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Activation of G12/G13 results in shape change and Rho/Rho-kinase-mediated myosin light chain phosphorylation in mouse platelets.

Klages B, Brandt U, Simon MI, Schultz G, Offermanns S - J. Cell Biol. (1999)

Bottom Line: Platelets lacking the alpha-subunit of the heterotrimeric G protein Gq do not aggregate and degranulate but still undergo shape change after activation through thromboxane-A2 (TXA2) or thrombin receptors.TXA2 receptor-mediated activation of G12/G13 resulted in tyrosine phosphorylation of pp72(syk) and stimulation of pp60(c-src) as well as in phosphorylation of myosin light chain (MLC) in Galphaq-deficient platelets.These data indicate that G12/G13 couple receptors to tyrosine kinases as well as to the Rho/Rho-kinase-mediated regulation of MLC phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Institut für Pharmakologie, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, 14195 Berlin, Germany.

ABSTRACT
Platelets respond to various stimuli with rapid changes in shape followed by aggregation and secretion of their granule contents. Platelets lacking the alpha-subunit of the heterotrimeric G protein Gq do not aggregate and degranulate but still undergo shape change after activation through thromboxane-A2 (TXA2) or thrombin receptors. In contrast to thrombin, the TXA2 mimetic U46619 led to the selective activation of G12 and G13 in Galphaq-deficient platelets indicating that these G proteins mediate TXA2 receptor-induced shape change. TXA2 receptor-mediated activation of G12/G13 resulted in tyrosine phosphorylation of pp72(syk) and stimulation of pp60(c-src) as well as in phosphorylation of myosin light chain (MLC) in Galphaq-deficient platelets. Both MLC phosphorylation and shape change induced through G12/G13 in the absence of Galphaq were inhibited by the C3 exoenzyme from Clostridium botulinum, by the Rho-kinase inhibitor Y-27632 and by cAMP-analogue Sp-5,6-DCl-cBIMPS. These data indicate that G12/G13 couple receptors to tyrosine kinases as well as to the Rho/Rho-kinase-mediated regulation of MLC phosphorylation. We provide evidence that G12/G13-mediated Rho/Rho-kinase-dependent regulation of MLC phosphorylation participates in receptor-induced platelet shape change.

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Effect of thrombin and U46619 on tyrosine phosphorylation and pp72syk and pp60c-src-activity in wild-type and Gαq-deficient platelets. (A) Wild-type and Gαq-deficient platelets  were incubated for 30 s in the absence (−) or presence of 5 U/ml  thrombin (Thr.) or 5 μM U46619 (TXA2). (B) Wild-type and  Gαq-deficient platelets were incubated for the indicated times in  the absence (−) or presence of 5 μM of the thromboxane A2 mimetic U46619 (T). Cells were lysed and cellular proteins were  separated by SDS-PAGE and blotted on nitrocellulose filters.  Phosphotyrosine was detected by an antiphosphotyrosine antibody. Shown are autoluminograms with the position of standard  proteins shown on the left. (C) Wild-type (left) and Gαq-deficient  platelets (right) were incubated with buffer (−) or 5 μM U46619  (+) for the indicated times. Platelets were lysed and incubated  with agarose conjugates of anti-pp72syk IgG (IP: Syk) or of anti-pp60c-src IgG (IP: Src), and immunoprecipitates were analyzed by  immunoblotting with antiphosphotyrosine antibodies (WB: P-Y),  anti-pp72syk antibodies (WB: Syk), anti-pp60c-src antibodies (WB:  Src), or were subjected to in vitro kinase assays using histone as a  substrate (Histone-P).
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Figure 6: Effect of thrombin and U46619 on tyrosine phosphorylation and pp72syk and pp60c-src-activity in wild-type and Gαq-deficient platelets. (A) Wild-type and Gαq-deficient platelets were incubated for 30 s in the absence (−) or presence of 5 U/ml thrombin (Thr.) or 5 μM U46619 (TXA2). (B) Wild-type and Gαq-deficient platelets were incubated for the indicated times in the absence (−) or presence of 5 μM of the thromboxane A2 mimetic U46619 (T). Cells were lysed and cellular proteins were separated by SDS-PAGE and blotted on nitrocellulose filters. Phosphotyrosine was detected by an antiphosphotyrosine antibody. Shown are autoluminograms with the position of standard proteins shown on the left. (C) Wild-type (left) and Gαq-deficient platelets (right) were incubated with buffer (−) or 5 μM U46619 (+) for the indicated times. Platelets were lysed and incubated with agarose conjugates of anti-pp72syk IgG (IP: Syk) or of anti-pp60c-src IgG (IP: Src), and immunoprecipitates were analyzed by immunoblotting with antiphosphotyrosine antibodies (WB: P-Y), anti-pp72syk antibodies (WB: Syk), anti-pp60c-src antibodies (WB: Src), or were subjected to in vitro kinase assays using histone as a substrate (Histone-P).

