title: Molecular basis for association of PIPKI gamma-p90 with clathrin adaptor AP-2.
abstract: Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) is an essential determinant in clathrin-mediated endocytosis (CME). In mammals three type I phosphatidylinositol-4-phosphate 5-kinase (PIPK) enzymes are expressed, with the I gamma-p90 isoform being highly expressed in the brain where it regulates synaptic vesicle (SV) exo-/endocytosis at nerve terminals. How precisely PI(4,5)P(2) metabolism is controlled spatially and temporally is still uncertain, but recent data indicate that direct interactions between type I PIPK and components of the endocytic machinery, in particular the AP-2 adaptor complex, are involved. Here we demonstrated that PIPKI gamma-p90 associates with both the mu and beta2 subunits of AP-2 via multiple sites. Crystallographic data show that a peptide derived from the splice insert of the human PIPKI gamma-p90 tail binds to a cognate recognition site on the sandwich subdomain of the beta2 appendage. Partly overlapping aromatic and hydrophobic residues within the same peptide also can engage the C-terminal sorting signal binding domain of AP-2mu, thereby potentially competing with the sorting of conventional YXXØ motif-containing cargo. Biochemical and structure-based mutagenesis analysis revealed that association of the tail domain of PIPKI gamma-p90 with AP-2 involves both of these sites. Accordingly the ability of overexpressed PIPKI gamma tail to impair endocytosis of SVs in primary neurons largely depends on its association with AP-2 beta and AP-2mu. Our data also suggest that interactions between AP-2 and the tail domain of PIPKI gamma-p90 may serve to regulate complex formation and enzymatic activity. We postulate a model according to which multiple interactions between PIPKI gamma-p90 and AP-2 lead to spatiotemporally controlled PI(4,5)P(2) synthesis during clathrin-mediated SV endocytosis.
authors.affiliation: Institute of Chemistry and Biochemistry, Department of Membrane Biochemistry, Freie Universität Berlin, 14195 Berlin, Germany.
authors.forename: Nina
authors.initials: N
authors.lastname: Kahlfeldt
authors.forename: Ardeschir
authors.initials: A
authors.lastname: Vahedi-Faridi
authors.forename: Seong Joo
authors.initials: SJ
authors.lastname: Koo
authors.forename: Johannes G
authors.initials: JG
authors.lastname: Schäfer
authors.forename: Georg
authors.initials: G
authors.lastname: Krainer
authors.forename: Sandro
authors.initials: S
authors.lastname: Keller
authors.forename: Wolfram
authors.initials: W
authors.lastname: Saenger
authors.forename: Michael
authors.initials: M
authors.lastname: Krauss
authors.forename: Volker
authors.initials: V
authors.lastname: Haucke
mesh_terms: D033962:Adaptor Protein Complex 2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002151:Calorimetry; D002966:Clathrin / Q000378:metabolism; D003461:Crystallography; D004705:Endocytosis / Q000502:physiology*; D004789:Enzyme Activation / Q000502:physiology; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007536:Isomerism; D016296:Mutagenesis; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs / Q000502:physiology; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar
chemical_list: D033962:Adaptor Protein Complex 2; D002966:Clathrin; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase