Disease relevance of ARF6

• After subcellular fractionation of cultured T98G human glioblastoma cells, ARF6 was almost entirely in the crude membrane fraction, whereas ARF-GEP(100), a 100-kDa protein detected with antipeptide antibodies, was cytosolic [1].
• We demonstrate here that ARFs 3 and 5, but not ARF6, are translocated to membranes in HeLa cell extracts that are engaged in translation of poliovirus RNA [2].
• Sustained activation of ARF6 significantly enhances the invasive capacity of melanoma as well as breast tumor cell lines, whereas dominant negative ARF6 abolishes basal cell invasive capacity as well as invasion induced by growth factors [3].
• By contrast, ARF6 activation of phospholipase D was not required for Chlamydia uptake [4].
• Consistent with these observations being relevant for cell motility, mutant forms of ARF6 that affect either actin rearrangement or recycling inhibit the motility of a breast cancer cell line [5].

High impact information on ARF6

• In vivo, ARF6, but not ARF1 or ARF5, spatially coincides with PI(4)P5Kalpha [6].
• This colocalization occurs in ruffling membranes formed upon AIF4 and EGF stimulation and is blocked by dominant-negative ARF6 [6].
• Phosphatidylinositol 4-phosphate 5-kinase alpha is a downstream effector of the small G protein ARF6 in membrane ruffle formation [6].
• Here we show that Arfaptin binds specifically to GTP-bound Arf1 and Arf6, but binds to Rac.GTP and Rac.GDP with similar affinities [7].
• A regulatory role for ARF6 in receptor-mediated endocytosis [8].

Biological context of ARF6

• Together, these data suggest that ARF6 activation stimulates two distinct signaling pathways, one leading to Rac activation, the other to changes in membrane phospholipid composition, and that both pathways are required for cell motility [9].
• One member of this family, ARF6, translocates on activation from intracellular compartments to the plasma membrane and has been implicated in regulated exocytosis in neuroendocrine cells [10].
• In addition to a role in endocytosis, ARF6 has also been shown to regulate assembly of the actin cytoskeleton [11].
• However, unlike ARF1, release of membrane-bound ARF6 to the cytosol requires hydrolysis of GTP that is sensitive to the level of magnesium [12].
• However, the expression of ARF6 decreased during adipogenesis and monocyte differentiation [13].

Anatomical context of ARF6

• Our paper suggests that ARF6 specifies delivery and insertion of recycling membranes to regions of dynamic reorganization of the plasma membrane through interaction with the vesicle-tethering exocyst complex [14].
• The delivery of ARF6 and membrane to defined sites along the PM may provide components necessary for remodeling the cell surface and the underlying actin cytoskeleton [15].
• To study the effector function of the ADP- ribosylation factor (ARF) 6 GTP-binding protein, we transfected HeLa cells with wild-type, epitope-tagged ARF6 [16].
• Importantly, the protrusions observed in cells transfected with ARF6 were distinct from the enhanced stress fibers and membrane ruffles observed in cells transfected with RhoA and Rac1, respectively [16].
• Distribution of ARF6 between membrane and cytosol is regulated by its GTPase cycle [12].

Associations of ARF6 with chemical compounds

• Aluminum fluoride stimulates surface protrusions in cells overexpressing the ARF6 GTPase [16].
• The new human BFA-insensitive GEP may function with ARF6 in specific endocytic processes [1].
• Centaurin-alpha1 is an in vivo phosphatidylinositol 3,4,5-trisphosphate-dependent GTPase-activating protein for ARF6 that is involved in actin cytoskeleton organization [17].
• LH/CG R activation also promotes the binding of a photoaffinity GTP analog to a protein that migrates on one- and two-dimensional polyacrylamide gel electrophoresis with ARF6 [18].
• Moreover, treatment of cells with butan-1-ol or phospholipase D1 antisense oligonucleotides inhibits translocation of PKCdelta, -epsilon, and -zeta but had no effect on the association of PKCalpha or ARF6 with phospholipase D1 [19].

Physical interactions of ARF6

• Here, we show that GTP-bound ARF6 interacts with Sec10, a subunit of the exocyst complex involved in docking of vesicles with the plasma membrane [14].
• Examples of other G protein-coupled receptors assessed in comparison display alternative pathways of protein kinase C- or ARF6-dependent activation of PLD2 [20].
• Furthermore, we demonstrated using glutathione S-transferase (GST)-fusion proteins of receptor domains that ARF1 and ARF6 bind to the third intracellular loop (i3) and the carboxy terminal tail (ct) of the 5-HT2AR [21].
• Furthermore, we observed that ARF6 interacts directly with POR1 and that this interaction was GTP dependent [22].
• Mutation of residues S472 or E473 to A in the cytoplasmic tail of CPE obliterated its binding to ARF6, and internalization from the plasma membrane of Tac-CPE25 mutated at S472 or E473 was significantly reduced [23].
• Taken together, our findings reveal a novel function of endogenously expressed ARF6 and demonstrate that by interacting with Rac1, this small GTPase is a central regulator of the signaling pathways leading to actin remodeling [24].

Enzymatic interactions of ARF6

• We have previously shown that ARNO localizes to the plasma membrane in vivo and efficiently catalyzes ARF6 nucleotide exchange in vitro [11].
• The ability of GRP1 to catalyze GTP/GDP exchange on ARF6 was confirmed using recombinant proteins in a cell-free system [25].

Co-localisations of ARF6

• Endogenous Rac1 colocalized with ARF6 at the plasma membrane and on the ARF6 recycling endosome in untransfected HeLa and primary human fibroblast cells [26].
• Like EFA6, which will be referred to as EFA6A from now on, EFA6B is involved in membrane recycling and colocalizes with ARF6 in actin-rich membrane ruffles and microvilli-like protrusions on the dorsal cell surface in transfected baby hamster kidney cells [27].

Regulatory relationships of ARF6

• Whereas AlF treatment blocked internalization, CD treatment blocked the recycling of wild-type ARF6 and Tac back to the PM; these blocks were mimicked by expression of ARF6 mutants Q67L and T27N, which were predicted to be in either the GTP- or GDP-bound state, respectively [15].
• Together, these results provide the first direct evidence that ARF6 plays a role in calcium-regulated exocytosis in neuroendocrine cells, and suggest that ARF6-stimulated PLD1 activation at the plasma membrane and consequent changes in membrane phospholipid composition are critical for formation of the exocytotic fusion pore [28].
• In this study, we have analyzed whether HERC1 may also regulate ARF6 activity [29].
• Our results demonstrate that by catalyzing nucleotide exchange on ARF6 at the plasma membrane and by regulating Rac1 activation, EFA6 coordinates endocytosis with cytoskeletal rearrangements [30].
• The possibility that the plasma membrane-associated ARNO participates in the exocytotic pathway by activating ARF6 and downstream PLD is discussed [31].

Other interactions of ARF6

• Previously shown to indirectly activate the ARF1 GTPase, aluminum fluoride (AIF) treatment of ARF6-transfected cells resulted in a redistribution of both ARF6 and actin to discrete sites on the plasma membrane, which became increasingly protrusive over time [16].
• Similar experiments with mutants of ARF5 and ARF6 showed that these other ARF family members had little or no effect on AP-3 [32].
• We also examined possible interaction of PAG3 with ARFs and showed evidence that at least one of them, ARF6, seems to be an intracellular substrate for GAP activity of PAG3 [33].
• Crosstalk between ARF6 and protein kinase Calpha in Fc(gamma)RI-mediated activation of phospholipase D1 [19].
• It is therefore likely that ARNO functions in plasma membrane events by modulating the activity of ARF6 in vivo [34].

Analytical, diagnostic and therapeutic context of ARF6

• Sequence analysis of the human ARF6 gene indicates it spans 4004 bp, contains a single 98-bp intron within the 5'-untranslated region, and is localized to chromosome 14q21 [35].
• Using a combination of SDS-PAGE and quantitative enhanced chemiluminescence Western blot analysis, the quantification of the ARF 6 distribution in membrane and cytoplasmic fractions of proximal tubules was made and its predominance in membrane fractions was demonstrated [36].
• Here we demonstrate that at the lowest detectable levels of protein expression by cryoimmunogold electron microscopy, ARF6 localized predominantly to an intracellular compartment at the pericentriolar region of the cell [37].
• By immuno-electron microscopy, the wild-type ARF6 protein is observed along the plasma membrane and associated with endosomes, and overexpression of ARF6 does not appear to alter the morphology of the peripheral membrane system [38].
• Assignment of the human ADP-ribosylation factor 6 (ARF6) gene to chromosome 7q22.1 by radiation hybrid mapping [39].

References