The AP-2 Adaptor beta 2 Appendage Scaffolds Alternate Cargo Endocytosis

摘要

The independently folded appendages of the large alpha and beta 2 subunits of the endocytic adaptor protein (AP)-2 complex coordinate proper assembly and operation of endocytic components during clathrin-mediated endocytosis. The beta 2 subunit appendage contains a common binding site for beta-arrestin or the autosomal recessive hypercholesterolemia (ARH) protein. To determine the importance of this interaction surface in living cells, we used small interfering RNA-based gene silencing. The effect of extinguishing beta 2 subunit expression on the internalization of transferrin is considerably weaker than an AP-2 alpha subunit knockdown. We show the mild sorting defect is due to fortuitous substitution of the beta 2 chain with the closely related endogenous beta 1 subunit of the AP-1 adaptor complex. Simultaneous silencing of both beta 1 and beta 2 subunit transcripts recapitulates the strong alpha subunit RNA interference (RNAi) phenotype and results in loss of ARH from endocytic clathrin coats. An RNAi-insensitive beta 2-yellow fluorescent protein (YFP) expressed in the beta 1 + beta 2-silenced background restores cellular AP-2 levels, robust transferrin internalization, and ARH colocalization with cell surface clathrin. The importance of the beta appendage platform subdomain over clathrin for precise deposition of ARH at clathrin assembly zones is revealed by a beta 2-YFP with a disrupted ARH binding interface, which does not restore ARH colocalization with clathrin. We also show a beta-arrestin 1 mutant, which engages coated structures in the absence of any G protein-coupled receptor stimulation, colocalizes with beta 2-YFP and clathrin even in the absence of an operational clathrin binding sequence. These findings argue against ARH and beta-arrestin binding to a site upon the beta 2 appendage platform that is later obstructed by polymerized clathrin. We conclude that ARH and beta-arrestin depend on a privileged beta 2 appendage site for proper cargo recruitment to clathrin bud sites.