ADP-ribosylation factors (ARFs)1 are a group of structurally related GTP-binding proteins that form a subset of the Ras superfamily. ARFs are ubiquitous in eukaryotic cells with an amino acid sequence that is highly conserved across diverse species, suggesting a fundamental role in cellular physiology. Initially discovered as cofactors of the cholera toxin-catalyzed ADP-ribosylation of G␣ s (1), ARFs appear to be critical to vesicular trafficking in various subcellular compartments of the cell (2). More recently, members of the ARF family have been shown to activate phospholipase D (PLD) in several cellular systems as well as in isolated membranes (3-5). The ARF family has been divided into three classes based on size, sequence homology, gene structure, and phylogenetic analysis. Class I ARFs (ARFs 1-3) were initially identified as components of vesicles that originate from the Golgi (6, 7) and the endoplasmic reticulum (8), whereas class III ARF6, which is the most structurally divergent member of the family, has more recently been implicated in the exocytotic and endocytotic pathways at the plasma membrane (9 -12). Little is known about class II ARF4 and ARF5. Like other G proteins, ARFs cycle between a GDP-bound and a GTP-bound conformation. The GTP-induced conformational change is the "on" signal that permits the ARF proteins to bind to and activate specific protein effectors. Isolated ARFs have little detectable GTPase activity and exchange bound nucleotide very slowly. In cells, their GTPase cycle requires an interaction with GTPase-activating proteins and guanine-nucleotide exchange factors (GEFs) which catalyze the nucleotide exchange activity on ARF. The identification of ARF1 GEFs was facilitated by the discovery that the fungal metabolite brefeldin A (BFA) disrupts Golgi trafficking by inhibiting a Golgi-associated ARF1 exchange factor (13). Several GEF activities have been described, but the breakthrough toward the identification of GEFs acting on ARF proteins was the cloning of two related BFA-sensitive ARF1 GEFs-encoding genes in yeast Saccharomyces cerevisiae, Gea1 and Gea2 (14). This lead to the discovery of cytohesin-1 (15), ARNO (ARF nucleotidebinding-site opener, Ref. 16), and GRP1 (17) which promote guanine nucleotide exchange on ARF1 by a BFA-insensitive catalytic mechanism. Subsequent studies demonstrated that ARNO, GRP1, and cytohesin-1 can also promotes GDP/GTP exchange on ARF6 in both cell free and intact cell assays

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Regulated secretion requires both calcium and MgATP. Studies in diverse secretory systems indicate that ATP is required to prime the exocytotic apparatus whereas Ca2+ triggers the final ATP-independent fusion event. In this paper, we examine the possible role of trimeric G proteins in these two steps of exocytosis in chromaffin cells. We show that in the presence of low concentrations of Mg2+, mastoparan selectively stimulates G proteins associated with purified chromaffin granule membranes. Under similar conditions in permeabilized chromaffin cells, mastoparan inhibits ATP-dependent secretion but is unable to trigger ATP-independent release. This inhibitory effect of mastoparan on secretion was specifically reversed by anti-Galphao antibodies and a synthetic peptide corresponding to the carboxyl terminus of Galphao. In contrast, mastoparan required millimolar Mg2+ for the activation of plasma membrane-bound G proteins and stimulation of ATP-independent secretion in permeabilized chromaffin cells. The latter effect was completely inhibited by anti-Galphai3. By confocal immunofluorescence and immunoreplica analysis, we provide evidence that in chromaffin cells Go is preferentially associated with secretory granules, while Gi3 is essentially present on the plasma membrane. Our findings suggest that these two trimeric G proteins act in series in the exocytotic pathway in chromaffin cells: a secretory granule-associated Go protein controls the ATP-dependent priming reaction, whereas a plasma membrane-bound Gi3 protein is involved in the late calcium-dependent fusion step, which does not require ATP.

Phosducin and related proteins have been identified as ubiquitous regulators of signalling mediated by L LQ Q subunits of trimeric G proteins. To explore a role for phosducin in regulated exocytosis, we have examined the distribution and putative function of phosducin-like protein (PhLP) in adrenal medullary chromaffin cells. The full-length cDNA encoding the short splice variant of PhLP (PhLPs) was cloned from cultured chromaffin cells. Native PhLPs was found associated with plasma membranes and detected in the subplasmalemmal area of resting chromaffin cells by confocal immunofluorescence analysis. Stimulation with secretagogues triggered a massive redistribution of PhLPs into the cytoplasm. When microinjected into individual chromaffin cells, recombinant PhLPs inhibited catecholamine secretion evoked by a depolarizing concentration of K + without affecting calcium mobilization. Thus, PhLPs may participate directly in the regulation of calcium-evoked exocytosis. ß