Decoupling of Activation and Effector Binding Underlies ARF6 Priming of Fast Endocytic Recycling

Abstract

The small GTP-binding protein ADP-ribosylation factor 6 (ARF6) controls the endocytic recycling pathway of several plasma membrane receptors. We analyzed the localization and GDP/GTP cycle of GFP-tagged ARF6 by total internal reflection fluorescent microscopy. We found that ARF6-GFP associates with clathrin-coated pits (CCPs) at the plasma membrane in a GTP-dependent manner in a mechanism requiring the adaptor protein complex AP-2. In CCP, GTP-ARF6 mediates the recruitment of the ARF-binding domain of downstream effectors including JNK-interacting proteins 3 and 4 (JIP3 and JIP4) after the burst recruitment of the clathrin uncoating component auxilin. ARF6 does not contribute to receptor-mediated clathrin-dependent endocytosis. In contrast, we found that interaction of ARF6 and JIPs on endocytic vesicles is required for trafficking of the transferrin receptor in the fast, microtubule-dependent endocytic recycling pathway. Our findings unravel a novel mechanism of separation of ARF6 activation and effector function, ensuring that fast recycling may be determined at the level of receptor incorporation into CCPs.

BRAG2/GEP100/IQSec1 Interacts with Clathrin and Regulates α5β1 Integrin Endocytosis through Activation of ADP Ribosylation Factor 5 (Arf5)

“…Arf5-GFP was more diffusely distributed on the plasma membrane but could also be observed in puncta that colocalized with clathrin. In agreement with a recent report (24), Arf6-GFP also displayed colocalization with clathrin-positive structures (Fig. 6, G-I).…”

“…Montagnac et al (24) recently reported that Arf6 concentrates in clathrin-coated pits but that it is not necessary for endocytosis of transferrin receptors. Using a fluorescently tagged fragment of the Arf6 effector protein, JIP3, these authors demonstrated that Arf6 is active in clathrin-coated vesicles, but is not accessible to JIP3 until a later time when the vesicles uncoat (24).…”

EFA6 controls Arf1 and Arf6 activation through a negative feedback loop

“…Notably, a separation of Arf6 activation and effector functions has been observed in endocytic receptor recycling, in which a dormant pool of Arf6-GTP was observed in clathrin-coated pits that became mobilized only later in fast endocytic events (44). This finding would be consistent with the proposed role of EFA6 in sustaining a basal amount of Arf6-GTP.…”

A Role for Cargo in Arf-dependent Adaptor Recruitment

“…A model for Arf6 acting at sites quite distant from its site of activation has recently been proposed (67), but the actions of Arf6 at the cell surface differ substantially from those of Arfs 1-5 on endomembranes so it is not clear how comparable these studies will prove to be. We currently lack direct evidence of specific cargo-GEF-Arf complex formation and must also consider additional components acting between the cargo and the Arf GEFs.…”