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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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Receptor-dependent activation of G proteins  in membranes of wild-type  and Gαq-deficient platelets.  Membranes from wild-type  (A) and Gαq-deficient platelets (B) were photolabeled  with [α-32P]GTP azidoanilide in the absence (−) or  presence of 5 μM U46619 or  5 U/ml thrombin (+). Membranes were solubilized and  G protein α-subunits (Gα12,  Gα13, Gαq, and Gαi) were  immunoprecipitated as described under Materials and  Methods. Anti-Gα12, anti-Gα13, anti-Gαq/11 antisera,  and an antiserum recognizing  Gαi1, Gαi2, and Gαi3 were used. Precipitated proteins were subjected to SDS-PAGE. Shown are autoradiograms of dried SDS  gels with the position of the 43-kD standard protein shown on the  left.
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Figure 5: Receptor-dependent activation of G proteins in membranes of wild-type and Gαq-deficient platelets. Membranes from wild-type (A) and Gαq-deficient platelets (B) were photolabeled with [α-32P]GTP azidoanilide in the absence (−) or presence of 5 μM U46619 or 5 U/ml thrombin (+). Membranes were solubilized and G protein α-subunits (Gα12, Gα13, Gαq, and Gαi) were immunoprecipitated as described under Materials and Methods. Anti-Gα12, anti-Gα13, anti-Gαq/11 antisera, and an antiserum recognizing Gαi1, Gαi2, and Gαi3 were used. Precipitated proteins were subjected to SDS-PAGE. Shown are autoradiograms of dried SDS gels with the position of the 43-kD standard protein shown on the left.

Mentions: To identify the G proteins mediating receptor-induced platelet shape change we studied the coupling of TXA2 and thrombin receptors to heterotrimeric G proteins in wild-type and Gαq-deficient mouse platelets. Receptors for both, thrombin and TXA2, have been shown to be able to couple to members of the Gq, G12, and Gi families (Shenker et al., 1991; Hung et al., 1992; Offermanns et al., 1994; Ushikubi et al., 1994). In membranes of human platelets, receptors activated by thrombin couple to Gq, G12, G13, and Gi, whereas TXA2 receptors only activate Gq, G12, and G13 (Offermanns et al., 1994; Brass et al., 1997). Photolabeling of receptor-activated G proteins in mouse platelet membranes and subsequent immunoprecipitation of individual G protein α-subunits showed that in wild-type mouse platelets, activated TXA2 and thrombin receptors couple to Gq, G12, and G13, whereas Gi was only activated through the thrombin receptor (Fig. 5 A). Similarly, in membranes from Gαq-deficient platelets, only G12 and G13 were activated through the TXA2 receptor, whereas activated thrombin receptors coupled to G12, G13, and Gi (Fig. 5 B and data not shown). Coupling of thrombin receptors to Gi in murine platelets corresponded with the ability of thrombin to decrease cAMP levels in wild-type as well as in Gαq-deficient platelets, whereas activation of TXA2 receptors had no effect on adenylyl cyclase activity in wild-type or Gαq-deficient platelets (data not shown). These data clearly demonstrate that in Gαq-deficient platelets thrombin-receptors couple to G12, G13, and Gi, whereas only G12 and G13 are activated through TXA2 receptors. Consequently, effects which can still be induced by TXA2 receptor agonists in Gαq-deficient platelets like the shape change response are mediated by G12 and/or G13. TXA2-activated Gαq-deficient platelets therefore represent a model to study of G12/G13-mediated signaling processes.


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)

Receptor-dependent activation of G proteins  in membranes of wild-type  and Gαq-deficient platelets.  Membranes from wild-type  (A) and Gαq-deficient platelets (B) were photolabeled  with [α-32P]GTP azidoanilide in the absence (−) or  presence of 5 μM U46619 or  5 U/ml thrombin (+). Membranes were solubilized and  G protein α-subunits (Gα12,  Gα13, Gαq, and Gαi) were  immunoprecipitated as described under Materials and  Methods. Anti-Gα12, anti-Gα13, anti-Gαq/11 antisera,  and an antiserum recognizing  Gαi1, Gαi2, and Gαi3 were used. Precipitated proteins were subjected to SDS-PAGE. Shown are autoradiograms of dried SDS  gels with the position of the 43-kD standard protein shown on the  left.
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Figure 5: Receptor-dependent activation of G proteins in membranes of wild-type and Gαq-deficient platelets. Membranes from wild-type (A) and Gαq-deficient platelets (B) were photolabeled with [α-32P]GTP azidoanilide in the absence (−) or presence of 5 μM U46619 or 5 U/ml thrombin (+). Membranes were solubilized and G protein α-subunits (Gα12, Gα13, Gαq, and Gαi) were immunoprecipitated as described under Materials and Methods. Anti-Gα12, anti-Gα13, anti-Gαq/11 antisera, and an antiserum recognizing Gαi1, Gαi2, and Gαi3 were used. Precipitated proteins were subjected to SDS-PAGE. Shown are autoradiograms of dried SDS gels with the position of the 43-kD standard protein shown on the left.
Mentions: To identify the G proteins mediating receptor-induced platelet shape change we studied the coupling of TXA2 and thrombin receptors to heterotrimeric G proteins in wild-type and Gαq-deficient mouse platelets. Receptors for both, thrombin and TXA2, have been shown to be able to couple to members of the Gq, G12, and Gi families (Shenker et al., 1991; Hung et al., 1992; Offermanns et al., 1994; Ushikubi et al., 1994). In membranes of human platelets, receptors activated by thrombin couple to Gq, G12, G13, and Gi, whereas TXA2 receptors only activate Gq, G12, and G13 (Offermanns et al., 1994; Brass et al., 1997). Photolabeling of receptor-activated G proteins in mouse platelet membranes and subsequent immunoprecipitation of individual G protein α-subunits showed that in wild-type mouse platelets, activated TXA2 and thrombin receptors couple to Gq, G12, and G13, whereas Gi was only activated through the thrombin receptor (Fig. 5 A). Similarly, in membranes from Gαq-deficient platelets, only G12 and G13 were activated through the TXA2 receptor, whereas activated thrombin receptors coupled to G12, G13, and Gi (Fig. 5 B and data not shown). Coupling of thrombin receptors to Gi in murine platelets corresponded with the ability of thrombin to decrease cAMP levels in wild-type as well as in Gαq-deficient platelets, whereas activation of TXA2 receptors had no effect on adenylyl cyclase activity in wild-type or Gαq-deficient platelets (data not shown). These data clearly demonstrate that in Gαq-deficient platelets thrombin-receptors couple to G12, G13, and Gi, whereas only G12 and G13 are activated through TXA2 receptors. Consequently, effects which can still be induced by TXA2 receptor agonists in Gαq-deficient platelets like the shape change response are mediated by G12 and/or G13. TXA2-activated Gαq-deficient platelets therefore represent a model to study of G12/G13-mediated signaling processes.

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