Temporal dispersion of activation of phospholipase C-β1 and -γ isoforms by angiotensin II in vascular smooth muscle cells. Role of α(q/11), α12, and βγ G protein subunits

Masuko Fukai, Kathy K. Griendling, Marjorie Akers, P. Reid Lyons, R. Wayne Alexander

Research output: Contribution to journalArticle

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Abstract

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-γ and G protein-dependent PLC-β activation pathways have been reported. The expression of PLC-β1, furthermore, has not been consistently demonstrated in VSMCs. Here we identified the PLC subtypes and subunits of heterotrimeric G proteins involved in AT1 R-PLC coupling using cultured rat VSMCs. Western analysis revealed the expression of PLC-β1, - γ1, and -δ1 in VSMCs. Ang II-stimulated inositol trisphosphate (IP3) formation measured at 15 s, which corresponds to the peak response, was significantly in. hibited by electroporation of antibodies against PLC-β1, but not by anti-PLC-γ and -δ antibodies. Electroporation of anti-Gα(q/11) and -Gα12 antibodies also showed significant inhibition of the Ang II- induced IP3 generation at 15 s, while anti-Gα(i) and Gα13 antibodies were ineffective. Furthermore, in VSMCs electroporated with anti-Gβ antibody and cells stably transfected with the plasmid encoding the Gβγ-binding region of the carboxyl terminus of β-adrenergic receptor kinase1, 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 s, a time period which temporally correlated with PLC-γ tyrosine phosphorylation in response to Ang II. Moreover, electroporation of anti-PLC-γ antibody markedly inhibited the IP3 production measured at 30 s, indicating that tyrosine phosphorylation of PLC-γ contributes mainly to the later phase of PLC activation. Thus, these results suggest that: 1) AT1 receptors sequentially couple to PLC-β1 via a heterotrimeric G protein and to PLC-γ via a downstream tyrosine kinase; 2) the initial AT1 receptor-PLC-β1 coupling is mediated by Gα(q/11)βγ and Gα12βγ; 3) Gβγ acts as a signal transducer for activation of PLC in VSMCs. The sequential coupling of AT1 receptors to PLC-β1 and PLC-γ, as well as dual coupling of AT1 receptors to distinct Gα proteins, suggests a novel mechanism for a temporally controlled, highly organized and convergent Ang II-signaling network in VSMCs.

Original languageEnglish (US)
Pages (from-to)19772-19777
Number of pages6
JournalJournal of Biological Chemistry
Volume273
Issue number31
DOIs
StatePublished - Jul 31 1998
Externally publishedYes

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Protein Subunits
Type C Phospholipases
Vascular Smooth Muscle
GTP-Binding Proteins
Angiotensin II
Smooth Muscle Myocytes
Muscle
Protein Isoforms
Chemical activation
Cells
Phosphorylation
Electroporation
Antibodies
Tyrosine
Heterotrimeric GTP-Binding Proteins
Protein-Tyrosine Kinases
Signal transduction
Gastrin-Secreting Cells
Angiotensin Type 1 Receptor
Genistein

ASJC Scopus subject areas

  • Biochemistry

Cite this

Temporal dispersion of activation of phospholipase C-β1 and -γ isoforms by angiotensin II in vascular smooth muscle cells. Role of α(q/11), α12, and βγ G protein subunits. / Fukai, Masuko; Griendling, Kathy K.; Akers, Marjorie; Lyons, P. Reid; Alexander, R. Wayne.

In: Journal of Biological Chemistry, Vol. 273, No. 31, 31.07.1998, p. 19772-19777.

Research output: Contribution to journalArticle

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abstract = "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-γ and G protein-dependent PLC-β activation pathways have been reported. The expression of PLC-β1, furthermore, has not been consistently demonstrated in VSMCs. Here we identified the PLC subtypes and subunits of heterotrimeric G proteins involved in AT1 R-PLC coupling using cultured rat VSMCs. Western analysis revealed the expression of PLC-β1, - γ1, and -δ1 in VSMCs. Ang II-stimulated inositol trisphosphate (IP3) formation measured at 15 s, which corresponds to the peak response, was significantly in. hibited by electroporation of antibodies against PLC-β1, but not by anti-PLC-γ and -δ antibodies. Electroporation of anti-Gα(q/11) and -Gα12 antibodies also showed significant inhibition of the Ang II- induced IP3 generation at 15 s, while anti-Gα(i) and Gα13 antibodies were ineffective. Furthermore, in VSMCs electroporated with anti-Gβ antibody and cells stably transfected with the plasmid encoding the Gβγ-binding region of the carboxyl terminus of β-adrenergic receptor kinase1, 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 s, a time period which temporally correlated with PLC-γ tyrosine phosphorylation in response to Ang II. Moreover, electroporation of anti-PLC-γ antibody markedly inhibited the IP3 production measured at 30 s, indicating that tyrosine phosphorylation of PLC-γ contributes mainly to the later phase of PLC activation. Thus, these results suggest that: 1) AT1 receptors sequentially couple to PLC-β1 via a heterotrimeric G protein and to PLC-γ via a downstream tyrosine kinase; 2) the initial AT1 receptor-PLC-β1 coupling is mediated by Gα(q/11)βγ and Gα12βγ; 3) Gβγ acts as a signal transducer for activation of PLC in VSMCs. The sequential coupling of AT1 receptors to PLC-β1 and PLC-γ, as well as dual coupling of AT1 receptors to distinct Gα proteins, suggests a novel mechanism for a temporally controlled, highly organized and convergent Ang II-signaling network in VSMCs.",
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AU - Griendling, Kathy K.

AU - Akers, Marjorie

AU - Lyons, P. Reid

AU - Alexander, R. Wayne

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AB - 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-γ and G protein-dependent PLC-β activation pathways have been reported. The expression of PLC-β1, furthermore, has not been consistently demonstrated in VSMCs. Here we identified the PLC subtypes and subunits of heterotrimeric G proteins involved in AT1 R-PLC coupling using cultured rat VSMCs. Western analysis revealed the expression of PLC-β1, - γ1, and -δ1 in VSMCs. Ang II-stimulated inositol trisphosphate (IP3) formation measured at 15 s, which corresponds to the peak response, was significantly in. hibited by electroporation of antibodies against PLC-β1, but not by anti-PLC-γ and -δ antibodies. Electroporation of anti-Gα(q/11) and -Gα12 antibodies also showed significant inhibition of the Ang II- induced IP3 generation at 15 s, while anti-Gα(i) and Gα13 antibodies were ineffective. Furthermore, in VSMCs electroporated with anti-Gβ antibody and cells stably transfected with the plasmid encoding the Gβγ-binding region of the carboxyl terminus of β-adrenergic receptor kinase1, 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 s, a time period which temporally correlated with PLC-γ tyrosine phosphorylation in response to Ang II. Moreover, electroporation of anti-PLC-γ antibody markedly inhibited the IP3 production measured at 30 s, indicating that tyrosine phosphorylation of PLC-γ contributes mainly to the later phase of PLC activation. Thus, these results suggest that: 1) AT1 receptors sequentially couple to PLC-β1 via a heterotrimeric G protein and to PLC-γ via a downstream tyrosine kinase; 2) the initial AT1 receptor-PLC-β1 coupling is mediated by Gα(q/11)βγ and Gα12βγ; 3) Gβγ acts as a signal transducer for activation of PLC in VSMCs. The sequential coupling of AT1 receptors to PLC-β1 and PLC-γ, as well as dual coupling of AT1 receptors to distinct Gα proteins, suggests a novel mechanism for a temporally controlled, highly organized and convergent Ang II-signaling network in VSMCs.

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