Type Iγ661 Phosphatidylinositol Phosphate Kinase Directly Interacts with AP2 and Regulates Endocytosis

Abstract

Clathrin-coated vesicles mediate sorting and intracellular transport of membrane-bound proteins. The formation of these coats is initiated by the assembly of adaptor proteins (AP), which specifically bind to membrane cargo proteins via recognition of endocytic sorting motifs. The lipid signaling molecule phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is critical for this process, as it serves as both a targeting and regulatory factor. PI(4,5)P2 is synthesized by type I phosphatidylinositol phosphate kinases (PIPKI). We have discovered a direct interaction between the μ2-subunit of the AP2 complex and PIPKIγ661 via a yeast two-hybrid screen. This interaction was confirmed using both the μ2-subunit in glutathione S-transferase pulldowns and via coimmunoprecipitation of endogenous PIPKIγ661 with the AP2 complex from HEK293 cells. The interaction is mediated, in vivo, by a tyrosine-based motif in the 26-amino acid tail of PIPKIγ661. Because AP2 regulates endocytosis of transferrin receptor from the plasma membrane, we also examined a role for PIPKIγ661 using a flow cytometry endocytosis assay. We observed that stable expression of wild type PIPKIγ661 in Madin-Darby canine kidney cells enhanced transferrin uptake, whereas stable expression of kinase-dead PIPKIγ661 had an inhibitory effect. Neither condition affected the overall cellular level of PI(4,5)P2. RNA interference-based knockdown of PIPKIγ661 in HeLa cells also had an inhibitory effect on transferrin endocytosis using the same assay system. Collectively, this evidence implies an important role for PIPKIγ661 in the AP2-mediated endocytosis of transferrin. Clathrin-coated vesicles mediate sorting and intracellular transport of membrane-bound proteins. The formation of these coats is initiated by the assembly of adaptor proteins (AP), which specifically bind to membrane cargo proteins via recognition of endocytic sorting motifs. The lipid signaling molecule phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is critical for this process, as it serves as both a targeting and regulatory factor. PI(4,5)P2 is synthesized by type I phosphatidylinositol phosphate kinases (PIPKI). We have discovered a direct interaction between the μ2-subunit of the AP2 complex and PIPKIγ661 via a yeast two-hybrid screen. This interaction was confirmed using both the μ2-subunit in glutathione S-transferase pulldowns and via coimmunoprecipitation of endogenous PIPKIγ661 with the AP2 complex from HEK293 cells. The interaction is mediated, in vivo, by a tyrosine-based motif in the 26-amino acid tail of PIPKIγ661. Because AP2 regulates endocytosis of transferrin receptor from the plasma membrane, we also examined a role for PIPKIγ661 using a flow cytometry endocytosis assay. We observed that stable expression of wild type PIPKIγ661 in Madin-Darby canine kidney cells enhanced transferrin uptake, whereas stable expression of kinase-dead PIPKIγ661 had an inhibitory effect. Neither condition affected the overall cellular level of PI(4,5)P2. RNA interference-based knockdown of PIPKIγ661 in HeLa cells also had an inhibitory effect on transferrin endocytosis using the same assay system. Collectively, this evidence implies an important role for PIPKIγ661 in the AP2-mediated endocytosis of transferrin. The surface expression level of plasma membrane receptors is highly regulated via balanced vesicular trafficking to and from the plasma membrane. An important aspect of this regulation is the internalization of receptors by the clathrin-mediated endocytosis pathway. This process not only internalizes and delivers extracellular ligands to the endosomal transport system, but it also serves to recycle receptors or target them for degradation. Assembly of the clathrin coat is a highly orchestrated process that requires the action of several distinct proteins (1.Brodsky F.M. Chen C.Y. Knuehl C. Towler M.C. Wakeham D.E. Annu. Rev. Cell Dev. Biol. 2001; 17: 517-568Crossref PubMed Scopus (540) Google Scholar), including G-proteins, protein and lipid kinases and phosphatases, and various adaptor proteins, such as the adaptin protein (AP) 3The abbreviations used are: AP, adaptor protein; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PIPKI, type I phosphatidylinositol phosphate kinases; MDCK, Madin-Darby canine kidney; RNAi, RNA interference; HA, hemagglutinin; siRNA, small interfering RNA; PBS, phosphate-buffered saline; GST, glutathione S-transferase; HPLC, high performance liquid chromatography; WT, wild type; KD, kinase-dead. complexes. These complexes specifically bind to integral membrane cargo proteins and serve both as protein docking sites and as nucleation points for assembly of the clathrin lattice. Several AP complexes have been identified, with each complex having a distinct function (2.Robinson M.S. Bonifacino J.S. Curr. Opin. Cell Biol. 2001; 13: 444-453Crossref PubMed Scopus (444) Google Scholar). For example, AP1B is thought to modulate sorting to the basolateral membrane in epithelial cells, whereas AP2 is thought to regulate receptor-mediated endocytosis from the plasma membrane (3.Folsch H. Ohno H. Bonifacino J.S. Mellman I. Cell. 1999; 99: 189-198Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar, 4.Gan Y. McGraw T.E. Rodriguez-Boulan E. Nat. Cell Biol. 2002; 4: 605-609Crossref PubMed Scopus (167) Google Scholar, 5.Kirchhausen T. Annu. Rev. Cell Dev. Biol. 1999; 15: 705-732Crossref PubMed Scopus (425) Google Scholar, 6.Folsch H. Pypaert M. Schu P. Mellman I. J. Cell Biol. 2001; 152: 595-606Crossref PubMed Scopus (213) Google Scholar). The AP complexes consist of four distinct subunits. The crystal structure of the AP2 complex has recently been solved and consists of two large subunits, α and β2, a medium subunit, μ2, and a small subunit, σ2 (7.Collins B.M. McCoy A.J. Kent H.M. Evans P.R. Owen D.J. Cell. 2002; 109: 523-535Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar). The μ2-subunit is critical for the function of the complex as it specifically binds to membrane cargo proteins via tyrosine-based sorting motifs within their cytoplasmic domains (8.Bonifacino J.S. Traub L.M. Annu. Rev. Biochem. 2003; 72: 395-447Crossref PubMed Scopus (1664) Google Scholar). This subunit consists of an N-terminal domain, which is buried within the core of the AP2 complex, joined by a flexible linker to a solvent exposed C-terminal domain. In the AP2 crystal, the μ2-subunit C-terminal domain was found to be buried within a pocket formed by the other three subunits of the complex (7.Collins B.M. McCoy A.J. Kent H.M. Evans P.R. Owen D.J. Cell. 2002; 109: 523-535Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar). In this “closed” conformation, the μ-subunit is thought be unable to interact with membrane-bound cargo proteins. The current model for the AP2 cargo binding suggests that the complex shifts into an “open” conformation upon binding to the plasma membrane in the presence of cargo (7.Collins B.M. McCoy A.J. Kent H.M. Evans P.R. Owen D.J. Cell. 2002; 109: 523-535Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar, 9.Kirchhausen T. Cell. 2002; 109: 413-416Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The displaced μ-subunit can then bind to cargo and dock to the membrane. Assembly of the AP2 complex onto membranes is mediated by a specific phospholipid, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) (10.Gaidarov I. Keen J.H. J. Cell Biol. 1999; 146: 755-764Crossref PubMed Scopus (234) Google Scholar). PI(4,5)P2 also regulates numerous proteins that participate in clathrin-mediated endocytosis, including AP180, epsin, and dynamin (11.Martin T.F. Curr. Opin. Cell Biol. 2001; 13: 493-499Crossref PubMed Scopus (328) Google Scholar). In addition, PI(4,5)P2 is an important regulatory factor for modulation of the actin cytoskeleton and subsequently vesicular transport (12.Qualmann B. Kessels M.M. Kelly R.B. J. Cell Biol. 2000; 150: F111-F116Crossref PubMed Scopus (357) Google Scholar, 13.Qualmann B. Kelly R.B. J. Cell Biol. 2000; 148: 1047-1062Crossref PubMed Scopus (253) Google Scholar). Two PI(4,5)P2 binding sites have been identified within the AP2 complex. One binding site is located on the and is to dock the AP2 complex to membranes T. Annu. Rev. Cell Dev. Biol. 1999; 15: 705-732Crossref PubMed Scopus (425) Google Scholar, B.M. McCoy A.J. Kent H.M. Evans P.R. Owen D.J. Cell. 2002; 109: 523-535Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar). The other binding site is located on the μ2-subunit and formed by a of J. Cell Biol. 2002; PubMed Scopus Google Scholar). The AP2 structure suggests that the μ2-subunit binds PI(4,5)P2 only AP complex is to the membrane and the μ2-subunit is to cargo (7.Collins B.M. McCoy A.J. Kent H.M. Evans P.R. Owen D.J. Cell. 2002; 109: 523-535Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar, T.F. Annu. Rev. Cell Dev. Biol. PubMed Scopus Google Scholar). This model has recently been using an in with surface M. B. M. C. Owen D.J. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). This of evidence the critical role for PI(4,5)P2 in both the targeting of AP2 to endocytic sites and for the interaction the plasma membrane upon of the complex into the PI(4,5)P2 is synthesized the plasma membrane by type I phosphatidylinositol phosphate kinases (PIPKI). PI(4,5)P2, important not only for regulation of vesicular but also have in and T.F. Annu. Rev. Cell Dev. Biol. PubMed Scopus Google Scholar, J.H. PubMed Scopus Google Scholar, Curr. Opin. Cell Biol. PubMed Scopus Google Scholar). three of the and using the have been in clathrin-mediated has that expression of in cells in endocytosis of the factor whereas expression of endocytosis J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). found that expression had a effect on transferrin receptor endocytosis in HeLa cells and that knockdown of expression had an inhibitory effect M. H. J. Cell Biol. 2003; PubMed Scopus Google Scholar). has been as the of PI(4,5)P2 in and this role has been by observed in endocytosis is M. T.F. P. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, M. P. PubMed Scopus Google Scholar). the by which specifically to sites of endocytosis for of PI(4,5)P2 The is in cells, in two and which only by a 26-amino acid C-terminal We have discovered a direct interaction between PIPKIγ661 and the μ2-subunit of AP2 via a yeast two-hybrid the C-terminal of PIPKIγ661 as we that this interaction is direct and in PIPKIγ661 and specifically expression of we have also found that this interaction has direct on the endocytosis of the transferrin receptor from the plasma membrane. These that PIPKIγ661 be an important regulatory factor in clathrin-mediated and and expression A.J. 2002; PubMed Scopus Google Scholar, A.J. J. Cell Biol. 2003; PubMed Scopus Google Scholar). C-terminal also A.J. J. Cell Biol. 2003; PubMed Scopus Google Scholar). The PIPKIγ661 using the and the and their and The was A.J. J. Cell Biol. 2003; PubMed Scopus Google Scholar). The μ2-subunit yeast two-hybrid was from a of of this subunit was by of the μ2-subunit with an site and an The was into and expression Cell and and Madin-Darby canine kidney cells using medium with HEK293 cells via phosphate with or of each expression The cells used for Cell cells with various PIPKIγ661 which the of the The cells in medium and to for stable The medium was with to as in expression of transferrin receptor was from was from was from and was from was from and and from was from was from was and as A.J. 2002; PubMed Scopus Google Scholar). was and as Chen J. