Institute of Chemistry and Biochemistry, Department of Membrane Biochemistry, Freie Universität Berlin, 14195 Berlin, Germany.
J Biol Chem. 2010 Jan 22;285(4):2734-49. doi: 10.1074/jbc.M109.074906. Epub 2009 Nov 10.
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.
磷脂酰肌醇 4,5-二磷酸（PI(4,5)P(2)）是网格蛋白介导的胞吞作用（CME）的重要决定因素。在哺乳动物中，表达了三种类型 I 磷脂酰肌醇-4-磷酸 5-激酶（PIPK）酶，其中 Iγ-p90 同工型在大脑中高度表达，在那里它调节神经末梢处的突触小泡（SV）外/内吞作用。PI(4,5)P(2)代谢如何在空间和时间上精确控制仍不确定，但最近的数据表明，I 型 PIPK 与内吞作用机制的成分之间的直接相互作用，特别是 AP-2 衔接复合物，参与其中。在这里，我们证明 PIPKIγ-p90 通过多个位点与 AP-2 的μ和β2 亚基结合。晶体学数据显示，来自人 PIPKIγ-p90 尾巴拼接插入的肽结合到β2 附属物夹心亚域的同源识别位点。同一肽中的部分重叠芳香族和疏水性残基也可以与 AP-2μ的 C 端分选信号结合域结合，从而可能与包含常规 YXXØ 基序的货物的分选竞争。生化和基于结构的诱变分析表明，PIPKIγ-p90 尾巴与 AP-2 的结合涉及这两个位点。因此，过表达的 PIPKIγ 尾巴干扰原代神经元中 SV 内吞的能力在很大程度上取决于它与 AP-2β和 AP-2μ的结合。我们的数据还表明，AP-2 和 PIPKIγ-p90 尾巴域之间的相互作用可能有助于调节复合物的形成和酶活性。我们提出了一个模型，根据该模型，PIPKIγ-p90 与 AP-2 之间的多种相互作用导致网格蛋白介导的 SV 内吞作用过程中时空控制的 PI(4,5)P(2)合成。