Mentions: Agonist-induced platelet activation results in tyrosine phosphorylation of multiple proteins (Ferrell and Martin, 1988; Nakamura and Yamamura, 1989; Golden and Brugge, 1989). Phosphorylation of these proteins occurs in three temporal phases which have been experimentally distinguished. Early tyrosine phosphorylation occurs by an integrin-independent mechanism, whereas the second and third wave of tyrosine phosphorylation depends on the aggregation of platelets through binding of fibrinogen to αIIbβ3-integrin (glycoprotein IIb-IIIa) (Clark et al., 1994b). In Gαq-deficient platelets that do not aggregate in response to thrombin or U46619, only a subset of proteins became tyrosine phosphorylated upon exposure of platelets to both stimuli compared with wild-type platelets (Fig. 6, A and B). Most prominently, a rapid tyrosine phosphorylation of a protein of ∼72 kD could be observed in Gαq-deficient platelets activated with thrombin and U46619. In contrast, several proteins with relative molecular masses of 40 and 95–130 kD which were tyrosine phosphorylated in wild-type platelets did not show increased tyrosine phosphorylation in activated Gαq-deficient platelets (Fig. 6, A and B).


Activation of G12/G13 results in shape change and Rho/Rho-kinase-mediated myosin light chain phosphorylation in mouse platelets.

Klages B, Brandt U, Simon MI, Schultz G, Offermanns S - J. Cell Biol. (1999)

