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?1A-adrenergic receptor induces activation of extracellular signal-regulated kinase 1/2 through endocytic pathway.

ABSTRACT: G protein-coupled receptors (GPCRs) activate mitogen-activated protein kinases through a number of distinct pathways in cells. Increasing evidence has suggested that endosomal signaling has an important role in receptor signal transduction. Here we investigated the involvement of endocytosis in ?(1A)-adrenergic receptor (?(1A)-AR)-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2). Agonist-mediated endocytic traffic of ?(1A)-AR was assessed by real-time imaging of living, stably transfected human embryonic kidney 293A cells (HEK-293A). ?(1A)-AR was internalized dynamically in cells with agonist stimulation, and actin filaments regulated the initial trafficking of ?(1A)-AR. ?(1A)-AR-induced activation of ERK1/2 but not p38 MAPK was sensitive to disruption of endocytosis, as demonstrated by 4°C chilling, dynamin mutation and treatment with cytochalasin D (actin depolymerizing agent). Activation of protein kinase C (PKC) and C-Raf by ?(1A)-AR was not affected by 4°C chilling or cytochalasin D treatment. U73122 (a phospholipase C [PLC] inhibitor) and Ro 31-8220 (a PKC inhibitor) inhibited ?(1B)-AR- but not ?(1A)-AR-induced ERK1/2 activation. These data suggest that the endocytic pathway is involved in ?(1A)-AR-induced ERK1/2 activation, which is independent of G(q)/PLC/PKC signaling.

SUBMITTER: Liu F

PROVIDER: S-EPMC3125289 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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α1A-adrenergic receptor induces activation of extracellular signal-regulated kinase 1/2 through endocytic pathway.

Liu Fei F He Kangmin K Yang Xinxing X Xu Ning N Liang Zhangyi Z Xu Ming M Zhao Xinsheng X Han Qide Q Zhang Youyi Y

PloS one 20110628 6

G protein-coupled receptors (GPCRs) activate mitogen-activated protein kinases through a number of distinct pathways in cells. Increasing evidence has suggested that endosomal signaling has an important role in receptor signal transduction. Here we investigated the involvement of endocytosis in α(1A)-adrenergic receptor (α(1A)-AR)-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2). Agonist-mediated endocytic traffic of α(1A)-AR was assessed by real-time imaging of living, s ...[more]

PMID: 21738688

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Project description:In cardiac myocytes, sustained (3 min) intracellular acidosis activates the ERK1/2 (extracellular-signal-regulated kinase 1/2) pathway and, through this pathway, increases sarcolemmal NHE (Na+/H+ exchanger) activity [Haworth, McCann, Snabaitis, Roberts and Avkiran (2003) J. Biol. Chem. 278, 31676-31684]. In the present study, we aimed to determine the time-dependence, pH-dependence and upstream signalling mechanisms of acidosis-induced ERK1/2 activation in ARVM (adult rat ventricular myocytes). Cultured ARVM were subjected to intracellular acidosis for up to 20 min by exposure to NH4Cl, followed by washout with a bicarbonate-free Tyrode solution containing the NHE1 inhibitor cariporide. After the desired duration of intracellular acidosis, the phosphorylation status of ERK1/2 and its downstream effector p90(RSK) (90 kDa ribosomal S6 kinase) were determined by Western blotting. This revealed a time-dependent transient phosphorylation of both ERK1/2 and p90(RSK) by intracellular acidosis (intracellular pH approximately 6.6), with maximum activation occurring at 3 min and a return to basal levels by 20 min. When the degree of intracellular acidosis was varied from approximately 6.8 to approximately 6.5, maximum ERK1/2 phosphorylation was observed at an intracellular pH of 6.64. Inhibition of MEK1/2 [MAPK (mitogen-activated protein kinase)/ERK kinase 1/2) by pre-treatment of ARVM with U0126 or adenoviral expression of dominant-negative D208A-MEK1 protein prevented the phosphorylation of ERK1/2 by sustained intracellular acidosis, as did inhibition of Raf-1 with GW 5074 or ZM 336372. Interference with Ras signalling by the adenoviral expression of dominant-negative N17-Ras protein or with FPT III (farnesyl protein transferase inhibitor III) also prevented acidosis-induced ERK1/2 phosphorylation, whereas inhibiting G-protein signalling [by adenoviral expression of RGS4 or Lsc, the RGS domain of p115 RhoGEF (guanine nucleotide-exchange factor)] or protein kinase C (with bisindolylmaleimide I) had no effect. Our data show that, in ARVM, sustained intracellular acidosis activates ERK1/2 through proximal activation of the classical Ras/Raf/MEK pathway.

Dataset Information

?1A-adrenergic receptor induces activation of extracellular signal-regulated kinase 1/2 through endocytic pathway.

Publications

α1A-adrenergic receptor induces activation of extracellular signal-regulated kinase 1/2 through endocytic pathway.

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