The role of tyrosine phosphorylation in G protein-coupled receptor mediated signal transduction: Platelets as a model system

Karen Marie Cichowski, University of Pennsylvania

Abstract

Physiological agonists cause platelets to form multicellular aggregates, secrete the contents of their storage granules and spread on extracellular matrix proteins. Many of these agents stimulate G protein-coupled receptors, and have been shown to utilize "classical" signaling pathways involving phospholipase C and adenylyl cyclase, to transduce their effects. However, platelet activation is also accompanied by a dramatic increase in protein tyrosine phosphorylation, suggesting that these receptors may couple to tyrosine kinases. We have therefore been using platelets, as a model system, to investigate a potential role for tyrosine kinases in G protein-coupled receptor mediated signal transduction, by identifying proteins which become phosphorylated in response to thrombin. Specifically, we have examined phosphorylation events that are independent of platelet aggregation, a process mediated by the $\alpha\sb{IIb}\beta\sb3$ integrin, in order to define signals that occur as a result of G protein-coupled receptor activation and not as a secondary consequence of integrin engagement. We found that $\rm \sb{p}21\sp{ras}$GAP, and several associated proteins became phosphorylated on tyrosine in response to thrombin, and that three Src-related kinases Fyn, Lyn and Yes, were associated with GAP in complexes, detectable after agonist stimulation. GAP's phosphorylation and its association with these kinases was not dependent on the formation of platelet aggregates, indicating that (1) thrombin alone is capable of stimulating GAP phosphorylation and that (2) these kinases may be responsible for transducing the effects of thrombin in platelets. To investigate the potential physiological significance of this finding we examined a downstream effector of Ras, MAP kinase, and its reported substrate cPLA$\sb2,$ and found that their phosphorylation patterns correlated with GAP phosphorylation, illustrating one mechanism by which tyrosine kinases may regulate arachidonate formation in platelets. We were also interested in identifying signals involved in platelet aggregation. Since Rho has been shown to be required for this process we examined the phosphorylation of Vav, a putative Rho exchange factor, and found that thrombin rapidly induced its phosphorylation on tyrosine, independent of aggregation. Interestingly, Vav also became phosphorylated as a result of integrin-mediated adhesion, which initiates the process of platelet spreading and the formation of stress fibers. These results demonstrate that thrombin alone can induce protein tyrosine phosphorylation in platelets, suggesting a role for tyrosine kinases in G protein-coupled receptor mediated signal transduction. Furthermore, by identifying these phosphoproteins and potential downstream effectors, we have begun to outline mechanisms by which tyrosine kinases may participate in platelet activation.

Subject Area

Molecular biology|Cellular biology

Recommended Citation

Cichowski, Karen Marie, "The role of tyrosine phosphorylation in G protein-coupled receptor mediated signal transduction: Platelets as a model system" (1996). Dissertations available from ProQuest. AAI9627902.
https://repository.upenn.edu/dissertations/AAI9627902

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