Activation of human D3 dopamine receptor inhibits P/Q-type calcium channels and secretory activity in AtT-20 cells.
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The D3 dopamine receptor is postulated to play an important role in the regulation of neurotransmitter secretion at both pre- and postsynaptic terminals. However, this hypothesis and the underlying mechanisms remain untested because of the lack of D3-selective ligands, paucity of appropriate model secretory systems, and the weak and inconsistent coupling of D3 receptors to classical signal transduction pathways. The absence of ligands that selectively discriminate between D3 and D2 receptors in vivo precludes the study of D3 receptor function in the brain and necessitates the use of heterologous expression systems. In this report we demonstrate that activation of the human D3 dopamine receptor expressed in the AtT-20 neuroendocrine cell line causes robust inhibition of P/Q-type calcium channels via pertussis toxin-sensitive G-proteins. In addition, using the vesicle trafficking dye FM1-43, we show that D3 receptor activation significantly inhibits spontaneous secretory activity in these cells. Our results not only support the hypothesis that the D3 receptor can regulate secretory activity but also provide insight into the underlying signaling mechanisms. We propose a functional model in which the D3 receptor tightly regulates neurotransmitter release at a synapse by only allowing the propagation of spikes above a certain frequency or burst-duration threshold. (+info)
Identification and localization of G protein subunits in human spermatozoa.
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Antibodies to alpha and beta subunits of guanine nucleotide regulatory proteins (G proteins) were used to identify which G proteins are present in mature human spermatozoa and to determine their subcellular localization. Immunoblots of membranes from spermatozoa demonstrate the presence of Galphai2, Galphai3, Galphaq/11 and Gbeta35 and the absence of Galphai1, Galpha0, Galphas, Galpha12, Galpha13, Galpha16, Galpha and Gbeta36. Indirect immunofluorescence demonstrates the presence of Galphaq/11 in the acrosome, with the highest proportion in the equatorial segment. Galphai2 is present in the acrosome, midpiece and tailpiece and Galphai3 in the postnuclear cap, midpiece and tailpiece. The Gbeta35 subunit is found mostly in the midpiece, with marginal labelling of the head, tailpiece and the equatorial segment of the acrosome. The distinct pattern of distribution of G proteins suggests that they may couple to receptors or effectors which also have discrete regions of localization in spermatozoa. These highly localized signal transduction pathways may regulate discrete functions, such as activation of the acrosome reaction, fusion with the oocyte and motility. (+info)
Gi-mediated tyrosine phosphorylation of Grb2 (growth-factor-receptor-bound protein 2)-bound dynamin-II by lysophosphatidic acid.
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Lysophosphatidic acid (LPA) is the prototypic G-protein-coupled receptor agonist that activates the Ras-mitogen-activated protein (MAP) kinase cascade through pertussis toxin (PTX)-sensitive Gi and enhanced tyrosine kinase activity. We recently detected a 100 kDa protein (p100) that binds to the C-terminal SH3 domain of growth-factor-receptor-bound protein 2 (Grb2) and becomes tyrosine phosphorylated in a PTX-sensitive manner in LPA-treated Rat-1 cells [Kranenburg, Verlaan, Hordijk and Moolenaar (1997) EMBO J. 16, 3097-3105]. Through glutathione S-transferase-Grb2 affinity purification and microsequencing, we have now identified p100 as dynamin-II, a GTPase that regulates clathrin-mediated endocytosis. We show that in Rat-1 cells, Grb2-bound dynamin-II is rapidly tyrosine phosphorylated in response to LPA in a PTX-sensitive manner. Thus, tyrosine phosphorylation of Grb2-bound dynamin-II may be a critical event in Gi-mediated activation of the Ras-MAP kinase cascade in fibroblasts. (+info)
Both Gs and Gi proteins are critically involved in isoproterenol-induced cardiomyocyte hypertrophy.
