The members of the ARF family of small GTP-binding proteins play important roles in the regulation of membrane traffic and recently have been shown to activate cellular phospholipase D, an important modulator of cell function and proliferation that is controlled by various types of extracellular signals. However, the effects of extracellular signals on the state of activation of ARF proteins has not been studied. Thus, although the effects of ARF on phospholipase D activation strongly suggest a signalling role for these proteins, the regulation of their activity by extracellular signals remains to be demonstrated. Very recent work in the Principal Investigator's laboratory has shown that ARF activation can be stimulated by extracellular signals. Using as a model cells that overexpress wild type human insulin receptors, we have shown that the activation of purified bovine ARF by plasma membranes is enhanced by insulin. Cells that overexpress mutated receptors do not show this behavior. Likewise, the isoprenoid Brefeldin A, an agent that disrupts intracellular membrane traffic and whose effects are linked to the inhibition of ARF activation, has been shown to block insulin-induced receptor internalization and activation of phospholipase D. A detailed investigation of the possible mechanisms by which insulin might induce ARF activation revealed that the insulin receptor and ARF co-precipitate with immobilized insulin-agarose or with specific anti-insulin receptor antibodies. This co-precipitation was inhibited by guanine nucleotides in a manner reminiscent of the interactions of heterotrimeric G proteins with their specific receptors. It is proposed to continue these studies to determine the nature of the interactions of ARF proteins with the insulin receptor signalling system. The proposed studies will focus on the following aspects: a) characterization of the interactions between ARF and the insulin receptor using in vitro ARF activation and binding reconstitution assays recently developed in this laboratory; b) analysis of the interactions between the receptor and ARF using a yeast two-hybrid system; c) development of a reconstitution assay to study ARF activation by growth factors in vitro; d) development of a cell-free reconstitution assay to study the role of ARF proteins in insulin- and growth factor-mediated activation of phospholipase D; and e) test the role of ARF proteins in receptor-mediated PLD activation using dominant activated and negative ARF mutants in vitro and in cells that express the mutated ARF protein.
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