cyclase isoenzymes in CHO and B82 cells.
      ), with AC7 excluded from membrane rafts (
      • Crossthwaite A.J.
      • Seebacher T.
      • Masada N.
      • Ciruela A.
      • Dufraux K.
      • Schultz J.E.
      • Cooper D.M.
      The cytosolic domains of Ca2+-sensitive adenylyl cyclases dictate their targeting to plasma membrane lipid rafts.
      ). In contrast, the AC6 isoform is enriched in membrane rafts, and this localization is known to be functionally important (
      • Pontier S.M.
      • Percherancier Y.
      • Galandrin S.
      • Breit A.
      • Galés C.
      • Bouvier M.
      Cholesterol-dependent separation of the β2-adrenergic receptor from its partners determines signaling efficacy: insight into nanoscale organization of signal transduction.
      ,
      • Crossthwaite A.J.
      • Seebacher T.
      • Masada N.
      • Ciruela A.
      • Dufraux K.
      • Schultz J.E.
      • Cooper D.M.
      The cytosolic domains of Ca2+-sensitive adenylyl cyclases dictate their targeting to plasma membrane lipid rafts.
      ,
      • Thangavel M.
      • Liu X.
      • Sun S.Q.
      • Kaminsky J.
      • Ostrom R.S.
      The C1 and C2 domains target human type 6 adenylyl cyclase to lipid rafts and caveolae.
      ). We have shown that in CHO-K1 cells expressing the β3a-AR, filipin treatment not only enhances PTX sensitivity but also causes a right shift of the concentration-response curve to CL316243. On the other hand, filipin treatment of cells expressing the β3b-AR increases basal and maximal cAMP accumulation. In cardiomyocytes and S49 lymphoma cells expressing low levels of endogenous β-ARs, on the order of 30 fmol/mg protein, the molar ratio of receptor/Gαs protein/AC has been estimated as 1:100:3 (
      • Ostrom R.S.
      • Post S.R.
      • Insel P.A.
      Stoichiometry and compartmentation in G protein-coupled receptor signaling: implications for therapeutic interventions involving G(s).
      ). Our recombinant CHO-K1 cells have a 30-fold higher abundance of receptors, so AC6 is almost certainly the limiting step in cAMP accumulation, and the enrichment of AC6 in membrane rafts may be another key difference between β3a-AR and β3b-AR responses. In the presence of filipin, the β3a-AR may display reduced responsiveness because it loses its co-localization with AC6, whereas the β3b-AR becomes more responsive because the AC6 is redistributed throughout the membrane and has higher availability. Another key question is why the β3a-AR does not couple to Gαi/o even though these subunits are also enriched in membrane rafts (
      • Razani B.
      • Woodman S.E.
      • Lisanti M.P.
      Caveolae. From cell biology to animal physiology.
      ). We suggest that despite their close proximity within rafts, coupling may be suppressed because the activity of Gαi/o is inhibited by interaction with caveolin (
      • Xu W.
      • Yoon S.I.
      • Huang P.
      • Wang Y.
      • Chen C.
      • Chong P.L.
      • Liu-Chen L.Y.
      Localization of the κ-opioid receptor in lipid rafts.
      ). In the presence of filipin, the β3a-AR would not only lose co-localization with AC6 but also gain the ability to interact with Gαi/o that is no longer associated with caveolin.
      We have shown that the effects of filipin III treatment or caveolin-1 knockdown observed in CHO-K1 cells expressing the β3a-AR are seen also in cultured brown adipocytes. These cells express the β3-AR at ∼400 fmol/mg of protein (
      • Sillence M.N.
      • Moore N.G.
      • Pegg G.G.
      • Lindsay D.B.
      Ligand binding properties of putative β3-adrenoceptors compared in brown adipose tissue and in skeletal muscle membranes.
      ,
      • Adli H.
      • Bazin R.
      • Vassy R.
      • Perret G.Y.
      Effects of triiodothyronine administration on the adenylyl cyclase system in brown adipose tissue of rat.
      ,
      • Malo A.
      • Puerta M.
      Oestradiol and progesterone change β3-adrenergic receptor affinity and density in brown adipocytes.