Effect of thrombin and U46619 on tyrosine phosphorylation and pp72syk and pp60c-src-activity in wild-type and Gαq-deficient platelets. (A) Wild-type and Gαq-deficient platelets  were incubated for 30 s in the absence (−) or presence of 5 U/ml  thrombin (Thr.) or 5 μM U46619 (TXA2). (B) Wild-type and  Gαq-deficient platelets were incubated for the indicated times in  the absence (−) or presence of 5 μM of the thromboxane A2 mimetic U46619 (T). Cells were lysed and cellular proteins were  separated by SDS-PAGE and blotted on nitrocellulose filters.  Phosphotyrosine was detected by an antiphosphotyrosine antibody. Shown are autoluminograms with the position of standard  proteins shown on the left. (C) Wild-type (left) and Gαq-deficient  platelets (right) were incubated with buffer (−) or 5 μM U46619  (+) for the indicated times. Platelets were lysed and incubated  with agarose conjugates of anti-pp72syk IgG (IP: Syk) or of anti-pp60c-src IgG (IP: Src), and immunoprecipitates were analyzed by  immunoblotting with antiphosphotyrosine antibodies (WB: P-Y),  anti-pp72syk antibodies (WB: Syk), anti-pp60c-src antibodies (WB:  Src), or were subjected to in vitro kinase assays using histone as a  substrate (Histone-P).
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Figure 6: Effect of thrombin and U46619 on tyrosine phosphorylation and pp72syk and pp60c-src-activity in wild-type and Gαq-deficient platelets. (A) Wild-type and Gαq-deficient platelets were incubated for 30 s in the absence (−) or presence of 5 U/ml thrombin (Thr.) or 5 μM U46619 (TXA2). (B) Wild-type and Gαq-deficient platelets were incubated for the indicated times in the absence (−) or presence of 5 μM of the thromboxane A2 mimetic U46619 (T). Cells were lysed and cellular proteins were separated by SDS-PAGE and blotted on nitrocellulose filters. Phosphotyrosine was detected by an antiphosphotyrosine antibody. Shown are autoluminograms with the position of standard proteins shown on the left. (C) Wild-type (left) and Gαq-deficient platelets (right) were incubated with buffer (−) or 5 μM U46619 (+) for the indicated times. Platelets were lysed and incubated with agarose conjugates of anti-pp72syk IgG (IP: Syk) or of anti-pp60c-src IgG (IP: Src), and immunoprecipitates were analyzed by immunoblotting with antiphosphotyrosine antibodies (WB: P-Y), anti-pp72syk antibodies (WB: Syk), anti-pp60c-src antibodies (WB: Src), or were subjected to in vitro kinase assays using histone as a substrate (Histone-P).
Mentions: Agonist-induced platelet activation results in tyrosine phosphorylation of multiple proteins (Ferrell and Martin, 1988; Nakamura and Yamamura, 1989; Golden and Brugge, 1989). Phosphorylation of these proteins occurs in three temporal phases which have been experimentally distinguished. Early tyrosine phosphorylation occurs by an integrin-independent mechanism, whereas the second and third wave of tyrosine phosphorylation depends on the aggregation of platelets through binding of fibrinogen to αIIbβ3-integrin (glycoprotein IIb-IIIa) (Clark et al., 1994b). In Gαq-deficient platelets that do not aggregate in response to thrombin or U46619, only a subset of proteins became tyrosine phosphorylated upon exposure of platelets to both stimuli compared with wild-type platelets (Fig. 6, A and B). Most prominently, a rapid tyrosine phosphorylation of a protein of ∼72 kD could be observed in Gαq-deficient platelets activated with thrombin and U46619. In contrast, several proteins with relative molecular masses of 40 and 95–130 kD which were tyrosine phosphorylated in wild-type platelets did not show increased tyrosine phosphorylation in activated Gαq-deficient platelets (Fig. 6, A and B).

Bottom Line: Platelets lacking the alpha-subunit of the heterotrimeric G protein Gq do not aggregate and degranulate but still undergo shape change after activation through thromboxane-A2 (TXA2) or thrombin receptors.TXA2 receptor-mediated activation of G12/G13 resulted in tyrosine phosphorylation of pp72(syk) and stimulation of pp60(c-src) as well as in phosphorylation of myosin light chain (MLC) in Galphaq-deficient platelets.These data indicate that G12/G13 couple receptors to tyrosine kinases as well as to the Rho/Rho-kinase-mediated regulation of MLC phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Institut für Pharmakologie, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, 14195 Berlin, Germany.

ABSTRACT
Platelets respond to various stimuli with rapid changes in shape followed by aggregation and secretion of their granule contents. Platelets lacking the alpha-subunit of the heterotrimeric G protein Gq do not aggregate and degranulate but still undergo shape change after activation through thromboxane-A2 (TXA2) or thrombin receptors. In contrast to thrombin, the TXA2 mimetic U46619 led to the selective activation of G12 and G13 in Galphaq-deficient platelets indicating that these G proteins mediate TXA2 receptor-induced shape change. TXA2 receptor-mediated activation of G12/G13 resulted in tyrosine phosphorylation of pp72(syk) and stimulation of pp60(c-src) as well as in phosphorylation of myosin light chain (MLC) in Galphaq-deficient platelets. Both MLC phosphorylation and shape change induced through G12/G13 in the absence of Galphaq were inhibited by the C3 exoenzyme from Clostridium botulinum, by the Rho-kinase inhibitor Y-27632 and by cAMP-analogue Sp-5,6-DCl-cBIMPS. These data indicate that G12/G13 couple receptors to tyrosine kinases as well as to the Rho/Rho-kinase-mediated regulation of MLC phosphorylation. We provide evidence that G12/G13-mediated Rho/Rho-kinase-dependent regulation of MLC phosphorylation participates in receptor-induced platelet shape change.

Show MeSH
Related in: MedlinePlus