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Journal Biol. Chem., 1998, 273: 19772-19777
Presenter: E. S. Werstiuk
Presenter's Summary:
Angiotensin II (Ang II) is an important endogenous mediator of vascular smooth muscle (VSMC) contraction and proliferation. These effects of Ang II are mediated predominantly by the AT1 receptor, (a 7-transmembrane spanning-heterotrimeric G protein-coupled receptor). The intracellular signalling pathways activated by this receptor, however are quite complex (reviewed by Griendling et al., 1996). Ang II binding leads to rapid and transient activation of the phosphatidylinositol-specific phospholipase C (PLC) to produce inositol trisphosphate (IP3), and diacylglycerol. In VSMC this is followed by a prolonged activation of phospholipase D, which hydrolyses phosphatidylcholine and ultimately produces diacylglycerol. More recently, it has also been shown that in VSMCs Ang II occupancy of the AT1 receptors also stimulates tyrosine phosphorylation of a number of intracellular proteins, including pp60src (Ishida et al., 1995), PLC-gamma (Marrero et al., 1994), and this leads to the stimulation of intracellular pathways such as the mitogen-activated protein kinase pathway (MAPK) (reviewed by Duff et al. 1995; and Griendling et al., 1997). AT1 receptors in other systems have been shown to activate the isozyme PLC-beta, via Galphaq/11 protein and lead to rapid (5 - 15 s) IP3 production. In rat VSMC, however this enzyme has been difficult to detect. PLC-gamma has been identified in VSMCs and shown to be phosphorylated in response to Ang II, but the time course of IP3 formation is much longer (30 s - 1 min). The objectives of the present study were to clarify the roles of PLC isozymes and tyrosine kinases in the Ang II-induced PLC activation, and to identify the subunits of heterotrimeric G-proteins involved in this process.
1) In cultured rat VSMCs, Western-immunoblot analysis using monoclonal antibodies against PLC-beta1, -gamma1, and -1 demonstrated the presence of all three PLC isozymes. Binding was detected at 150-kDa, 145-kDa and 85-kDa, corresponding to the relative molecular mass of the three different PLC isozymes.
2) The type of PLC isozymes involved in Ang II-stimulated AT1 receptor-PLC coupling were assessed by measuring Ang II-stimulated IP3 formation in myo-[H3]inositol labelled VSMCs, electroporated with specific antibodies against PLC-beta1, -gamma1, and -1. Ang II-stimulated IP3 formation was found to peak following 15 s of Ang II-stimulation. In cells with no electroporation Ang II-stimulated IP3 production increased by 126 ± 8%. In cells electroporated with no antibody, IP3 formation stimulated by Ang II increased by 119 ± 7%. When cells were electroporated with non-immune rabbit IgG, (mock electroporation) Ang II-stimulated IP3 formation decreased by 20% compared to cells electroporated with no antibodies. Electroporation of VSMCs with antibodies against PLC-gamma1, or -1 had no significant effect on the Ang II-stimulated IP3 formation measured at 15 s. In contrast, electroporation with an antibody against PLC-beta1 inhibited Ang II-stimulated IP3 production significantly. (83 ± 5% inhibition, p < 0.05) when compared to Ang II-stimulated IP3 formation in mock electroporated cells. These results indicated that PLC-beta1 is the isozyme which participates in the initial AT1 receptor-PLC coupling.
3) The role of tyrosine kinases in the Ang II-stimulated IP3 formation was evaluated by treating myo-[H3]inositol labelled VSMCs with or without genistein (a tyrosine kinase inhibitor), and measuring the time course of the Ang II-stimulated IP 3 formation. Genistein, at 100 µM inhibited IP 3 formation only after 30 s, but had no effect on Ang II-stimulated IP 3 production at 15 s. The time course of genistein's effect temporally correlated with the Ang II-induced tyrosine phosphorylation of PLC-gamma, as demonstrated by Western immunoblot analysis. Ang II-stimulated IP 3 production measured at 30 s was effectively blocked by electroporation with antibodies against PLC-gamma. Theses results suggested that PLC-gamma participates in the later phases of AT1 receptor-PLC coupling.
