Eukaryotic cells use clathrin mediated endocytosis (CME) to internalize transmembrane protein cargo. CME is orchestrated by the clathrin adaptor protein 2 (AP2), which couples the structural coat protein clathrin to the plasma membrane and endocytic cargo. Prior to this work, it was known that AP2 must undergo a conformational change to bind cargo and clathrin, yet how this activation occurred was unclear. Apart from the core components of endocytosis (membrane, cargo, and clathrin) no other factors have been shown to modulate the conformation of AP2. My work expands our understanding of how cells tune AP2 activity at endocytic sites through the discovery of two new mechanisms 1) direct inactivation of phosphorylated AP2 complexes and 2) an endocytic checkpoint that ensures cargo incorporation.Our lab previously identified NECAPs as candidate inactivators of AP2. Here we developed methods to reconstitute AP2 conformational changes in vitro and show that adaptin ear-binding coat-associated proteins (NECAPs) directly inactivate AP2 complexes. This requires the enigmatic AP2 µ2 T156 phosphorylation, implicating AP2 phosphorylation in AP2 inactivation. We determined a cryo-EM structure of AP2-NECAP, revealing a ‘clamp’-like mechanism by which NECAP inactivated AP2 complexes. Mutations from a C. elegans genetic screen for the AP2-NECAP interface are explained by the structure using vertebrate proteins, showing that inactivation of phosphorylated AP2 is a highly conserved pathway in AP2 regulation We then characterized the AP2 activator domain (APA) found in the muniscin family of proteins. Muniscins are membrane-binding proteins that arrive early to endocytic sites, localized to the rim of growing endocytic pits. We found that the APA directly alters the conformation of membrane-bound AP2. Cryo-EM reveals that this results in a novel conformation of AP2, which we call primed. APA binding occurs prior to cargo binding, as addition of peptides containing the canonical YxxΦ cargo motif releases the APA and rearranges AP2 into the conformation adopted in vesicles. We propose that AP2 priming serves as a cargo checkpoint, where muniscins concentrate AP2 at the rim of endocytic vesicles until cargo is bound, at which point the AP2 is released into the growing bud.