Phosphoinositides determine specificity of the guanine-nucleotide exchange activity of cytohesin-1 for ADP-ribosylation factors derived from a mammalian expression system.
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 beta2 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 (PtdInsP3). In contrast, cytohesin-1-dependent GTP binding of ARF1 is significantly enhanced by PtdInsP3. We conclude that the membrane phospholipid PtdInsP3 determines the specificity of the GEF activity of cytohesin-1.
Knorr
T
T
Laboratorium für Molekulaire Biologie, Gezentrum der Universität München, Germany.
Nagel
W
W
Kolanus
W
W
D020823:ADP-Ribosylation Factor 1 / Q000235:genetics / Q000378:metabolism*; D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism*; D002451:Cell Compartmentation; D006147:Guanine / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism*; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000378:metabolism
D000089982:ADP-Ribosylation Factor 6; D015815:Cell Adhesion Molecules; D020662:Guanine Nucleotide Exchange Factors; D007074:Immunoglobulin G; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; C100723:cytohesin-1; C055525:phosphatidylinositol 3-phosphate; D006147:Guanine; D006160:Guanosine Triphosphate; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human