The guanine nucleotide exchange factor ARNO mediates the activation of ARF and phospholipase D by insulin.
Phospholipase D (PLD) is involved in many signaling pathways. In most systems, the activity of PLD is primarily regulated by the members of the ADP-Ribosylation Factor (ARF) family of GTPases, but the mechanism of activation of PLD and ARF by extracellular signals has not been fully established. Here we tested the hypothesis that ARF-guanine nucleotide exchange factors (ARF-GEFs) of the cytohesin/ARNO family mediate the activation of ARF and PLD by insulin.



Wild type ARNO transiently transfected in HIRcB cells was translocated to the plasma membrane in an insulin-dependent manner and promoted the translocation of ARF to the membranes. ARNO mutants: DeltaCC-ARNO and CC-ARNO were partially translocated to the membranes while DeltaPH-ARNO and PH-ARNO could not be translocated to the membranes. Sec7 domain mutants of ARNO did not facilitate the ARF translocation. Overexpression of wild type ARNO significantly increased insulin-stimulated PLD activity, and mutations in the Sec7 and PH domains, or deletion of the PH or CC domains inhibited the effects of insulin.



Small ARF-GEFs of the cytohesin/ARNO family mediate the activation of ARF and PLD by the insulin receptor.
Li
Hai-Sheng
HS
Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA. hal9001@pitt.edu
Shome
Kuntala
K
Rojas
Raúl
R
Rizzo
Megan A
MA
Vasudevan
Chandrasekaran
C
Fluharty
Eric
E
Santy
Lorraine C
LC
Casanova
James E
JE
Romero
Guillermo
G
D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007328:Insulin / Q000494:pharmacology*; D018613:Microscopy, Confocal; D009154:Mutation; D010739:Phospholipase D / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport / Q000187:drug effects; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection
D020662:Guanine Nucleotide Exchange Factors; D007328:Insulin; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D011972:Receptor, Insulin; D010739:Phospholipase D; D020727:ADP-Ribosylation Factors