Characterization of the endogenous GIT1–βPIX complex, and identification of its association to membranes

Abstract G protein-coupled receptor kinase interactors (GITs) are adaptor proteins with ADP-ribosylating factor – GTPase-activating protein (ARF–GAP) activity that form complexes with the p21-activated kinase-interacting exchange factor (PIX) guanine nucleotide exchanging factors for Rac and Cdc42. In this study we have characterized the endogenous GIT1/p95-APP1/Cat1 (GIT1)– PIX complexes in neuronal and non-neuronal cells. In COS7 cells, immunocytochemical analysis shows the localization of endogenous GIT1 in the perinuclear region of the cell, as well as at the cell periphery, where GIT1 co-localizes with filamentous actin. The perinuclear localization of endogenous GIT1 was confirmed in avian fibroblasts. In COS7 cells, immunoprecipitation and microsequencing experiments with either anti-GIT1 or anti- β PIX antibodies unequivocally show that β PIX is uniquely associated with GIT1 in lysates from these cells, while GIT2/PKL/p95-APP2/Cat2 (GIT2) is undetectable in the endogenous complexes. Moreover, this analysis demonstrates that β PIX is the limiting factor for the formation of the endogenous complexes, since a small fraction of GIT1 can be co-immunoprecipitated with most β PIX from these cells. Saponin treatment of unfixed cells indicates that β PIX-bound GIT1 is preferentially retained in the saponin-resistant fraction when compared to β PIX-free GIT1. Moreover, analysis by tissue fractionation shows that a significant fraction of the endogenous GIT1– β PIX complex is firmly associated to membranes from brain homogenates. Our findings show the specific localization of the complex at intracellular membranes, and indicate a correlation between the association of GIT1 to β PIX, and the localization of the endogenous complex at membranes.