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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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Determination of F-actin content. Wild-type and Gαq-deficient platelets were preincubated for 30 min in the absence  (c) or presence of 10 μM Y-27632 (Y) or were pretreated for 2 h  with 50 μg/ml C3 exoenzyme (C3). Thereafter, platelets were incubated for 10 s in the absence (−) or presence of 5 μM U46619  (+), fixed, and then incubated with fluorescein isothiocyanate  (FITC)-phalloidin. Platelets incubated for 2 h in C3 exoenzyme  buffer alone showed full increase in F-actin content in response  to U46619 (data not shown). F-actin content was determined as  described in Materials and Methods. Shown are means ± SD of  triplicates.
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Figure 4: Determination of F-actin content. Wild-type and Gαq-deficient platelets were preincubated for 30 min in the absence (c) or presence of 10 μM Y-27632 (Y) or were pretreated for 2 h with 50 μg/ml C3 exoenzyme (C3). Thereafter, platelets were incubated for 10 s in the absence (−) or presence of 5 μM U46619 (+), fixed, and then incubated with fluorescein isothiocyanate (FITC)-phalloidin. Platelets incubated for 2 h in C3 exoenzyme buffer alone showed full increase in F-actin content in response to U46619 (data not shown). F-actin content was determined as described in Materials and Methods. Shown are means ± SD of triplicates.

Mentions: Since platelet shape change including protrusion of filopodia and lamellipodia is accompanied by actin polymerization (Siess, 1989; Wurzinger, 1990; Fox, 1993) we measured F-actin content in wild-type and Gαq-deficient platelets (Fig. 4). U46619 induced an increase in F-actin content of both, wild-type and Gαq-deficient platelets, which could be completely blocked by Y-27632. Reduction of the amount of functional Rho by pretreatment with 50 μg/ml C3 exoenzyme for 2 h markedly reduced the effect of U46619 in wild-type and Gαq-deficient platelets.


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)

Determination of F-actin content. Wild-type and Gαq-deficient platelets were preincubated for 30 min in the absence  (c) or presence of 10 μM Y-27632 (Y) or were pretreated for 2 h  with 50 μg/ml C3 exoenzyme (C3). Thereafter, platelets were incubated for 10 s in the absence (−) or presence of 5 μM U46619  (+), fixed, and then incubated with fluorescein isothiocyanate  (FITC)-phalloidin. Platelets incubated for 2 h in C3 exoenzyme  buffer alone showed full increase in F-actin content in response  to U46619 (data not shown). F-actin content was determined as  described in Materials and Methods. Shown are means ± SD of  triplicates.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132941&req=5

Figure 4: Determination of F-actin content. Wild-type and Gαq-deficient platelets were preincubated for 30 min in the absence (c) or presence of 10 μM Y-27632 (Y) or were pretreated for 2 h with 50 μg/ml C3 exoenzyme (C3). Thereafter, platelets were incubated for 10 s in the absence (−) or presence of 5 μM U46619 (+), fixed, and then incubated with fluorescein isothiocyanate (FITC)-phalloidin. Platelets incubated for 2 h in C3 exoenzyme buffer alone showed full increase in F-actin content in response to U46619 (data not shown). F-actin content was determined as described in Materials and Methods. Shown are means ± SD of triplicates.
Mentions: Since platelet shape change including protrusion of filopodia and lamellipodia is accompanied by actin polymerization (Siess, 1989; Wurzinger, 1990; Fox, 1993) we measured F-actin content in wild-type and Gαq-deficient platelets (Fig. 4). U46619 induced an increase in F-actin content of both, wild-type and Gαq-deficient platelets, which could be completely blocked by Y-27632. Reduction of the amount of functional Rho by pretreatment with 50 μg/ml C3 exoenzyme for 2 h markedly reduced the effect of U46619 in wild-type and Gαq-deficient platelets.

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