Open AccessPhosphoinositides determine specificity of the guanine‐nucleotide exchange activity of cytohesin‐1 for ADP‐ribosylation factors derived from a mammalian expression system
Author(s) -
Knorr Thomas,
Nagel Wolfgang,
Kolanus Waldemar
Publication year - 2000
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.2000.01416.x
Subject(s) - adp ribosylation factor , guanine nucleotide exchange factor , microbiology and biotechnology , rab , intracellular , gtpase , phosphatidylinositol , biology , small gtpase , cytoskeleton , cytoplasm , proximity ligation assay , chemistry , biochemistry , signal transduction , receptor , cell , endoplasmic reticulum , golgi apparatus
ADP‐ribosylation factors (ARFs) are small Ras‐like GTPases which play important roles in intracellular vesicle transport and in the remodeling of the actin cytoskeleton. Guanine nucleotide exchange factors (GEFs) for ARFs have recently been identified. One of them, cytohesin‐1, a 47‐kDa cytoplasmic protein acts as an inside‐out signaling molecule and regulates binding of the β2 integrin leukocyte function antigen 1 (LFA‐1) to its ligand intercellular adhesion molecule 1 (ICAM‐1). In this study, we address the regulation of the GEF activity of cytohesin‐1 by phosphoinositides, using mammalian expression of functional ARF–Ig chimeras. The fusion proteins, which can be quantitatively immunoprecipitated on protein A–Sepharose, target to the expected intracellular compartments, and they are readily induced to bind GTP in vitro . We show that both ARF1–Ig and ARF6–Ig chimeras are activated in vitro by cytohesin‐1. However, GEF activity towards ARF6 is strongly suppressed by phosphatidylinositol‐(3,4,5)‐trisphosphate (PtdIns P 3 ). In contrast, cytohesin‐1‐dependent GTP binding of ARF1 is significantly enhanced by PtdIns P 3 . We conclude that the membrane phospholipid PtdIns P 3 determines the specificity of the GEF activity of cytohesin‐1.