Methods to Investigate the Roles of β-Arrestin-Dependent RalGDS Activation in GPCR-Stimulated Membrane Blebbing

Abstract

G protein-coupled receptors (GPCRs) comprise the largest family of integral membrane proteins, which in addition to signaling via heterotrimeric G proteins can activate small G proteins both directly and indirectly. The activation of a variety of GPCRs leads to the translocation of Ral GDP dissociation stimulator (RalGDS) to the plasma membrane, where it functions as a guanine nucleotide exchange factor of RalA to promote membrane blebbing. The translocation of RalGDS is β-arrestin-dependent and can be inhibited by either the expression of the β-arrestin1 amino-terminal domain or the expression of RalGDS clone 284 (amino acid residues 616–768 of RalGDS). We describe here a methodology for assessing GPCR-dependent stimulation of RalGDS plasma membrane translocation.