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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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U46619-induced MLC phosphorylation in wild-type and Gαq-deficient  platelets. (A) Wild-type and Gαq-deficient  platelets were incubated for the indicated  time periods with buffer (c) or 5 μM  U46619 (U). (B and C) Platelets were preincubated for 20 min without or with the  synthetic cyclic nucleotides 8-pCPT-cGMP (cGMP) or Sp-5,6-DCl-cBIMPS  (cAMP) at the indicated concentrations.  Thereafter, platelets were incubated for  10 s with buffer (c) or 5 μM U46619 (U).  (D and E) Platelets were preincubated for  30 min without or with the Rho-kinase inhibitor Y-27632 at the indicated concentrations (30 or 100 μM for wild-type and  10 or 30 μM for Gαq-deficient platelets).  Incubation was conducted for 10 s with  buffer (c) or 5 μM U46619 (U). (F) Wild-type and Gαq-deficient platelets were preincubated for 120 min without (−) or with  50 μg/ml C3 exoenzyme (+). Thereafter,  platelets were incubated for 10 s with buffer (c) or 5 μM U46619 (U). Reactions were stopped by addition of perchloric acid, and phosphorylation of MLC was determined using urea/glycin gels as described in Materials and Methods. Shown are autoluminograms of anti-MLC immunoblots. Phosphorylation of MLC results in a faster mobility (lower position) of MLC on urea/glycin gels.
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Figure 7: U46619-induced MLC phosphorylation in wild-type and Gαq-deficient platelets. (A) Wild-type and Gαq-deficient platelets were incubated for the indicated time periods with buffer (c) or 5 μM U46619 (U). (B and C) Platelets were preincubated for 20 min without or with the synthetic cyclic nucleotides 8-pCPT-cGMP (cGMP) or Sp-5,6-DCl-cBIMPS (cAMP) at the indicated concentrations. Thereafter, platelets were incubated for 10 s with buffer (c) or 5 μM U46619 (U). (D and E) Platelets were preincubated for 30 min without or with the Rho-kinase inhibitor Y-27632 at the indicated concentrations (30 or 100 μM for wild-type and 10 or 30 μM for Gαq-deficient platelets). Incubation was conducted for 10 s with buffer (c) or 5 μM U46619 (U). (F) Wild-type and Gαq-deficient platelets were preincubated for 120 min without (−) or with 50 μg/ml C3 exoenzyme (+). Thereafter, platelets were incubated for 10 s with buffer (c) or 5 μM U46619 (U). Reactions were stopped by addition of perchloric acid, and phosphorylation of MLC was determined using urea/glycin gels as described in Materials and Methods. Shown are autoluminograms of anti-MLC immunoblots. Phosphorylation of MLC results in a faster mobility (lower position) of MLC on urea/glycin gels.

Mentions: MLC phosphorylation has been suggested to be involved in early processes during platelet activation (Daniel et al., 1984). To test whether TXA2 receptor-G12/G13– mediated signaling in Gαq-deficient platelets resulted in MLC phosphorylation, we activated platelets with U46619 for different times and separated phosphorylated and unphosphorylated MLC on urea/glycin polyacrylamide gels. Separated proteins were blotted onto nitrocellulose filters and MLC was detected using a specific antiserum. Fig. 7 A shows that U46619 caused phosphorylation of the total detectable pool of MLC in wild-type platelets within 10 s. Interestingly, a rapid and apparently complete phosphorylation of MLC was also observed in Gαq-deficient platelets activated by U46619. Chelation of extracellular Ca2+ by EGTA or preincubation of platelets with various tyrosine kinase inhibitors had no effect on U46619-induced MLC phosphorylation in wild-type and Gαq-deficient platelets (data not shown). Although the cAMP analogue Sp-5,6-DCl-cBIMPS completely inhibited MLC phosphorylation in wild-type and Gαq-deficient platelets the cGMP analogue 8-pCPT-cGMP was without effect (Fig. 7, B and C). In smooth muscle cells and fibroblasts, the phosphorylation state of MLC has been shown to be under dual control of the Ca2+/calmodulin-activated myosin light chain kinase (MLCK) as well as of myosin-phosphatase (Somlyo and Somlyo, 1994; Burridge and Chrzanowska-Wodnicka, 1996). Myosin-phosphatase has been demonstrated to be regulated by Rho/Rho-kinase (Kimura et al., 1996; Narumiya et al., 1997). Since U46619-induced platelet shape change was blocked by the Rho-kinase inhibitor Y-27632 and was greatly inhibited after reduction of the amount of active Rho by C3 exoenzyme (Figs. 1 and 2) we tested the effect of C3 exoenzyme and Y-27632 on U46619-induced phosphorylation of MLC. Fig. 7, D–F shows that Y-27632 blocked and C3 exoenzyme markedly inhibited U46619-induced MLC-phosphorylation in wild-type as well as in Gαq-deficient platelets. Incomplete inhibition of MLC phosphorylation by C3 exoenzyme was most likely due to incomplete inactivation of Rho by C3 exoenzyme (see Fig. 3). Y-27632 exerted its inhibitory effect on receptor-induced MLC phosphorylation with higher potency in Gαq-deficient platelets compared with wild-type platelets (Fig. 7, D and E). Similarly, the effect of C3 exoenzyme appeared to be more pronounced in the absence of Gαq (Fig. 7 F). These data indicate that activation of G12/G13 through the TXA2 receptor results in MLC phosphorylation and that this process involves Rho/Rho-kinase. The data also provide further evidence for the concept that MLC phosphorylation underlies platelet shape change.


