title: Regulation of clathrin-mediated endocytosis by hierarchical allosteric activation of AP2.
abstract: The critical initiation phase of clathrin-mediated endocytosis (CME) determines where and when endocytosis occurs. Heterotetrameric adaptor protein 2 (AP2) complexes, which initiate clathrin-coated pit (CCP) assembly, are activated by conformational changes in response to phosphatidylinositol-4,5-bisphosphate (PIP2) and cargo binding at multiple sites. However, the functional hierarchy of interactions and how these conformational changes relate to distinct steps in CCP formation in living cells remains unknown. We used quantitative live-cell analyses to measure discrete early stages of CME and show how sequential, allosterically regulated conformational changes activate AP2 to drive both nucleation and subsequent stabilization of nascent CCPs. Our data establish that cargoes containing Yxxφ motif, but not dileucine motif, play a critical role in the earliest stages of AP2 activation and CCP nucleation. Interestingly, these cargo and PIP2 interactions are not conserved in yeast. Thus, we speculate that AP2 has evolved as a key regulatory node to coordinate CCP formation and cargo sorting and ensure high spatial and temporal regulation of CME.
authors.affiliation: Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390.
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Kadlecova
authors.affiliation: Institute for Genomics and Evolutionary Medicine, Temple University, Philadelphia, PA 19122.
authors.forename: Stephanie J
authors.initials: SJ
authors.lastname: Spielman
authors.affiliation: Department of Physics and Astronomy, University of Denver, Denver, CO 80208.
authors.forename: Dinah
authors.initials: D
authors.lastname: Loerke
authors.affiliation: Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390.
authors.forename: Aparna
authors.initials: A
authors.lastname: Mohanakrishnan
authors.affiliation: Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390.
authors.forename: Dana Kim
authors.identifier: http://orcid.org/0000-0002-3980-4239
authors.initials: DK
authors.lastname: Reed
authors.affiliation: Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390 sandra.schmid@utsouthwestern.edu.
authors.forename: Sandra L
authors.identifier: http://orcid.org/0000-0002-1690-7024
authors.initials: SL
authors.lastname: Schmid
mesh_terms: D033962:Adaptor Protein Complex 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D033965:Adaptor Protein Complex alpha Subunits / Q000235:genetics / Q000378:metabolism; D033983:Adaptor Protein Complex mu Subunits / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D002460:Cell Line; D002966:Clathrin / Q000378:metabolism*; D022163:Clathrin-Coated Vesicles / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D004705:Endocytosis; D006801:Humans; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D055550:Protein Stability; D021381:Protein Transport; D034622:RNA Interference; D055213:Retinal Pigment Epithelium / Q000378:metabolism*; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013997:Time Factors; D014162:Transfection
chemical_list: D033962:Adaptor Protein Complex 2; D033965:Adaptor Protein Complex alpha Subunits; D033983:Adaptor Protein Complex mu Subunits; D002966:Clathrin; D019269:Phosphatidylinositol 4,5-Diphosphate; C439124:adaptor protein complex 2, alpha 2 subunit; C438885:adaptor protein complex 2, mu 1 subunit; C451304:AAK1 protein, human; D017346:Protein Serine-Threonine Kinases