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). from and with and subsequently and in and Cell with of protein and of the as The by or and to was used for on and in in or expression into cells and using the or using as the PIPKIγ661 was via with and as J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). was with with in of and for or The with of four by and via was used as a for other pulldowns with the proteins in the same cells in of was for by from For transferrin cells with medium for The cells then with transferrin in binding medium for the cells three with PBS, three with acid acid and and three with by and with of the cells used to expression by flow cells with and then with The of from cells and used to The other of the cells was in of for of transferrin using a flow of transferrin from cells. RNA cells in medium The cells into to The cells with a using or used as the cells in the same The cells then used for transferrin and of PI(4,5)P2 cells with and the and as PubMed Scopus Google Scholar). The in to by The using a and a of phosphate The level of cellular PI(4,5)P2 was with a liquid flow using as a and with with in for and with in for The cells with in for with the for and with in The cells then with for and with in The to in using a with a or using a on a to an M. for as using A.J. 2002; PubMed Scopus Google Scholar). the between AP2 and PIPKIγ661 cells in with of each expression of the cells in medium for and then with of transferrin for The cells subsequently and as PIPKIγ661 with the of AP2 in and in a yeast two-hybrid using the C-terminal of PIPKIγ661 as A.J. 2002; PubMed Scopus Google Scholar). This a direct with the μ2-subunit of The from a the μ2-subunit with the of the that this interaction was a was Because the μ2-subunit is in E. a was This was by of the of the N-terminal domain and it with The the linker domain and the C-terminal domain. The was then by PIPKIγ661 with in the presence of in PIPKIγ661 with but not with In vivo, AP2 serves as an adaptor for binding to several other proteins in clathrin-mediated to that this interaction was in vivo, we endogenous AP2 from HEK293 cells using a specific for The complexes and by a was in the but not in the A.J. 2002; PubMed Scopus Google Scholar). The using the specific for was to that only the was by This to as the both PIPKIγ661 and as in the Because PI(4,5)P2 has been to be an integral of the endocytic process, we to was for this interaction in type and PIPKIγ661 into HEK293 cells via AP2 was then using specific for the in both wild type and kinase-dead PIPKIγ661 with AP2 in In addition, PIPKIγ661 to with the AP2 complex with as with PIPKIγ661. The endogenous coimmunoprecipitation that AP2 with the of by the coimmunoprecipitation was used to which of the two with AP2 in HEK293 cells with PIPKIγ661 or and endogenous AP2 was with the in only PIPKIγ661 was of binding to AP2 in This that the in binding site for was in the C-terminal of PIPKIγ661. the specific binding three of PIPKIγ661 in the same coimmunoprecipitation A.J. J. Cell Biol. 2003; PubMed Scopus Google Scholar). in in of to observed in the on PIPKIγ661 the with of the between the and is a sorting motif These motifs consist of a sorting to a (8.Bonifacino J.S. Traub L.M. Annu. Rev. Biochem. 2003; 72: 395-447Crossref PubMed Scopus (1664) Google Scholar). an Ohno H. T. Bonifacino J.S. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, H. M.C. Bonifacino J.S. J. Biol. Full Text Full Text PDF PubMed Scopus Google has evidence the sorting motif for each of the of the AP complexes. these as a several with the of PIPKIγ661 binding to to binding was also as a of these was into HEK293 cells and endogenous AP2 was with a PIPKIγ661 was then by with an The observed in the by Ohno H. T. Bonifacino J.S. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). of or to the in of the The was to have to binding in the Ohno H. T. Bonifacino J.S. J. Biol. Full Text Full Text PDF PubMed Scopus Google and of to the had effect on AP2 of to the not PIPKIγ661 binding to These in confirmed via in was used to or The was with to in PIPKIγ661 was specifically by whereas of the with These that PIPKIγ661 a sorting motif that is by the μ2-subunit of AP2 and the direct interaction between these two proteins. by Ohno H. T. Bonifacino J.S. J. Biol. Full Text Full Text PDF PubMed Scopus Google has also that of the within sorting motifs the of such motifs with the We have that of PIPKIγ661 is by in a A.J. 2002; PubMed Scopus Google Scholar, A.J. J. Cell Biol. 2003; PubMed Scopus Google Scholar). of this the between PIPKIγ661 and the this we in pulldowns by with PIPKIγ661 or as