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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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ADP ribosylation  of Rho in C3-treated platelets. (A) Platelets from wild-type and Gαq-deficient animals were preincubated for 2 h  in the absence (−) or presence of the indicated concentrations of C3 exoenzyme.  Thereafter, platelets were  lysed and subjected to C3-exoenzyme catalyzed (32P)  ADP ribosylation as described under Materials and  Methods. Shown is an autoradiogram with the position  of the 21-kD standard protein on the left. (B) Densitometric evaluation of bands  shown in A. Bars represent  mean of two independent experiments as percent of control.
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Figure 3: ADP ribosylation of Rho in C3-treated platelets. (A) Platelets from wild-type and Gαq-deficient animals were preincubated for 2 h in the absence (−) or presence of the indicated concentrations of C3 exoenzyme. Thereafter, platelets were lysed and subjected to C3-exoenzyme catalyzed (32P) ADP ribosylation as described under Materials and Methods. Shown is an autoradiogram with the position of the 21-kD standard protein on the left. (B) Densitometric evaluation of bands shown in A. Bars represent mean of two independent experiments as percent of control.

Mentions: We have recently shown that Gαq-deficient platelets do not aggregate and secrete their granule contents in response to various stimuli indicating that Gq-mediated activation of phospholipase C represents the central early signal transduction process leading to full platelet activation. However, Gq-deficient platelets were still able to undergo ligand-induced platelet shape change. This suggests that G proteins other than Gq mediate the platelet shape change response. Shape change induced by the TXA2 analogue U46619 could be observed in Gαq-deficient platelets by scanning electron microscopy of single cells (Fig. 1, A–D) as well as by measuring the light transmission of a platelet suspension (Fig. 2). Shape change induced by U46619 in Gαq-deficient platelets and wild-type platelets was blocked by the cAMP analogue Sp-5,6-DCl-cBIMPS but not by the cGMP analogue 8-pCPT-cGMP, whereas both cyclic nucleotides blocked aggregation in wild-type platelets (Fig. 2, A and B). Similar results were observed with thrombin-activated wild-type and Gαq-deficient platelets (data not shown). Preincubation of platelets with the recently described Rho-kinase inhibitor Y-27632 (Uehata et al., 1997) blocked U46619-induced shape change both in wild-type and Gαq-deficient platelets (Fig. 1, E–H and Fig. 2, C and D). To assess the role of Rho in agonist-induced platelet shape change we preincubated platelets for 2 h with 50 μg/ml C3 exoenzyme which ADP ribosylates and inactivates the small GTPase Rho (Morii et al., 1992). This C3 exoenzyme concentration and preincubation time resulted in ADP-ribosylation of 70–75% of endogenous Rho as determined by the inability of C3 exoenzyme to [32P]ADP-ribosylate Rho in subsequently prepared cell lysates (Fig. 3). Longer preincubation times and higher C3 exoenzyme concentrations further increased the ADP-ribosylated fraction of Rho (Fig. 3), but resulted in preactivation of platelets (data not shown). C3-pretreated platelets showed markedly reduced shape change in response to U46619 with only partial spheration and occasional filopodia formation (Fig. 1, I–L and Fig. 2 E).


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)

ADP ribosylation  of Rho in C3-treated platelets. (A) Platelets from wild-type and Gαq-deficient animals were preincubated for 2 h  in the absence (−) or presence of the indicated concentrations of C3 exoenzyme.  Thereafter, platelets were  lysed and subjected to C3-exoenzyme catalyzed (32P)  ADP ribosylation as described under Materials and  Methods. Shown is an autoradiogram with the position  of the 21-kD standard protein on the left. (B) Densitometric evaluation of bands  shown in A. Bars represent  mean of two independent experiments as percent of control.
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Figure 3: ADP ribosylation of Rho in C3-treated platelets. (A) Platelets from wild-type and Gαq-deficient animals were preincubated for 2 h in the absence (−) or presence of the indicated concentrations of C3 exoenzyme. Thereafter, platelets were lysed and subjected to C3-exoenzyme catalyzed (32P) ADP ribosylation as described under Materials and Methods. Shown is an autoradiogram with the position of the 21-kD standard protein on the left. (B) Densitometric evaluation of bands shown in A. Bars represent mean of two independent experiments as percent of control.
Mentions: We have recently shown that Gαq-deficient platelets do not aggregate and secrete their granule contents in response to various stimuli indicating that Gq-mediated activation of phospholipase C represents the central early signal transduction process leading to full platelet activation. However, Gq-deficient platelets were still able to undergo ligand-induced platelet shape change. This suggests that G proteins other than Gq mediate the platelet shape change response. Shape change induced by the TXA2 analogue U46619 could be observed in Gαq-deficient platelets by scanning electron microscopy of single cells (Fig. 1, A–D) as well as by measuring the light transmission of a platelet suspension (Fig. 2). Shape change induced by U46619 in Gαq-deficient platelets and wild-type platelets was blocked by the cAMP analogue Sp-5,6-DCl-cBIMPS but not by the cGMP analogue 8-pCPT-cGMP, whereas both cyclic nucleotides blocked aggregation in wild-type platelets (Fig. 2, A and B). Similar results were observed with thrombin-activated wild-type and Gαq-deficient platelets (data not shown). Preincubation of platelets with the recently described Rho-kinase inhibitor Y-27632 (Uehata et al., 1997) blocked U46619-induced shape change both in wild-type and Gαq-deficient platelets (Fig. 1, E–H and Fig. 2, C and D). To assess the role of Rho in agonist-induced platelet shape change we preincubated platelets for 2 h with 50 μg/ml C3 exoenzyme which ADP ribosylates and inactivates the small GTPase Rho (Morii et al., 1992). This C3 exoenzyme concentration and preincubation time resulted in ADP-ribosylation of 70–75% of endogenous Rho as determined by the inability of C3 exoenzyme to [32P]ADP-ribosylate Rho in subsequently prepared cell lysates (Fig. 3). Longer preincubation times and higher C3 exoenzyme concentrations further increased the ADP-ribosylated fraction of Rho (Fig. 3), but resulted in preactivation of platelets (data not shown). C3-pretreated platelets showed markedly reduced shape change in response to U46619 with only partial spheration and occasional filopodia formation (Fig. 1, I–L and Fig. 2 E).

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