A Phosphotyrosine Switch for Cargo Sequestration at Clathrin-Coated Buds [Signal Transduction]

May 5th, 2014 by Chakraborty, S., Umasankar, P. K., Preston, G. M., Khandelwal, P., Apodaca, G., Watkins, S. C., Traub, L. M.

The AP-2 clathrin adaptor complex oversees endocytic cargo selection in two parallel but independent manners. First, by physically engaging peptide-based endocytic sorting signals, a subset of clathrin-dependent transmembrane cargoes are directly collected into assembling buds. Synchronously, by interacting with an assortment of clathrin-associated sorting proteins (CLASPs) that independently select different integral membrane cargoes for inclusion within the incipient bud, AP-2 handles additional cargo capture indirectly. The distal platform subdomain of the AP-2 β2-subunit appendage is a privileged CLASP-binding surface that recognizes a cognate, short α-helical interaction motif. This signal, found in the CLASPs β-arrestin and ARH, docks into an elongated groove on the β2 appendage platform. Tyr888 is a critical constituent of this spatially-confined β2 appendage contact interface, and is phosphorylated in numerous high-throughput proteomic studies. We find a phosphomimetic Y888E substitution does not interfere with incorporation of expressed β2-YFP subunit into AP-2, nor alter AP-2 deposition at surface clathrin-coated structures. The Y888E mutation does not affect interactions involving the sandwich subdomain of the β2 appendage, indicating the mutated appendage is folded and operational. Yet the Y888E, but not Y888F, switch selectively uncouples interactions with ARH and β-arrestin. Phyogenetic conservation of Tyr888 suggests this residue can reversibly control occupancy of the β2 platform-binding site and hence, cargo sorting.