Inhibition of clathrin‐mediated endocytosis by knockdown of AP‐2 leads to alterations in the plasma membrane proteome

In eukaryotic cells, clathrin‐mediated endocytosis (CME) is a central pathway for the internalization of proteins from the cell surface, thereby contributing to the maintenance of the plasma membrane protein composition. A key component for the formation of endocytic clathrin‐coated vesicles (CCVs) is AP‐2, as it sequesters cargo membrane proteins, recruits a multitude of other endocytic factors and initiates clathrin polymerization. Here, we inhibited CME by depletion of AP‐2 and explored the consequences for the plasma membrane proteome. Quantitative analysis revealed accumulation of major constituents of the endosomal‐lysosomal system reflecting a block in retrieval by compensatory CME. The noticeable enrichment of integrins and blockage of their turnover resulted in severely impaired cell migration. Rare proteins such as the anti‐cancer drug target CA9 and tumor markers (CD73, CD164, CD302) were significantly enriched. The AP‐2 knockdown attenuated the global endocytic capacity, but clathrin‐independent entry pathways were still operating, as indicated by persistent internalization of specific membrane‐spanning and GPI‐anchored receptors (PVR, IGF1R, CD55, TNAP). We hypothesize that blocking AP‐2 function and thus inhibiting CME may be a novel approach to identify new druggable targets, or to increase their residence time at the plasma membrane, thereby increasing the probability for efficient therapeutic intervention. Knockdown of the endocytic clathrin adaptor AP‐2 μ‐subunit was used to inhibit clathrin‐mediated endocytosis (CME). As a result, the plasma membrane proteome was fundamentally altered, including strong accumulation of endo‐lysosomal proteins, tumor markers and other clinically relevant transmembrane proteins. Inspection of individual proteins showed that the blockage of CME affected a subset of GPI‐anchored proteins and integrins, the latter leading to a strong defect in cell migration.