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␥-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␥-p90 associates with both the and ␤2 subunits of AP-2 via multiple sites. Crystallographic data show that a peptide derived from the splice insert of the human PIPKI␥-p90 tail binds to a cognate recognition site on the sandwich subdomain of the ␤2 appendage. Partly overlapping aromatic and hydrophobic residues within the same peptide also can engage the C-terminal sorting signal binding domain of AP-2, 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␥-p90 with AP-2 involves both of these sites. Accordingly the ability of overexpressed PIPKI␥ tail to impair endocytosis of SVs in primary neurons largely depends on its association with AP-2␤ and AP-2. Our data also suggest that interactions between AP-2 and the tail domain of PIPKI␥-p90 may serve to regulate complex formation and enzymatic activity. We postulate a model according to which multiple interactions between PIPKI␥-p90 and AP-2 lead to spatiotemporally controlled PI(4,5)P 2 synthesis during clathrin-mediated SV endocytosis.Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) 5 plays a key role in a variety of cell physiological pathways including but not limited to CME, neurotransmitter release (31) and SV cycling, phagocytosis, cell signaling and proliferation, and regulation of the actin cytoskeleton, as well as nuclear functions (1-7). In mammals these diverse roles of PI(4,5)P 2 are regulated by only three type I phosphatidylinositol-4-phosphate 5-kinase (PIPK) isozymes. Although PIPKI␣ and -␤ are ubiquitous proteins, the type I␥ isozyme is most highly expressed in the brain where it localizes to synaptic sites (8). PIPKI␥ is also present at focal adhesion sites in non-neuronal cells (9, 10).An overwhelming body of data implicates PI(4,5)P 2 in CME and in the exo-/endocytic cycling of SV membranes (2-4). Recruitment of clathrin adaptors, including epsins, CALM/ AP180, Dab2, HIP1/1R, and AP-2, to the plasma membrane strictly requires the presence of PI(4,5)P 2 , which associates directly with cognate recognition motifs and domains within these proteins. AP-2 is a heterotetrameric complex composed of ␣, ␤2, 2, and 2 subunits that fold into a largely ␣-helical solenoid (termed the "core") with two appendage domains joined to the core by long flexible linkers (11). Membrane...