Rab35 establishes the EHD1-association site by coordinating two distinct effectors during neurite outgrowth.

Endocytic recycling is a process in which molecules that have been internalized are recycled back to the plasma membrane, and although it is crucial for regulating various cellular events, the molecular nexus underlying this process remains poorly understood. Here we report a molecular link between two gatekeepers for endocytic recycling, the molecular switch Rab35 and the molecular scissors EHD1, that is mediated by two distinct Rab35 effectors during neurite outgrowth of PC12 cells. Rab35 forms a tripartite complex with MICAL-L1 and centaurin-β2/ACAP2 and recruits them to perinuclear Arf6-positive endosomes in response to nerve growth factor stimulation. MICAL-L1 and centaurin-β2 then cooperatively recruit EHD1 to the same compartment by functioning as a scaffold for EHD1 and as an inactivator of Arf6, respectively. We propose that Rab35 regulates the formation of an EHD1-association site on Arf6-positive endosomes by integrating the functions of two distinct Rab35 effectors for successful neurite outgrowth.