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Activation of beta-adrenoreceptors induces cardiomyocyte hypertrophy. In the present study, we examined isoproterenol-evoked intracellular signal transduction pathways leading to activation of extracellular signal-regulated kinases (ERKs) and cardiomyocyte hypertrophy. Inhibitors for cAMP and protein kinase A (PKA) abolished isoproterenol-evoked ERK activation, suggesting that Gs protein is involved in the activation. Inhibition of Gi protein by pertussis toxin, however, also suppressed isoproterenol-induced ERK activation. Overexpression of the Gbetagamma subunit binding domain of the beta-adrenoreceptor kinase 1 and of COOH-terminal Src kinase, which inhibit functions of Gbetagamma and the Src family tyrosine kinases, respectively, also inhibited isoproterenol-induced ERK activation. Overexpression of dominant-negative mutants of Ras and Raf-1 kinase and of the beta-adrenoreceptor mutant that lacks phosphorylation sites by PKA abolished isoproterenol-stimulated ERK activation. The isoproterenol-induced increase in protein synthesis was also suppressed by inhibitors for PKA, Gi, tyrosine kinases, or Ras. These results suggest that isoproterenol induces ERK activation and cardiomyocyte hypertrophy through two different G proteins, Gs and Gi. cAMP-dependent PKA activation through Gs may phosphorylate the beta-adrenoreceptor, leading to coupling of the receptor from Gs to Gi. Activation of Gi activates ERKs through Gbetagamma, Src family tyrosine kinases, Ras, and Raf-1 kinase. (+info)
A decrease in the amount and function of inhibitory GTP-binding protein in the resistance small artery from spontaneously hypertensive rats.
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The inhibitory GTP-binding protein (Gi protein) plays an important role in regulation of vascular tone. Many studies have implicated the role of Gi protein in conduit vessels. However, the physiological role of Gi protein in the control of peripheral microvascular tone in hypertension has not been established yet. Therefore, we investigated the concentration of Gi protein in the peripheral resistance arteries and aorta in the spontaneously hypertensive rats (SHR), normotensive Wistar-Kyoto rats (WKY) and renovascular hypertensive rats (RHR), using immunohistochemical methods semiquantitatively. Changes in the function of Gi protein in relation to alpha2-adrenoceptor were also investigated by microcannulation techniques. We have shown that the amount of alpha2 subunits of Gi protein in the cremaster small artery was significantly lower in SHR aged 4 weeks and older than in age-matched WKY and that there were no significant differences between RHR and WKY. We also demonstrated that the function of Gi protein in relation to alpha2-adrenoceptor was already lower in SHR before the onset of hypertension. The quantitative and functional decline in Gi protein in the smooth muscle cells of peripheral small arteries were observed in SHR even before the onset of hypertension, whereas rats with secondary hypertension did not exhibit this finding. (+info)
The beta3-adrenergic receptor activates mitogen-activated protein kinase in adipocytes through a Gi-dependent mechanism.
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Promiscuous coupling between G protein-coupled receptors and multiple species of heterotrimeric G proteins provides a potential mechanism for expanding the diversity of G protein-coupled receptor signaling. We have examined the mechanism and functional consequences of dual Gs/Gi protein coupling of the beta3-adrenergic receptor (beta3AR) in 3T3-F442A adipocytes. The beta3AR selective agonist disodium (R, R)-5-[2[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1, 3-benzodioxole-2,2-dicarboxylate (CL316,243) stimulated a dose-dependent increase in cAMP production in adipocyte plasma membrane preparations, and pretreatment of cells with pertussis toxin resulted in a further 2-fold increase in cAMP production by CL316,243. CL316,243 (5 microM) stimulated the incorporation of 8-azido-[32P]GTP into Galphas (1.57 +/- 0.12; n = 3) and Galphai (1. 68 +/- 0.13; n = 4) in adipocyte plasma membranes, directly demonstrating that beta3AR stimulation results in Gi-GTP exchange. The beta3AR-stimulated increase in 8-azido-[32P]GTP labeling of Galphai was equivalent to that obtained with the A1-adenosine receptor agonist N6-cyclopentyladenosine (1.56 +/- 0.07; n = 4), whereas inclusion of unlabeled GTP (100 microM) eliminated all binding. Stimulation of the beta3AR in 3T3-F442A adipocytes led to a 2-3-fold activation of mitogen-activated protein (MAP) kinase, as measured by extracellular signal-regulated kinase-1 and -2 (ERK1/2) phosphorylation. Pretreatment of cells with pertussis toxin (PTX) eliminated MAP kinase activation by beta3AR, demonstrating that this response required receptor coupling to Gi. Expression of the human beta3AR in HEK-293 cells reconstituted the PTX-sensitive stimulation of MAP kinase, demonstrating that this phenomenon is not exclusive to adipocytes or to the rodent beta3AR. ERK1/2 activation by the beta3AR was insensitive to the cAMP-dependent protein kinase inhibitor H-89 but was abolished by genistein and AG1478. These data indicate that constitutive beta3AR coupling to Gi proteins serves both to restrain Gs-mediated activation of adenylyl cyclase and to initiate additional signal transduction pathways, including the ERK1/2 MAP kinase cascade. (+info)
Fyn kinase-directed activation of SH2 domain-containing protein-tyrosine phosphatase SHP-2 by Gi protein-coupled receptors in Madin-Darby canine kidney cells.
