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Tyrosine 308 is necessary for ligand-directed Gs protein-biased signaling of ?2-adrenoceptor.

ABSTRACT: Interaction of a given G protein-coupled receptor to multiple different G proteins is a widespread phenomenon. For instance, ?2-adrenoceptor (?2-AR) couples dually to Gs and Gi proteins. Previous studies have shown that cAMP-dependent protein kinase (PKA)-mediated phosphorylation of ?2-AR causes a switch in receptor coupling from Gs to Gi. More recent studies have demonstrated that phosphorylation of ?2-AR by G protein-coupled receptor kinases, particularly GRK2, markedly enhances the Gi coupling. We have previously shown that although most ?2-AR agonists cause both Gs and Gi activation, (R,R')-fenoterol preferentially activates ?2-AR-Gs signaling. However, the structural basis for this functional selectivity remains elusive. Here, using docking simulation and site-directed mutagenesis, we defined Tyr-308 as the key amino acid residue on ?2-AR essential for Gs-biased signaling. Following stimulation with a ?2-AR-Gs-biased agonist (R,R')-4'-aminofenoterol, the Gi disruptor pertussis toxin produced no effects on the receptor-mediated ERK phosphorylation in HEK293 cells nor on the contractile response in cardiomyocytes expressing the wild-type ?2-AR. Interestingly, Y308F substitution on ?2-AR enabled (R,R')-4'-aminofenoterol to activate Gi and to produce these responses in a pertussis toxin-sensitive manner without altering ?2-AR phosphorylation by PKA or G protein-coupled receptor kinases. These results indicate that, in addition to the phosphorylation status, the intrinsic structural feature of ?2-AR plays a crucial role in the receptor coupling selectivity to G proteins. We conclude that specific interactions between the ligand and the Tyr-308 residue of ?2-AR stabilize receptor conformations favoring the receptor-Gs protein coupling and subsequently result in Gs-biased agonism.

SUBMITTER: Woo AY

PROVIDER: S-EPMC4094047 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Tyrosine 308 is necessary for ligand-directed Gs protein-biased signaling of β2-adrenoceptor.

Woo Anthony Yiu-Ho AY Jozwiak Krzysztof K Toll Lawrence L Tanga Mary J MJ Kozocas Joseph A JA Jimenez Lucita L Huang Ying Y Song Ying Y Plazinska Anita A Pajak Karolina K Paul Rajib K RK Bernier Michel M Wainer Irving W IW Xiao Rui-Ping RP

The Journal of biological chemistry 20140515 28

Interaction of a given G protein-coupled receptor to multiple different G proteins is a widespread phenomenon. For instance, β2-adrenoceptor (β2-AR) couples dually to Gs and Gi proteins. Previous studies have shown that cAMP-dependent protein kinase (PKA)-mediated phosphorylation of β2-AR causes a switch in receptor coupling from Gs to Gi. More recent studies have demonstrated that phosphorylation of β2-AR by G protein-coupled receptor kinases, particularly GRK2, markedly enhances the Gi couplin ...[more]

PMID: 24831005

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Project description:Mitochondrial dysfunction is a common mediator of disease and organ injury. Although recent studies show that inducing mitochondrial biogenesis (MB) stimulates cell repair and regeneration, only a limited number of chemicals are known to induce MB. To examine the impact of the β-adrenoceptor (β-AR) signaling pathway on MB, primary renal proximal tubule cells (RPTC) and adult feline cardiomyocytes were exposed for 24 h to multiple β-AR agonists: isoproterenol (nonselective β-AR agonist), (±)-(R*,R*)-[4-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]phenoxy] acetic acid sodium hydrate (BRL 37344) (selective β(3)-AR agonist), and formoterol (selective β(2)-AR agonist). The Seahorse Biosciences (North Billerica, MA) extracellular flux analyzer was used to quantify carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP)-uncoupled oxygen consumption rate (OCR), a marker of maximal electron transport chain activity. Isoproterenol and BRL 37244 did not alter mitochondrial respiration at any of the concentrations examined. Formoterol exposure resulted in increases in both FCCP-uncoupled OCR and mitochondrial DNA (mtDNA) copy number. The effect of formoterol on OCR in RPTC was inhibited by the β-AR antagonist propranolol and the β(2)-AR inverse agonist 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol hydrochloride (ICI-118,551). Mice exposed to formoterol for 24 or 72 h exhibited increases in kidney and heart mtDNA copy number, peroxisome proliferator-activated receptor γ coactivator 1α, and multiple genes involved in the mitochondrial electron transport chain (F0 subunit 6 of transmembrane F-type ATP synthase, NADH dehydrogenase subunit 1, NADH dehydrogenase subunit 6, and NADH dehydrogenase [ubiquinone] 1β subcomplex subunit 8). Cheminformatic modeling, virtual chemical library screening, and experimental validation identified nisoxetine from the Sigma Library of Pharmacologically Active Compounds and two compounds from the ChemBridge DIVERSet that increased mitochondrial respiratory capacity. These data provide compelling evidence for the use and development of β(2)-AR ligands for therapeutic MB.

Project description:Background and objectiveSympathetic activity plays an important role in the proliferation and differentiation of stem cells, and it changes over time, thereby exerting differential effects on various stem cell types. Aging causes sympathetic hyperactivity in aged tissues and blunts sympathetic nerves regulation, and sympathetic abnormalities play a role in aging-related diseases. Currently, the effect of sympathetic activity on skeletal muscle stem cells, namely satellite cells (SCs), is unclear. This study aimed to investigate the effects of skeletal muscle sympathetic activity on SC aging and skeletal muscle repair.Materials and methodsTo evaluate skeletal muscle and fibrotic areas, numbers of SCs and myonuclei per muscle fiber, β2-adrenoceptor (β2-ADR) expression, muscle repair, and sympathetic innervation in skeletal muscle, aged mice, young mice that underwent chemical sympathectomy (CS) were utilized. Mice with a tibialis anterior muscle injury were treated by barium chloride (BaCl2) and clenbuterol (CLB) in vivo. SCs or C2C12 cells were differentiated into myotubes and treated with or without CLB. Immunofluorescence, western blot, sirius red, and hematoxylin-eosin were used to evaluate SCs, myogenic repair and differentiation.ResultsThe number of SCs, sympathetic activity, and reparability of muscle injury in aged mice were significantly decreased, compared with those in young mice. The above characteristics of young mice that underwent CS were similar to those of aged mice. While CLB promoted the repair of muscle injury in aged and CS mice and ameliorated the reduction in the SC number and sympathetic activity, the effects of CLB on the SCs and sympathetic nerves in young mice were not significant. CLB inhibited the myogenic differentiation of C2C12 cells in vitro. We further found that NF-κB and ERK1/2 signaling pathways were activated during myogenic differentiation, and this process could be inhibited by CLB.ConclusionNormal sympathetic activity promoted the stemness of SCs to thereby maintain a steady state. It also could maintain total and self-renewing number of SCs and maintain a quiescent state, which was correlated with skeletal SCs via β2-ADR. Normal sympathetic activity was also beneficial for the myogenic repair of injured skeletal muscle.

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Tyrosine 308 is necessary for ligand-directed Gs protein-biased signaling of ?2-adrenoceptor.

Publications

Tyrosine 308 is necessary for ligand-directed Gs protein-biased signaling of β2-adrenoceptor.

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