ACTIVATION OF THE PLATELET FIBRINOGEN RECEPTOR

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The cytoplasmic domains of the platelet integrin GPIIb-IIIa (or alphaIIbBeta3) play a critical role in receiving signals from agonist- stimulated platelets, resulting in a activated conformation of GPIIb-IIIa. We have therefore attempted to identify proteins that bind to the cytoplasmic domains and regulated GPIIb-IIIa activation. We have obtained the complete sequence of a novel approximately 25 kDa protein that binds to the GPIIb cytoplasmic domain, using the yeast two-hybrid system. This protein is homologous to the regulatory subunits calmodulin and calcineurin B, has two EF hand domains and binds Ca/45, is expressed in platelets, and binds to antibody-captured, native GPIIb-IIIa. We have therefore named this protein "CIB) for Ca2+ and integrin binding protein. In specific aim #1, we propose to further explore the structure and function of CIB, especially as related to integrin function. In specific aim #2, we propose to map signal transduction pathways leading to (1) the activation and (2) the maintenance of activation of GPIIb-IIIa. To map pathways for GPIIb-IIIa activation, we will use CHO cells expressing constitutively active R-Ras, since GPIIb-IIIa becomes active in these cells as reported recently by Ruosiahti and coworkers and reproduced by us. Specifically we will (a) further determine if other molecules closely related to R-Ras (e.g. TC21 and others) also activate GPIIb-IIIa in these cells; (b) microinject R-Ras protein and other molecules identified that activate GPIIb-IIIa, to determine whether they immediately and therefore more directly affect GPIIb-IIIa function, as opposed to potentially inducing synthesis of other proteins that actually induce the activation; and (c) dissect the signaling pathway between R-Ras and the integrin by expressing dominant negative versions of likely signaling molecules downstream of R-Ras. (d) Finally, to map signaling pathways necessary for the maintenance of GPIIb-IIIa activation we will use CHO cells expressing mutant GPIIb-IIIa that exists in an energy dependent, constitutively active state with approaches similar to those described above. Thus these studies will allow us to dissect "inside-out" integrin signaling pathways on a molecular level, with approaches that cannot be used in the anucleate platelet.