Role of Carboxyterminal Hypervariable Region in Rap1b Function

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DESCRIPTION (provided by applicant): Rap1b is a ubiquitous, membrane associated low molecular weight GTPase that has been shown to be important for integrin-mediated platelet aggregation and adhesion. The mechanism of rap1b action appears to be through a signaling cascade that leads to activation of integrins. Mice deficient in rap1b have abnormal platelet responses to all agonists tested. As with all low molecular weight GTPases, the membrane association of rap1b is important for its function. The purpose of the work in this proposal is to explore the membrane interactions of rap1b in more detail in order to better understand its mechanism of action. The attachment of rap1b to the membrane is through its C-terminal hypervariable region, a region that contains polybasic sequences believed to interact with membrane phospholipids, a post-translationally added prenyl group that associates with membranes, and a hydrophobic C-terminal carboxymethyl group that also associates with the membrane. The hypervariable region is also the site of phosphorylation by cyclic AMP (cAMP)-dependent protein kinase (PKA) when platelets are inhibited by agents that increase cAMP levels. Using various hypervariable region mutations that affect the structure of the polybasic cluster and the sites of carboxymethylation, prenylation, and phosphorylation, we will examine the role of these various moeities on the subcellular localization and function of rap1b, the interaction of the hypervariable region with specific phoshoplipids, and the interaction of the hypervariable region with other proteins. The results of these studies will lead to an improved understand of how GTPases work in general, how rap1b functions in platelets to regulate integrin activation, and may lead to new methods for inhibiting platelet function. PUBLIC HEALTH RELEVANCE: Platelets are the first responders when there is an injury to a blood vessel. The overall goal of this application is to understand how platelets respond to such an injury, as well as the mechanisms involved with their response. These findings will provide insight into how the body heals bleeding injuries, as well as the underlying cause of blood clotting conditions.