Hetero-oligomerization between β2- and β3-Adrenergic Receptors Generates a β-Adrenergic Signaling Unit with Distinct Functional Properties

Breit, Andreas; Lagacé, Monique; Bouvier, Michel

doi:10.1074/jbc.m313310200

Cited by 142 publications

(115 citation statements)

References 76 publications

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“…The half-maximal effective concentrations of isoproterenol required to induce PKA activation (EC 50 value) by endogenously expressed GPCR was determined to be 5.2 Ϯ 3.5 nM (Fig. 5B Right), in the range of values obtained by in vitro cAMP measurements of isoproterenol-treated ␤ 2 ARexpressing HEK293 cells (13.9 Ϯ 3.9 nM) (40).…”

Section: Use Of the Pka Sensor To Monitor Protein Complex Dynamics Inmentioning

confidence: 99%

Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo

Stefan

Aquin²,

Berger³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

174

179

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The G protein-coupled receptor (GPCR) superfamily represents the most important class of pharmaceutical targets. Therefore, the characterization of receptor cascades and their ligands is a prerequisite to discovering novel drugs. Quantification of agonist-induced second messengers and downstream-coupled kinase activities is central to characterization of GPCRs or other pathways that converge on GPCR-mediated signaling. Furthermore, there is a need for simple, cell-based assays that would report on direct or indirect actions on GPCR-mediated effectors of signaling. More generally, there is a demand for sensitive assays to quantify alterations of protein complexes in vivo. We describe the development of a Renilla luciferase (Rluc)-based protein fragment complementation assay (PCA) that was designed specifically to investigate dynamic protein complexes. We demonstrate these features for GPCR-induced disassembly of protein kinase A (PKA) regulatory and catalytic subunits, a key effector of GPCR signaling. Taken together, our observations show that the PCA allows for direct and accurate measurements of live changes of absolute values of protein complex assembly and disassembly as well as cellular imaging and dynamic localization of protein complexes. Moreover, the Rluc-PCA has a sufficiently high signal-tobackground ratio to identify endogenously expressed G␣s proteincoupled receptors. We provide pharmacological evidence that the phosphodiesterase-4 family selectively down-regulates constitutive ␤-2 adrenergic-but not vasopressin-2 receptor-mediated PKA activities. Our results show that the sensitivity of the Rluc-PCA simplifies the recording of pharmacological profiles of GPCR-based candidate drugs and could be extended to high-throughput screens to identify novel direct modulators of PKA or upstream components of GPCR signaling cascades.G protein-coupled receptor ͉ complementation assays ͉ protein-protein interactions ͉ protein fragment G protein-coupled receptors (GPCRs) represent the largest family of cell-surface molecules involved in signal transmission. GPCRs play roles in a broad range of biological processes through regulating the majority of cell-to-cell and cell-toenvironment communication, and, consequently, their dysfunction manifests in numerous diseases (1, 2). The GPCR family has enormous pharmacological importance, as demonstrated by the fact that Ͼ30% of approved drugs elicit their therapeutic effect by selectively acting on known members of this family (3). The human genome harbors Ͼ800 putative GPCRs including a considerable number with unknown physiological function or ligands. GPCR cascades hence remain a major focus of molecular pharmacology (4, 5).Signal transduction by GPCRs is mediated by activation of protein kinases (4), among which the most intensively studied is the cAMP-dependent protein kinase A (PKA) (6). Various extracellular signals converge on the cAMP/PKA pathway through ligand binding to GPCRs. The adenylyl cyclase then converts ATP to the ubiquitous second messenger cAMP. Intrac...

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Section: Use Of the Pka Sensor To Monitor Protein Complex Dynamics Inmentioning

confidence: 99%

Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo

Stefan

Aquin²,

Berger³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

174

179

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“…tors form heterodimers with the β 2 AR (13,14). We next examined whether activation of the EP 1 receptor or the β 2 AR altered the BRET signal.…”

Section: Figurementioning

confidence: 99%

“…BRET was carried out in living cells expressing 2 receptors having carboxyterminally fused Renilla luciferase (Rluc) or yellow fluorescent protein (YFP). As described in detail elsewhere (11)(12)(13), this approach takes advantage of a signal produced by the energy donor moiety (Rluc) acting on the energy acceptor (YFP) when the two are in very close proximity. As shown in Figure 5B, studies carried out in HEK 293 cells transfected to express β 2 AR-Rluc and EP 1 -YFP (see Methods) indeed revealed a BRET signal, as indicated by the perturbation of the emission spectrum at 530 nM.…”

Section: Introductionmentioning

confidence: 99%

Airway smooth muscle prostaglandin-EP1 receptors directly modulate 2-adrenergic receptors within a unique heterodimeric complex