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SHP-2, an SH2 domain-containing protein-tyrosine phosphatase, plays an important role in receptor tyrosine kinase-regulated cell proliferation and differentiation. Little is known about the activation mechanisms and the participation of SHP-2 in the activity of G protein-coupled receptors lacking intrinsic tyrosine kinase activity. We show that the activity of SHP-2 (but not SHP-1) is specifically stimulated by the selective alpha2A-adrenergic receptor agonist UK14304 and by lysophosphatidic acid (LPA) in Madin-Darby canine kidney (MDCK) cells. UK14304 and LPA promote the tyrosine phosphorylation of SHP-2 and its association with Grb2. The agonist-induced direct interaction of Grb2 with SHP-2 is mediated by the SH2 domain of Grb2 and the tyrosine phosphorylation of SHP-2. Rapid activation of Src family kinase by UK14304 preceded the SHP-2 activation. Among the Src family members (Src, Fyn, Lck, Yes, and Lyn) present in MDCK cells, Fyn was the only one specifically associated with SHP-2, and the physical interaction between them, which requires the Src family kinase activity, was increased in response to the agonists. Pertussis toxin, PP1 (a selective Src family kinase inhibitor), or overexpression of a catalytically inactive mutant of Fyn blocked the UK14304- or LPA-stimulated activity of SHP-2, SHP-2 tyrosine phosphorylation, and SHP-2 association with Grb2. Therefore, we have demonstrated for the first time that the activation of SHP-2 by these Gi protein-coupled receptors requires Fyn kinase and that there is a specific physical interaction of Fyn kinase with SHP-2 in MDCK cells. (+info)
Interaction of amiodarone and triiodothyronine on the expression of beta-adrenoceptors in brown adipose tissue of rat.
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1. This study was undertaken to evaluate in vivo the influence of amiodarone on the effects of triiodothyronine (T3) in brown adipose tissue (BAT) which are independent of thyroid hormone synthesis and of the conversion of thyroxine (T4) to T3. Thyroidectomized rats were given a replacement dose of T3 (0.5 mg kg(-1) p.o. daily for 3 days) with or without amiodarone (50 mg kg(-1) p.o. daily for 1 week). 2. As assessed by RT-PCR, treatment of thyroidectomized rats with T3 caused a 2 fold decrease in beta3-adrenoceptor (beta3-AR) mRNA levels and a 2 fold increase in beta1-AR mRNA levels. 3. Binding studies using [3H]-CGP 12177 as a ligand showed that treatment of thyoidectomized rats with T3 resulted in a 70% decrease in beta3-AR number and in an 80% increase in beta1-AR in BAT membranes. 4. T3-treatment abolished the increase in BAT adenylyl cyclase (AC) activity induced by CGP12177 in thyroidectomized rats. It also decreased the amount of Gi protein (ADP-ribosylation) by 30%. 5. At variance with the literature on the heart, amiodarone administration did not inhibit the positive effect of T3 on beta1-AR expression in BAT in thyroidectomized rats. However, it antagonized the effect of T3 on beta3-AR number, but not on AC activity or on Gi expression. 6. These results indicate that the effects of thyroid hormones on the responsiveness of BAT to catecholamines involves both receptor and post-receptor mechanisms, they also suggest that interaction between amiodarone and thyroid hormones is highly tissue-specific and depends on the beta-AR subtype. (+info)