ARF PROTEINS MEDIATE INSULIN-DEPENDENT ACTIVATION OF PHOSPHOLIPASE-D

Background: ADP-ribosylation factors (ARFs) have been shown to activat e 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 GTP gamma S to purified bovine or recombinant ARF was enhanc ed in the presence of insulin. Membranes obtained from cells that over expressed a mutated, nonfunctional insulin receptor failed to stimulat e ARF activation. Insulin promoted the association of ARF proteins wit h membranes in the presence of GTP gamma S in permeabilized cells. Ins ulin activated PLD in permeabilized HIRcB cells by a process that requ ired GTP gamma S and ARF. Azido-gamma[P-32]-GTP labelling of immunopre cipitated receptors revealed the presence of a unique 19 kD band; ARF proteins are approximately this size, and analysis using specific mono clonal antibodies demonstrated that ARF proteins coimmunoprecipitated with the insulin receptor. Coimmunoprecipitation of ARF with the recep tor 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[P-32]-GTP or anti-ARF antibodies. Conclusi ons: The activation of ARF proteins is stimulated by insulin and this process plays an important role in insulin-mediated regulation of PLD.