Pull-down assay for analysis of integrin-mediated activation of Rap proteins in adherent platelets.

Rap1 GTPases operate as molecular switches by cycling between a GDP-bound inactive state and a GTP-bound active state and regulate several cellular pathways in response to different stimuli. Circulating blood platelets express high levels of Rap1 proteins, mainly Rap1b, which plays a critical role in platelet adhesion and activation. Rap1 is a key element in the inside-out signaling pathway leading to the conversion of integrins into the high-affinity state for their ligands. In platelets, Rap1b regulates inside-out activation of both integrin αIIbß3 and α2ß1. In addition, Rap1b is also involved in integrin outside-in signaling. Integrin-mediated platelet adhesion leads to accumulation of GTP-bound Rap1b, which promotes integrin-mediated processes such as spreading and clot retraction. Rap1b is thus a bidirectional regulator of platelet integrin function. Here we describe a method to analyze Rap1b activation induced by platelet adhesion via integrin α2ß1.

Rap1 regulation of RIAM and cell adhesion.

The small GTPase Rap1 has been involved in different cellular processes. Rap1 is known to increase cell adhesion by means of integrin activation, to induce cell spreading, and to regulate adherent junctions at cell-cell contacts. How Rap1 mediates these cell responses is poorly known, but currently developing evidence points to the involvement of different effector pathways. Recently, we described RIAM, a Rap1 interacting adaptor protein that regulates integrin activation and hence cell adhesion. RIAM is required for Rap1-induced adhesion and seems to control Rap1 localization at the plasma membrane, where Rap1 regulates integrin activation. In this chapter, we focus in the role of RIAM in regulating Rap1-mediated cell adhesion. We describe the method for studying the Rap1-RIAM interaction using in vitro and in vivo approaches such as yeast two hybrids, pull-down assays. and coimmunoprecipitation. The role of Rap1 and RIAM in integrin-mediated adhesion is studied by cell adhesion assays to immobilized integrin substrates and by changes in integrin activation as determined by activation epitope exposure. Finally, we describe an approach to determine the role of RIAM in regulating intracellular localization of active Rap1.

Rap-specific GTPase activating protein follows an alternative mechanism.

Rap1 is a small GTPase that is involved in signal transduction cascades. It is highly homologous to Ras but it is down-regulated by its own set of GTPase activating proteins (GAPs). To investigate the mechanism of the GTP-hydrolysis reaction catalyzed by Rap1GAP, a catalytically active fragment was expressed in Escherichia coli and characterized by kinetic and mutagenesis studies. The GTPase reaction of Rap1 is stimulated 10(5)-fold by Rap1GAP and has a k(cat) of 6 s(-1) at 25 degrees C. The catalytic effect of GAPs from Ras, Rho, and Rabs depends on a crucial arginine which is inserted into the active site. However, all seven highly conserved arginines of Rap1GAP can be mutated without dramatically reducing V(max) of the GTP-hydrolysis reaction. We found instead two lysines whose mutations reduce catalysis 25- and 100-fold, most likely by an affinity effect. Rap1GAP does also not supply the crucial glutamine that is missing in Rap proteins at position 61. The Rap1(G12V) mutant which in Ras reduces catalysis 10(6)-fold is shown to be efficiently down-regulated by Rap1GAP. As an alternative, Rap1(F64A) is shown by kinetic and cell biological studies to be a Rap1GAP-resistant mutant. This study supports the notion of a completely different mechanism of the Rap1GAP-catalyzed GTP-hydrolysis reaction on Rap1.