      ). In untreated brown adipocytes, CL316243 potency is 10-fold lower than in CHO-K1 cells, despite only a 2.5-fold lower β3-AR abundance, suggesting that the efficiency of cAMP generation is also decreased in the adipocytes. In CHO-β3a-AR cells, filipin III treatment reduced the pEC50 of CL316243 without affecting the maximum response. In contrast, filipin III treatment of brown adipocytes had no effect on the pEC50 of CL316243, but the maximum cAMP response was reduced. This is consistent with the overall lower potency of CL316243 in adipocytes, evidence that a 10-fold higher receptor occupancy is required in adipocytes to achieve a maximum cAMP response. When signaling efficiency is reduced further in the presence of filipin III, high concentrations of CL316243 cannot produce the same maximum response as that seen without filipin III. It has been shown that caveolin-1 knock-out mice have compromised cAMP accumulation in response to CL316243, due to composite effects on β3-AR abundance and AC activity (
      • Mattsson C.L.
      • Andersson E.R.
      • Nedergaard J.
      Differential involvement of caveolin-1 in brown adipocyte signaling. Impaired β3-adrenergic, but unaffected LPA, PDGF, and EGF receptor signaling.
      ). Our data extend these findings by demonstrating that both filipin III treatment and knockdown of caveolin-1 in brown adipocytes cause cAMP responses to become PTX-sensitive, indicating that the β3a-AR acquires coupling to Gαi/o proteins.
      In conclusion, our study demonstrates that the β3a-AR interacts with caveolin-1 and that the interaction affects functional coupling of the receptor to Gαi/o and Gαs subunits. Our work and that of others indicates that the reduced β3-AR signaling in mice lacking caveolin-1 is due to disruption of a signaling complex containing caveolin-1, the β3a-AR, Gαs, and adenylyl cyclase and not just due to reduced β3-AR abundance (
      • Ahmad F.
      • Lindh R.
      • Tang Y.
      • Ruishalme I.
      • Ost A.
      • Sahachartsiri B.
      • Strålfors P.
      • Degerman E.
      • Manganiello V.C.
      Differential regulation of adipocyte PDE3B in distinct membrane compartments by insulin and the β3-adrenergic receptor agonist CL316243. Effects of caveolin-1 knockdown on formation/maintenance of macromolecular signaling complexes.
      ,
      • Cohen A.W.
      • Schubert W.
      • Brasaemle D.L.
      • Scherer P.E.
      • Lisanti M.P.
      Caveolin-1 expression is essential for proper nonshivering thermogenesis in brown adipose tissue.
      ,
      • Mattsson C.L.
      • Andersson E.R.
      • Nedergaard J.
      Differential involvement of caveolin-1 in brown adipocyte signaling. Impaired β3-adrenergic, but unaffected LPA, PDGF, and EGF receptor signaling.
      ). Physiologically, the β3-AR plays a major role in white and brown adipocytes, where caveolin-1 association serves to potentiate receptor-mediated cAMP accumulation and downstream responses such as lipolysis or thermogenesis. For other receptors such as the β2-AR, raft localization and caveolin interaction undergo dynamic regulation due to requirements for strict spatial and temporal control of signaling (
      • Pontier S.M.
      • Percherancier Y.
      • Galandrin S.
      • Breit A.
      • Galés C.
      • Bouvier M.
      Cholesterol-dependent separation of the β2-adrenergic receptor from its partners determines signaling efficacy: insight into nanoscale organization of signal transduction.
      ). Thus, although membrane raft localization is a critical factor in organizing GPCR signaling complexes, the functional impact varies between receptors (
      • Bethani I.
      • Skånland S.S.
      • Dikic I.
      • Acker-Palmer A.
      Spatial organization of transmembrane receptor signaling.
      ). Continuing studies on GPCRs will provide novel insights into the determinants that dictate association of receptors with caveolins, promote localization in membrane rafts, and thereby modulate receptor signaling.

      Acknowledgments

      We thank Dr. Debbie Thurmond for providing the caveolin-1 siRNA constructs and Dr. Robin Anderson (with permission from Dr. T. Kurzchalia) for the caveolin-1+/+ and caveolin-1−/− mice.

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