RAS and the RAIN/RasIP1 effector.

Ras proteins function as signaling nodes that are activated by extracellular stimuli. On activation, Ras interacts with a spectrum of functionally diverse downstream effectors and stimulates a variety of downstream cytoplasmic signaling cascades that regulate cellular proliferation, differentiation, and apoptosis. In addition to the association of Ras with the plasma membrane, recent studies have established an association of Ras with Golgi membranes and showed that H-Ras and N-Ras are activated on endomembranes and signal to regulate downstream pathways. Whereas the effectors of signal transduction by activated, plasma membrane-localized Ras are well characterized, very little is known about the effectors used by Golgi-associated Ras. Recently, we have reported the identification of the first endomembrane Ras effector molecule, RAIN. This chapter details the methods used to study RAIN-Ras interaction and localization in vivo. In addition, we describe the tools and methods we have used to explore role of endogenous RAIN in endothelial cells.