ARF6 stimulates clathrin/AP-2 recruitment to synaptic membranes by activating phosphatidylinositol phosphate kinase type I gamma

Krauss M, Kinuta M, Wenk MR, De Camilli P, Takei K and Haucke V
Source: J Cell Biol
Publication Date: (2003)
Issue: 162(1): 113-124
Cells used in publication:
Neuron, hippo/cortical, rat
Species: rat
Tissue Origin: brain
The authors studied the role of ARF6 in synaptic vesicle (SV) trafficking. ARFs are guanine-nucleotide-binding proteins and are implicated in clathrin-coated budding events at the Golgi complex and cell periphery. They also trigger assembly of vesicle coats onto membranes by direct interaction with coat proteins. The role of ARF proteins in recruitment of clathrin/AP-2 coats at the plasma membrane and in SV recycling has remained unclear. Primary rat cortical neurons were transfected with an HA tagged activated form of ARF6 (ARF6(Q67L). This form mimics the GTP-bound stage of ARF6. Distribution of ARF6(Q67) was analyzed by confocal laser microscopy. Co-localization with the pre-synaptic marker synaptophsin was determined. ARF6(Q67L) appears to be concentrated at synapses supporting the other biochemical data in this publication.
Clathrin-mediated endocytosis of synaptic vesicle membranes involves the recruitment of clathrin and AP-2 adaptor complexes to the presynaptic plasma membrane. Phosphoinositides have been implicated in nucleating coat assembly by directly binding to several endocytotic proteins including AP-2 and AP180. Here, we show that the stimulatory effect of ATP and GTPgammaS on clathrin coat recruitment is mediated at least in part by increased levels of PIP2. We also provide evidence for a role of ADP-ribosylation factor 6 (ARF6) via direct stimulation of a synaptically enriched phosphatidylinositol 4-phosphate 5-kinase type Igamma (PIPKIgamma), in this effect. These data suggest a model according to which activation of PIPKIgamma by ARF6-GTP facilitates clathrin-coated pit assembly at the synapse.