4) The specific G protein subunits, which are involved in the AT1 receptor-PLC coupling were evaluated by electroporating the cells in the presence of antibodies against specific G protein subunits and measuring the Ang II-stimulated IP 3 production. Electroporation of VSMCs with antibodies to rabbit IgG (negative control), or Galphai or Galpha13 had no significant effects on the Ang II-stimulated IP 3 production measured at 15 s. In contrast, anti-Galphaq/11, or Galpha12 antibodies inhibited the Ang II response significantly (56 ± 4%, p < 0.05; and 62 ± 5%, p < 0.05 inhibition, respectively). This partial inhibition could not be improved by increasing the amount of the individual antibodies added, but complete (93%) inhibition was obtained when the two antibodies, anti-Galphaq/11, and Galpha12 were added together. The inhibitory effect of the anti-Galphaq/11, or Galpha12 antibodies was abolished when these were boiled ( 100 for 30 min) prior to electroporation, suggesting that active antibodies were required for this inhibition of the Ang II-stimulated IP 3 formation. Ang II-induced repones were also inhibited (75 ± 6%, p < 0.05) when VSMCs were electroporated with anti-Gbeta antibodies, and this inhibition was also abolished by boiling the antibody. These results suggested that the AT1 receptor-PLC coupling is mediated by Galphaq/11betagamma, and Galpha12betagamma heterotrimeric G proteins, and both, Galpha and Gbetagamma subunits may serve in the early phases of the AT1 receptor-mediated signal transduction.
5) The role of the Gbetagamma subunit in the early phase of AT1 receptor-mediated signal transduction was further confirmed by measuring Ang II-stimulated IP 3 production in VSMC stably transfected with the plasmid encoding the carboxyl-terminus of the beta-adrenergic receptor kinase (betaARK1ct, effectively a Gbetagamma antagonist). The efficacy of the transfection was shown by Northern analysis. Ang II-stimulated IP 3 production was significantly inhibited (43 ± 4%, p < 0.05) in cells stably over expressing betaARK1ct compared to the Ang II response in two different cell lines transfected with vector alone.
In this paper the authors present evidence in support of a novel mechanism for the temporal regulation of distinct intracellular signaling pathways, mediated by a single plasma-membrane bound receptor. This is achieved by sequential coupling of the AT1 receptor to two different effector-enzymes. Thus, Ang II binding to the AT1 receptors in rat VSMCs activate two distinct intracellular signal-transduction pathways in a sequential manner. The initial phase of Ang II-induced IP3 formation occurs by AT1 receptor coupling to PLC-beta1, via Galphaq/11, Galpha12 and the G protein beta-gamma-subunits; whereas the later phase is mediated by tyrosine phosphorylation and involves the activation of PLC-gamma. Thus, the initial rapid and transient IP3 production is due to PIP2 hydrolysis by PLC-beta1, and the later, more sustained IP3 is formed by the action of PLC-gamma on the same substrate. This AT1 receptor coupling to the two separate signaling pathways appears highly organized and tightly regulated, so the final result is a precise temporal control of IP3 generation.
1) The early phase of Ang II-stimulated IP3 is formation is mediated by the interaction of the AT1 receptor with Galphaq/11, and Galpha12 and Gbetagamma. (The authors' data support a role for all these three G protein subunits in the activation of PLC-beta1).
What regulates the interaction between the AT1 receptor and the three G-protein subunits? Is this simply G protein subunit availability in rat VSMCs?
Are all three G protein subunits necessary for PLC-beta1 activation?
2) The late phase Ang II-induced IP3 production is via PLC-gamma activation.
What regulates the interaction between the AT1 receptor and PLC-gamma?
Is tyrosine phosphorylation of PLC-gamma a prerequisite for its activation?
Is PLC-gamma phosphorylated by the AT1 receptor-associated pp60c-src?
3) The early-, and late- phase Ang II-induced repones are coordinated, so the two distinct pathways are sequential .
How is this step-wise, sequential regulation achieved?
Is this first phase dependent on the availability of the hetero trimeric G protein-subunits in the vicinity of the activated AT1 receptor in the plasma membrane?
