Characterization of the endogenous GIT1-betaPIX complex, and identification of its association to membranes.
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-betaPIX antibodies unequivocally show that betaPIX 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 betaPIX is the limiting factor for the formation of the endogenous complexes, since a small fraction of GIT1 can be co-immunoprecipitated with most betaPIX from these cells. Saponin treatment of unfixed cells indicates that betaPIX-bound GIT1 is preferentially retained in the saponin-resistant fraction when compared to betaPIX-free GIT1. Moreover, analysis by tissue fractionation shows that a significant fraction of the endogenous GIT1-betaPIX 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 betaPIX, and the localization of the endogenous complex at membranes.
Botrugno
Oronza A
OA
Cell Adhesion Unit, Department of Molecular Biology and Functional Genomics, San Raffaele Scientific Institute, Via Olgettina 58, I-20132 Milano, Italy.
Paris
Simona
S
Za
Lorena
L
Gualdoni
Sara
S
Cattaneo
Angela
A
Bachi
Angela
A
de Curtis
Ivan
I
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D002522:Chlorocebus aethiops; D005347:Fibroblasts / Q000166:cytology; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000378:metabolism; D010750:Phosphoproteins / Q000032:analysis / Q000737:chemistry; D011485:Protein Binding; D021381:Protein Transport; D064067:Rho Guanine Nucleotide Exchange Factors; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C485647:GIT1 protein, human; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D008024:Ligands; D046912:Multiprotein Complexes; D010750:Phosphoproteins; D064067:Rho Guanine Nucleotide Exchange Factors