title: Clathrin regulates the association of PIPKIgamma661 with the AP-2 adaptor beta2 appendage.
abstract: The AP-2 clathrin adaptor differs fundamentally from the related AP-1, AP-3, and AP-4 sorting complexes because membrane deposition does not depend directly on an Arf family GTPase. Instead phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) appears to act as the principal compartmental cue for AP-2 placement at the plasma membrane as well as for the docking of numerous other important clathrin coat components at the nascent bud site. This PtdIns(4,5)P(2) dependence makes type I phosphatidylinositol 4-phosphate 5-kinases (PIPKIs) lynchpin enzymes in the assembly of clathrin-coated structures at the cell surface. PIPKIgamma is the chief 5-kinase at nerve terminals, and here we show that the 26-amino acid, alternatively spliced C terminus of PIPKIgamma661 is an intrinsically unstructured polypeptide that binds directly to the sandwich subdomain of the AP-2 beta2 subunit appendage. An aromatic side chain-based, extended interaction motif that also includes the two bulky C-terminal residues of the short PIPKIgamma635 variant is necessary for beta2 appendage engagement. The clathrin heavy chain accesses the same contact surface on the AP-2 beta2 appendage, but because of additional clathrin binding sites located within the unstructured hinge segment of the beta2 subunit, clathrin binds the beta2 chain with a higher apparent affinity than PIPKIgamma661. A clathrin-regulated interaction with AP-2 could allow PIPKIgamma661 to be strategically positioned for regional PtdIns(4,5)P(2) generation during clathrin-coated vesicle assembly at the synapse.
authors.affiliation: Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261.
authors.forename: James R
authors.initials: JR
authors.lastname: Thieman
authors.affiliation: Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261.
authors.forename: Sanjay K
authors.initials: SK
authors.lastname: Mishra
authors.affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905.
authors.forename: Kun
authors.initials: K
authors.lastname: Ling
authors.affiliation: Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: Balraj
authors.initials: B
authors.lastname: Doray
authors.affiliation: Department of Pharmacology, University of Wisconsin School of Medicine, Madison, Wisconsin 53706.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Anderson
authors.affiliation: Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261. Electronic address: traub@pitt.edu.
authors.forename: Linton M
authors.initials: LM
authors.lastname: Traub
mesh_terms: D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism; D033962:Adaptor Protein Complex 2 / Q000235:genetics / Q000378:metabolism*; D017398:Alternative Splicing / Q000502:physiology; D000818:Animals; D002966:Clathrin / Q000235:genetics / Q000378:metabolism*; D022163:Clathrin-Coated Vesicles / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D019269:Phosphatidylinositol 4,5-Diphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000502:physiology; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D051381:Rats; D013574:Synaptosomes / Q000378:metabolism*
chemical_list: D033962:Adaptor Protein Complex 2; D002966:Clathrin; D019269:Phosphatidylinositol 4,5-Diphosphate; D021122:Protein Subunits; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase; D020727:ADP-Ribosylation Factors