Abstract The primary route of internalization from the plasma membrane is clathrin-mediated endocytosis (CME), which is required for vital cellular processes such as signaling, nutrient uptake, and development. While the molecular mechanisms underlying the late stages of vesicle formation are well studied, the mechanisms underpinning the earliest stages of endocytosis, including initiation and cargo selection, are poorly understood. Initiation and cargo binding are largely controlled by the AP2 clathrin adaptor complex, a core component of the vesicle coat that serves as a bridge between the rigid clathrin lattice and membrane-embedded cargo. However, it is unclear how AP2 discriminates between hundreds of potential cargo in a complex membrane environment, while also responding to spatial and temporal regulatory cues. While >50 proteins are proposed to regulate or be required for CME, and many physically interact with AP2, we have little mechanistic and structural data for how they are regulated during the earliest stages of endocytosis. We propose that multiple unique AP2 conformations, driven by association with regulatory factors, control higher-order AP2 functions such as cargo selection. Understanding the nature of the regulatory mechanisms controlling endocytosis is critical, as CME largely controls the localization of many medically-relevant proteins such as RTKs and GPCRs. This proposal therefore seeks to reconstitute and define the molecular mechanisms of AP2-mediated endocytic initiation and cargo sorting. Current models of endocytosis largely rely on biochemical experiments performed with soluble components and live cell imaging. Importantly, our methodology is focused on modifying all experimental approaches — cryo-EM, biochemical reconstitution, and single molecule fluorescence microscopy — to include a membrane, thereby addressing a critical need to develop mechanistic models in a near-native membrane environment. This approach is poised to provide an understanding of the role of membrane-induced allostery in driving regulatory decision-making during endocytic initiation. This proposal will focus on two broad areas of endocytic regulation — cargo selection mediated by the conserved Muniscin family proteins, and a quality control checkpoint controlled by a single phosphorylation mark on the μ2 subunit of AP2. As diseases associated with endocytic defects are likely caused by missorting of important trans-membrane cargo, our insights into the mechanisms of endocytic initiation and cargo selection will enable hypothesis-driven research into disease model systems and drug development.