{"doi":"10.17615/zcdr-5767","title":"The AP-2 Adaptor  2 Appendage Scaffolds Alternate Cargo Endocytosis","abstract":"The independently folded appendages of the large α and β2 subunits of the endocytic adaptor protein (AP)-2 complex coordinate proper assembly and operation of endocytic components during clathrin-mediated endocytosis. The β2 subunit appendage contains a common binding site for β-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 β2 subunit expression on the internalization of transferrin is considerably weaker than an AP-2 α subunit knockdown. We show the mild sorting defect is due to fortuitous substitution of the β2 chain with the closely related endogenous β1 subunit of the AP-1 adaptor complex. Simultaneous silencing of both β1 and β2 subunit transcripts recapitulates the strong α subunit RNA interference (RNAi) phenotype and results in loss of ARH from endocytic clathrin coats. An RNAi-insensitive β2-yellow fluorescent protein (YFP) expressed in the β1 + β2-silenced background restores cellular AP-2 levels, robust transferrin internalization, and ARH colocalization with cell surface clathrin. The importance of the β appendage platform subdomain over clathrin for precise deposition of ARH at clathrin assembly zones is revealed by a β2-YFP with a disrupted ARH binding interface, which does not restore ARH colocalization with clathrin. We also show a β-arrestin 1 mutant, which engages coated structures in the absence of any G protein-coupled receptor stimulation, colocalizes with β2-YFP and clathrin even in the absence of an operational clathrin binding sequence. These findings argue against ARH and β-arrestin binding to a site upon the β2 appendage platform that is later obstructed by polymerized clathrin. We conclude that ARH and β-arrestin depend on a privileged β2 appendage site for proper cargo recruitment to clathrin bud sites.","journal":"UNC Libraries","year":2020,"id":144672,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9574,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":512341,"name":"Eric T. Everett","orcid":"0000-0001-7801-5278","position":1,"is_corresponding":false},{"id":613794,"name":"Peter A. Keyel","orcid":"0000-0001-8637-8538","position":2,"is_corresponding":false},{"id":481126,"name":"John E. Heuser","orcid":"0000-0002-3593-8846","position":3,"is_corresponding":false},{"id":298670,"name":"Robyn Roth","orcid":"0000-0001-7823-776X","position":4,"is_corresponding":false},{"id":613795,"name":"J. R. Thieman","orcid":"0000-0003-3615-9489","position":5,"is_corresponding":false},{"id":613796,"name":"Linton M. Traub","orcid":"0000-0002-1303-0298","position":6,"is_corresponding":false},{"id":279286,"name":"Simon C. Watkins","orcid":"0000-0003-4092-1552","position":7,"is_corresponding":false},{"id":613793,"name":"Elif Erkan","orcid":"0009-0001-8514-4753","position":0,"is_corresponding":true}],"reference_count":0,"raw_metadata":null,"created_at":"2026-07-18T23:17:48.191325Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}