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)

U46619-induced MLC phosphorylation in wild-type and Gαq-deficient  platelets. (A) Wild-type and Gαq-deficient  platelets were incubated for the indicated  time periods with buffer (c) or 5 μM  U46619 (U). (B and C) Platelets were preincubated for 20 min without or with the  synthetic cyclic nucleotides 8-pCPT-cGMP (cGMP) or Sp-5,6-DCl-cBIMPS  (cAMP) at the indicated concentrations.  Thereafter, platelets were incubated for  10 s with buffer (c) or 5 μM U46619 (U).  (D and E) Platelets were preincubated for  30 min without or with the Rho-kinase inhibitor Y-27632 at the indicated concentrations (30 or 100 μM for wild-type and  10 or 30 μM for Gαq-deficient platelets).  Incubation was conducted for 10 s with  buffer (c) or 5 μM U46619 (U). (F) Wild-type and Gαq-deficient platelets were preincubated for 120 min without (−) or with  50 μg/ml C3 exoenzyme (+). Thereafter,  platelets were incubated for 10 s with buffer (c) or 5 μM U46619 (U). Reactions were stopped by addition of perchloric acid, and phosphorylation of MLC was determined using urea/glycin gels as described in Materials and Methods. Shown are autoluminograms of anti-MLC immunoblots. Phosphorylation of MLC results in a faster mobility (lower position) of MLC on urea/glycin gels.
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Related In: Results  -  Collection

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Figure 7: U46619-induced MLC phosphorylation in wild-type and Gαq-deficient platelets. (A) Wild-type and Gαq-deficient platelets were incubated for the indicated time periods with buffer (c) or 5 μM U46619 (U). (B and C) Platelets were preincubated for 20 min without or with the synthetic cyclic nucleotides 8-pCPT-cGMP (cGMP) or Sp-5,6-DCl-cBIMPS (cAMP) at the indicated concentrations. Thereafter, platelets were incubated for 10 s with buffer (c) or 5 μM U46619 (U). (D and E) Platelets were preincubated for 30 min without or with the Rho-kinase inhibitor Y-27632 at the indicated concentrations (30 or 100 μM for wild-type and 10 or 30 μM for Gαq-deficient platelets). Incubation was conducted for 10 s with buffer (c) or 5 μM U46619 (U). (F) Wild-type and Gαq-deficient platelets were preincubated for 120 min without (−) or with 50 μg/ml C3 exoenzyme (+). Thereafter, platelets were incubated for 10 s with buffer (c) or 5 μM U46619 (U). Reactions were stopped by addition of perchloric acid, and phosphorylation of MLC was determined using urea/glycin gels as described in Materials and Methods. Shown are autoluminograms of anti-MLC immunoblots. Phosphorylation of MLC results in a faster mobility (lower position) of MLC on urea/glycin gels.
Mentions: MLC phosphorylation has been suggested to be involved in early processes during platelet activation (Daniel et al., 1984). To test whether TXA2 receptor-G12/G13– mediated signaling in Gαq-deficient platelets resulted in MLC phosphorylation, we activated platelets with U46619 for different times and separated phosphorylated and unphosphorylated MLC on urea/glycin polyacrylamide gels. Separated proteins were blotted onto nitrocellulose filters and MLC was detected using a specific antiserum. Fig. 7 A shows that U46619 caused phosphorylation of the total detectable pool of MLC in wild-type platelets within 10 s. Interestingly, a rapid and apparently complete phosphorylation of MLC was also observed in Gαq-deficient platelets activated by U46619. Chelation of extracellular Ca2+ by EGTA or preincubation of platelets with various tyrosine kinase inhibitors had no effect on U46619-induced MLC phosphorylation in wild-type and Gαq-deficient platelets (data not shown). Although the cAMP analogue Sp-5,6-DCl-cBIMPS completely inhibited MLC phosphorylation in wild-type and Gαq-deficient platelets the cGMP analogue 8-pCPT-cGMP was without effect (Fig. 7, B and C). In smooth muscle cells and fibroblasts, the phosphorylation state of MLC has been shown to be under dual control of the Ca2+/calmodulin-activated myosin light chain kinase (MLCK) as well as of myosin-phosphatase (Somlyo and Somlyo, 1994; Burridge and Chrzanowska-Wodnicka, 1996). Myosin-phosphatase has been demonstrated to be regulated by Rho/Rho-kinase (Kimura et al., 1996; Narumiya et al., 1997). Since U46619-induced platelet shape change was blocked by the Rho-kinase inhibitor Y-27632 and was greatly inhibited after reduction of the amount of active Rho by C3 exoenzyme (Figs. 1 and 2) we tested the effect of C3 exoenzyme and Y-27632 on U46619-induced phosphorylation of MLC. Fig. 7, D–F shows that Y-27632 blocked and C3 exoenzyme markedly inhibited U46619-induced MLC-phosphorylation in wild-type as well as in Gαq-deficient platelets. Incomplete inhibition of MLC phosphorylation by C3 exoenzyme was most likely due to incomplete inactivation of Rho by C3 exoenzyme (see Fig. 3). Y-27632 exerted its inhibitory effect on receptor-induced MLC phosphorylation with higher potency in Gαq-deficient platelets compared with wild-type platelets (Fig. 7, D and E). Similarly, the effect of C3 exoenzyme appeared to be more pronounced in the absence of Gαq (Fig. 7 F). These data indicate that activation of G12/G13 through the TXA2 receptor results in MLC phosphorylation and that this process involves Rho/Rho-kinase. The data also provide further evidence for the concept that MLC phosphorylation underlies platelet shape change.

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