McGraw

Mihlbachler²,

Schwarb³

et al. 2006

Journal of Clinical Investigation

110

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Multiple and paradoxical effects of airway smooth muscle (ASM) 7-transmembrane-spanning receptors activated during asthma, or by treatment with bronchodilators such as β 2 -adrenergic receptor (β 2 AR) agonists, indicate extensive receptor crosstalk. We examined the signaling of the prostanoid-EP 1 receptor, since its endogenous agonist prostaglandin E 2 is abundant in the airway, but its functional implications are poorly defined. Activation of EP 1 failed to elicit ASM contraction in mouse trachea via this G αq -coupled receptor. However, EP 1 activation markedly reduced the bronchodilatory function of β 2 AR agonist, but not forskolin, indicating an early pathway interaction. Activation of EP 1 reduced β 2 AR-stimulated cAMP in ASM but did not promote or augment β 2 AR phosphorylation or alter β 2 AR trafficking. Bioluminescence resonant energy transfer showed EP 1 and β 2 AR formed heterodimers, which were further modified by EP 1 agonist. In cell membrane [ 35 S]GTPγS binding studies, the presence of the EP 1 component of the dimer uncoupled β 2 AR from G αs , an effect accentuated by EP 1 agonist activation. Thus alone, EP 1 does not appear to have a significant direct effect on airway tone but acts as a modulator of the β 2 AR, altering G αs coupling via steric interactions imposed by the EP 1 :β 2 AR heterodimeric signaling complex and ultimately affecting β 2 AR-mediated bronchial relaxation. This mechanism may contribute to β-agonist resistance found in asthma. IntroductionThe 7-transmembrane-spanning (7-TM-spanning) receptors represent the largest signaling family in the genome. We estimate that the lung expresses 25-50 7-TM receptors in airway epithelial cells, airway smooth muscle (ASM), pulmonary vasculature, alveolar walls, and resident immune cells (1). In regard to asthma, several 7-TM receptors play established roles in bronchoconstriction (e.g., M 3 -muscarinic receptor) and bronchodilation (e.g., β 2 -adrenergic receptor [β 2 AR]). Despite identification of the endogenous ligands and receptor localization, there are a number of 7-TM receptors expressed in the airway whose functions are unknown, or appear to function paradoxically, based on recombinantly expressed receptors in model cell systems. This lack of understanding of receptor function has impeded our ability to ascertain the role of these ligands (some of which are markedly increased in asthma) in the relaxation/contraction of ASM; thus the mechanistic basis of bronchial hyperreactivity and bronchoconstriction in asthma remains only partially understood. In many cases the basis for incomplete mechanistic information can be attributed to the nature of recombinant expression systems, which may not take

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“…15 This provided a new proteomic assay to study GPCR activation, which, unlike other cell-based assays (e.g., intracellular Ca ++ release or cAMP accumulation), is not dependent on the preference of the receptor for specific G proteins (G q/11 , G s , G i/o ; reviewed by Heding 16 ). It is important to note here that there exist also few GPCRs that are suggested to internalize via β-arrestin independent pathways 17 or that do not internalize at all, at least in some cultured cells (e.g., GABA B receptors, β3-adrenergic receptors), 18,19 and therefore, functional assays relying on β-arrestin translocation are not suitable for these receptors.…”

mentioning

confidence: 99%

High-Throughput Screening of G Protein-Coupled Receptor Antagonists Using a Bioluminescence Resonance Energy Transfer 1-Based β-Arrestin2 Recruitment Assay

Hamdan

Audet

Garneau³

et al. 2005

SLAS Discovery

Self Cite

169

143

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In this study, the authors developed HEK293 cell lines that stably coexpressed optimal amounts of β-arrestin2-Rluc and VENUS fusions of G protein-coupled receptors (GPCRs) belonging to both class A and class B receptors, which include receptors that interact transiently or stably with β-arrestins. This allowed the use of a bioluminescence resonance energy transfer (BRET) 1-β-arrestin2 translocation assay to quantify receptor activation or inhibition. One of the developed cell lines coexpressing CCR5-VENUS and β-arrestin2-Renilla luciferase was then used for high-throughput screening (HTS) for antagonists of the chemokine receptor CCR5, the primary co-receptor for HIV. A total of 26,000 compounds were screened for inhibition of the agonist-promoted β-arrestin2 recruitment to CCR5, and 12 compounds were found to specifically inhibit the agonist-induced β-arrestin2 recruitment to CCR5. Three of the potential hits were further tested using other functional assays, and their abilities to inhibit CCR5 agonist-promoted signaling were confirmed. This is the 1st study describing a BRET1-β-arrestin recruitment assay in stable mammalian cells and its successful application in HTS for GPCRs antagonists. (Journal of Biomolecular Screening 2005:463-475)

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Hetero-oligomerization between β2- and β3-Adrenergic Receptors Generates a β-Adrenergic Signaling Unit with Distinct Functional Properties

Cited by 142 publications

References 76 publications

Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo

Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo

Airway smooth muscle prostaglandin-EP1 receptors directly modulate 2-adrenergic receptors within a unique heterodimeric complex

High-Throughput Screening of G Protein-Coupled Receptor Antagonists Using a Bioluminescence Resonance Energy Transfer 1-Based β-Arrestin2 Recruitment Assay

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