Does the late phase require recruitment of pp60c-src to the Ang II-occupied AT1 receptor in the plasma membrane?
What is the "message"between the early-phase-, and the late-phase signal transduction pathways?
Activation of phospholipase C (PLC) is one of the earliest events in angiotensin II (Ang II) type 1 (AT1) receptor (R)-mediated signal transduction in vascular smooth muscle cells (VSMCs). The coupling mechanisms of AT1 Rs to PLC, however, are controversial, because both tyrosine phosphorylation of PLC-gamma and G-protein-dependent PLC-beta activation pathways have been reported. The expression of PLC-beta1, furthermore, has not been consistently demonstrated in VSMCs. Here we identified the PLC subtype and subunits of hetero trimeric G-proteins involved in AT1 R-PLC coupling using cultured rat VSMCs. Western analysis revealed the expression of PLC-beta1, -gamma1, and -1 in VSMCs. Ang II-stimulated inositol trisphosphate (IP3 ) formation measured at 15 s, which corresponds to the peak response, was significantly inhibited by electroporation of antibodies against PLC-beta1, but not by anti-PLC-gamma and - antibodies. Electroporation of anti-Galphaq/11 and -Galpha12 antibodies also showed significant inhibition of the Ang II-induced IP3 generation at 15 s, while anti-Galphai and Galpha13 antibodies were ineffective. Furthermore, in VSMCs electroporated with anti-Gbeta antibody and cells stably transfected with the plasmid encoding the Gbetagamma-binding region of the carboxyl terminus of beta-adrenergic receptor kinase, the peak Ang II-stimulated PLC activity (at 15 s) was significantly inhibited. The tyrosine kinase inhibitor, genistein, had no effect on the peak response to Ang II stimulation, but significantly inhibited IP3 production after 30 sec, a time period which temporally correlated with PLC-gamma tyrosine phosphorylation in response to Ang II. Moreover, electroporation of anti-PLC-gamma antibody markedly inhibited the IP3 production measured at 30 s, indicating that tyrosine phosphorylation of PLC-gamma contributes mainly to the later phase of PLC activation. Thus, these results suggest that: 1) AT1 receptors sequentially couple to PLC-beta1 via a hetero trimeric G protein and to PLC-gamma via a down-stream tyrosine kinase; 2) the initial AT1 receptor- PLC-beta1 coupling is mediated by Galphaq/11betagamma and Galpha12betagamma; 3) Gbetagamma acts as a signal transducer for activation of PLC in VSMCs. The sequential coupling of AT1 receptors to PLC-beta1 and PLC-gamma, as well as dual coupling of AT1 receptors to distinct Galpha proteins, suggests a novel mechanism for temporally controlled, highly organized and convergent Ang II-signalling network in VSMCs.
K.K. Griendling, B. Lassègue and R. W. Alexander. Angiotensin Receptors and their Therapeutic Implications. Ann. Rev. Pharmacol. Toxicol., 1996, 36:281-306.
J.L. Duff, M.B. Marrero. W.G. Paxton, B. Schieffer, K.E. Bernstein and B.C. Berk. Angiotensin II signal transduction and the mitogen-activated protein kinase pathway. Cardiovascular Research, 1995, 30 :511-517.
M. Ishida, M.B. Marrero, B. Schieffer, T. Ishida, K.E. Bernstein and B.C. Berk. Angiotensin II Activates pp60c-src in Vascular Smooth Muscle Cells. Circulation Research, 1995, 77:1053-1059.
M.B. Marrero, W.G. Paxton, J.L. Duff, B.C. Berk and K.E. Bernstein. Angiotensin II Stimulates Tyrosine Phosphorylation of Phospholipase C-gamma1 in Vascular Smooth Muscle Cells. J. Biol. Chem., 1994, 269: 10935-10939.
K.K. Griendling, M. Ushio-Fukai, B. Lassègue and R. W. Alexander Angiotensin II Signalling in Vascular Smooth Muscle. New Concepts. Hypertension, 1997, 29 [part 2 ]:366-373.
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