The glycoprotein (GP) IIb-IIIa complex mediates the binding of fibrinogen to platelets and thus plays a major role in inducing the formation of platelet aggregates. On unstimulated platelets, GPIIb-IIIa cannot bind fibrinogen. Upon activation of platelets, unidentified intracellular events cause a change in the extracellular domain of GPIIb-IIIa that allows it to bind fibrinogen. This is known as """"""""inside-out"""""""" signaling. Binding of fibrinogen leads to the induction of intracellular events such as phosphorylation of specific proteins on tyrosine residues, activation of calpain and hydrolysis of specific cytoskeletal proteins. This is referred to as """"""""outside-in"""""""" signaling. The way in which the two way signaling across GPIIb-IIIa occurs is not understood. GPIIb-IIIa is a member of the integrin family of adhesion receptors. In cultured cells, evidence has been provided that integrins can associate with the cytoskeleton and that this interaction regulates functional activities of the integrins. Little is known about the possibility that the cytoskeleton regulates the functional activities of integrins in other cells. In preliminary experiments, the investigators have provided evidence that 1) GPIIb-IIIa is associated with the membrane skeleton in unstimulated platelets, 2) the tyrosine kinase, pp60c-src, appears to be associated with the membrane skeleton of platelets that are not yet competent to bind fibrinogen, and several of the proteins that become rapidly phosphorylated on tyrosine residues when platelets are activated co-isolate with the membrane skeleton in detergent lysates; 3) binding of fibrinogen drives GPIIb-IIIa and associated membrane skeleton proteins into association with cytoplasmic actin filaments, GPIIb-IIIa clusters, additional cytoskeletal proteins become phosphorylated on tyrosine residues, and additional signaling molecules (e.g., protein kinase C and phosphoinositide 3-kinase) are recruited to the integrin- rich skeletal structure; 4) incubation of platelets with cytochalasins inhibits the two way signaling across the integrin. These findings are consistent with the hypothesis that the cytoskeleton regulates functional activities of GPIIb-IIIa in platelets. Experiments in this proposal are designed to identify the mechanism(s) by which GPIIb-IIIa associates with the cytoskeleton, and to use this information to directly test the hypothesis that the interaction(s) is essential for regulating functional activities of the GPIIb-IIIa complex.
The Specific Aims of the proposed research are 1) to characterize interactions between GPIIb-IIIa and cytoskeletal proteins with which the integrin is known to interact in vitro; 2) to identify additional proteins that interact with the cytoplasm domains of GPIIb-IIIa; and 3) to elucidate the role of cytoskeletal proteins in regulating two way signaling across GPIIb-IIIa. Dr. Fox anticipates that these studies will provide insight into mechanisms involved in signaling across a variety of integrins, and may lead to understanding of ways in which integrin functions could be inhibited.
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