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

Oronza A. Botrugno, Simona Paris, Lorena Za, Sara Gualdoni, Angela Cattaneo, Angela Bachi, Ivan de Curtis

Research output: Contribution to journalArticle

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-βPIX antibodies unequivocally show that βPIX 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 βPIX is the limiting factor for the formation of the endogenous complexes, since a small fraction of GIT1 can be co-immunoprecipitated with most βPIX from these cells. Saponin treatment of unfixed cells indicates that βPIX-bound GIT1 is preferentially retained in the saponin-resistant fraction when compared to βPIX-free GIT1. Moreover, analysis by tissue fractionation shows that a significant fraction of the endogenous GIT1-βPIX 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 βPIX, and the localization of the endogenous complex at membranes.

Original languageEnglish
Pages (from-to)35-46
Number of pages12
JournalEuropean Journal of Cell Biology
Volume85
Issue number1
DOIs
Publication statusPublished - Jan 11 2006

Keywords

  • ARF-GAP
  • Cell fractionation
  • Endogenous complex
  • GTPases
  • PIX

ASJC Scopus subject areas

  • Anatomy
  • Cell Biology

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