Expression of ARF6 mutants in neuroendocrine cells suggests a role for ARF6 in synaptic vesicle biogenesis

Abstract

ARF6 regulates membrane trafficking between the plasma membrane and endosomes. We investigated the role of ARF6 in synaptic vesicle biogenesis as this process occurs both at the plasma membrane and at endosomes. We used a synaptic vesicle marker protein, p-selectin-horseradish peroxidase (HRP), to follow the effects of ARF6 expression on synaptic vesicle biogenesis in PC12 neuroendocrine cells. Expression of a constitutively active ARF6 mutant increased, while expression of a nucleotide-free ARF6 mutant decreased, p-selectin-HRP levels in the synaptic vesicle peak. These results provide the first direct evidence for a role for ARF6 in synaptic vesicle biogenesis.

ARF6 regulates a plasma membrane pool of phosphatidylinositol(4,5)bisphosphate required for regulated exocytosis

“…Plasma membrane SNAP-25 is co-internalized with PIP 2 -containing, ARF6 Q67L -enriched membranes by a dynamin-independent pathway (unpublished data), but ARF6 Q67L -expressing cells also exhibit increased initial rates of transferrin uptake (unpublished data) presumed to be via clathrin- and dynamin-mediated endocytosis. ARF6 Q67L expression also accelerates the formation of synaptic vesicle-like endosomes in PC12 cells ( Powelka and Buckley, 2001 ) that are formed by a clathrin-dependent pathway ( Strasser et al, 1999 ). These results indicate that multiple endocytic pathways are enhanced by expression of the constitutively active ARF6 Q67L in PC12 cells.…”

ADP Ribosylation Factor 6 (ARF6) Promotes Acrosomal Exocytosis by Modulating Lipid Turnover and Rab3A Activation

“…Highly motile sperm were recovered after a swim-up separation for 1 h in HTF (5 6 sperm/ml. To promote capacitation of the motile fraction recovered from the swim-up procedure, sperm were incubated in HTF supplemented with 5 mg/ml bovine serum albumin for at least 3 h at 37°C in an atmosphere of 5% CO 2 , 95% air.…”

The Hop Cassette of the PAC1 Receptor Confers Coupling to Ca2+ Elevation Required for Pituitary Adenylate Cyclase-activating Polypeptide-evoked Neurosecretion

“…The requirement for a discrete region of the IC3 loop of the PAC1 receptor to activate Ca 2ϩ mobilization, Ca 2ϩ influx, and exocytotic secretion suggests the possibility that several components required for regulated exocytosis may be brought into close proximity through the hop-dependent formation of a specific signaling complex to trigger PACAP-dependent neurosecretion. Ronaldson and co-workers (25) have demonstrated that the small GTP-binding protein ARF6 (ADP-ribosylation factor-6) specifically interacts with the hop cassette to activate phospholipase D (24,25), and the role of ARF6 in membrane trafficking, synaptic vesicle biogenesis (66), and Ca 2ϩ -dependent large dense core vesicle exocytosis in both PC12 and chromaffin cells has been well established (67,68). ARF6 or a related GTPase may have a role in PAC1 hop -mediated exocytosis through formation of such a complex.…”