ARF proteins mediate insulin-dependent activation of phospholipase D.
BACKGROUND
ADP-ribosylation factors (ARFs) have been shown to activate phospholipase D (PLD), an enzyme modulated by extracellular signals, including several growth factors and, in particular, insulin. We have tested the hypothesis that ARF proteins are involved specifically in insulin-induced activation of PLD.


RESULTS
We found that in membranes obtained from HIRcB cells, a cell line derived from Rat-1 fibroblasts that overexpresses normal human insulin receptors, binding of the GTP analogue GTPgammaS to purified bovine or recombinant ARF was enhanced in the presence of insulin. Membranes obtained from cells that overexpressed a mutated, nonfunctional insulin receptor failed to stimulate ARF activation. Insulin promoted the association of ARF proteins with membranes in the presence of GTPgammaS in permeabilized cells. Insulin activated PLD in permeabilized HIRcB cells by a process that required GTPgammaS and ARF. Azido-gamma[32P]-GTP labelling of immunoprecipitated receptors revealed the presence of a unique 19 kD band; ARF proteins are approximately this size, and analysis using specific monoclonal antibodies demonstrated that ARF proteins coimmunoprecipitated with the insulin receptor. Coimmunoprecipitation of ARF with the receptor was inhibited by guanine nucleotides and stimulated by insulin. No evidence of the coprecipitation of ARF with mutant receptors could be obtained using azido-gamma[32P]-GTP or anti-ARF antibodies.


CONCLUSIONS
The activation of ARF proteins is stimulated by insulin and this process plays an important role in insulin-mediated regulation of PLD.
Shome
K
K
Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
Vasudevan
C
C
Romero
G
G
D020727:ADP-Ribosylation Factors; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D008954:Models, Biological; D010739:Phospholipase D / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism*; D015398:Signal Transduction
D007328:Insulin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D011972:Receptor, Insulin; D010739:Phospholipase D; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors