Arf6 negatively controls the rapid recycling of the ß2 adrenergic receptor.

ß2-adrenergic receptor (ß2AR), a member of the GPCR (G-protein coupled receptor) family, is internalized in a ligand- and ß-arrestin-dependent manner into early endosomes, and subsequently recycled back to the plasma membrane. Here, we report that ß-arrestin promotes the activation of the small G protein Arf6, which regulates the recycling and degradation of ß2AR. We demonstrate in vitro that the C-terminal region of ß-arrestin1 interacts directly and simultaneously with Arf6GDP and its specific exchange factor EFA6, to promote Arf6 activation. Similarly, the ligand-mediated activation of ß2AR leads to the formation of Arf6GTP in vivo in a ß-arrestin-dependent manner. Expression of either EFA6 or an activated Arf6 mutant caused accumulation of ß2AR in the degradation pathway. This phenotype could be rescued by the expression of an activated mutant of Rab4, suggesting that Arf6 acts upstream of Rab4. We propose a model in which Arf6 plays an essential role in ß2AR desensitization. The ligand-mediated stimulation of ß2AR relocates ß-arrestin to the plasma membrane, and triggers the activation of Arf6 by EFA6. The activation of Arf6 leads to accumulation of ß2AR in the degradation pathway, and negatively controls Rab4-dependent fast recycling to prevent the re-sensitization of ß2AR.

Golgi-localized GAP for Cdc42 functions downstream of ARF1 to control Arp2/3 complex and F-actin dynamics.

The small GTP-binding ADP-ribosylation factor 1 (ARF1) acts as a master regulator of Golgi structure and function through the recruitment and activation of various downstream effectors. It has been proposed that members of the Rho family of small GTPases also control Golgi function in coordination with ARF1, possibly through the regulation of Arp2/3 complex and actin polymerization on Golgi membranes. Here, we identify ARHGAP10--a novel Rho GTPase-activating protein (Rho-GAP) that is recruited to Golgi membranes through binding to GTP-ARF1. We show that ARHGAP10 functions preferentially as a GAP for Cdc42 and regulates the Arp2/3 complex and F-actin dynamics at the Golgi through the control of Cdc42 activity. Our results establish a role for ARHGAP10 in Golgi structure and function at the crossroads between ARF1 and Cdc42 signalling pathways.

The small G-protein Arf6GTP recruits the AP-2 adaptor complex to membranes.

The small GTP-binding protein ADP-ribosylation factor 6 (Arf6) is involved in plasma membrane/endosomes trafficking. However, precisely how the activation of Arf6 regulates vesicular transport is still unclear. Here, we show that, in vitro, recombinant Arf6GTP recruits purified clathrin-adaptor complex AP-2 (but not AP-1) onto phospholipid liposomes in the absence of phosphoinositides. We also show that phosphoinositides and Arf6 tightly cooperate to translocate AP-2 to the membrane. In vivo, Arf6GTP (but not Arf6GDP) was found associated to AP-2. The expression of the GTP-locked mutant of Arf6 leads to the plasma membrane redistribution of AP-2 in Arf6GTP-enriched areas. Finally, we demonstrated that the expression of the GTP-locked mutant of Arf6 inhibits transferrin receptor internalization without affecting its recycling. Altogether, our results demonstrated that Arf6GTP interacts specifically with AP-2 and promotes its membrane recruitment. These findings strongly suggest that Arf6 plays a major role in clathrin-mediated endocytosis by directly controlling the assembly of the AP-2/clathrin coat.