J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). in PIPKIγ661 with as with PIPKIγ661. This is with the of an within the sorting motif and also serve as a regulatory for the interaction between AP2 and PIPKIγ661. PIPKIγ661 and AP2 in we had that PIPKIγ661 with the AP2 complex both in and in we examined a with and AP2 with specific for and in cells and examined by in is to the plasma membrane and to sites of in cells. This also with the plasma membrane of endogenous and can be observed not only the plasma membrane, but also within the by the in the This the plasma membrane was not as PIPKIγ661 has been to serve several the plasma membrane, including regulation of assembly via direct interaction with A.J. 2002; PubMed Scopus Google Scholar, J. P. 2002; PubMed Scopus Google Scholar). is important to that the in cells, and we have that the interaction with AP2 is specific for only the PIPKIγ661 the of the interaction between PIPKIγ661 and PIPKIγ661 and PIPKIγ661 in cells. three to the plasma membrane in a not upon of clathrin-mediated endocytosis via with distinct in both and PIPKIγ661 with AP2 in vesicular upon with transferrin. in cells PIPKIγ661 the the plasma membrane and was not These both the of the interaction in and in and also the observed in transferrin The interaction between PIPKIγ661 and AP2 be a to targeting of PIPKIγ661 to sites of endocytosis for of PI(4,5)P2. This be for recognition of cargo proteins by the μ2-subunit upon assembly of the AP2 complex onto the plasma membrane, PIPKIγ661 the cargo binding site with the vesicular cytoplasmic observed in and be PIPKIγ661 with AP2 the of the endocytic PIPKIγ661 is evidence that endocytosis from the plasma membrane mediated by the AP2 complex is a process (11.Martin T.F. Curr. Opin. Cell Biol. 2001; 13: 493-499Crossref PubMed Scopus (328) Google Scholar, J. Biol. 2001; PubMed Scopus Google Scholar). The observed endogenous and the direct interaction between the μ-subunit of AP2 and PIPKIγ661 that this interaction have on AP2 this we stable wild type or kinase-dead PIPKIγ661. expression was by of from the of the cells to an endocytosis assay via with transferrin and the of transferrin was then via flow The from these in and that stable expression of wild type PIPKIγ661 in an of to cells. The effect was observed in cells kinase-dead with a of that these on transferrin endocytosis to a specific interaction with we also stable wild type or kinase-dead in expression of wild type or kinase-dead the same had effect on transferrin In addition, also using an stable PIPKIγ661 with a not These with both in and in interaction which that only the PIPKIγ661 the motif is of direct interaction with the μ-subunit of the AP2 complex. by H. Pypaert M. P. J. Cell Biol. PubMed Scopus Google has also a between trafficking of the AP2 complex and the protein the binding site for on PIPKIγ661 with the motif for the direct interaction with AP2 A.J. 2002; PubMed Scopus Google Scholar, J. P. 2002; PubMed Scopus Google Scholar). these two distinct for we a stable the in this not binding to the AP2 complex in this the interaction between PIPKIγ661 and as observed by coimmunoprecipitation This was confirmed by in expression of wild type PIPKIγ661 in a distinct with the plasma membrane in cells. of PIPKIγ661 on the other with a to that observed upon expression of PIPKIγ661 In endocytosis in expression of the also had a effect in cells, but to a that of wild type PIPKIγ661. This be the of a of with for binding to PIPKIγ661. of this also of P. J. Cell Biol. PubMed Scopus Google have recently that both and of in of the not be to the effect of we have observed in PIPKIγ661 PI(4,5)P2 in in PI(4,5)P2 be to the observed we cellular PI(4,5)P2 via and The PIPKIγ661 and stable in the presence of and protein expression was for the same as in the transferrin The cellular and by The PI(4,5)P2 was identified using a PI(4,5)P2 and by using an flow of expression was by of cells in expression of or not have a effect on the cellular PI(4,5)P2 Because the PI(4,5)P2 not affected by or it is a modulation of highly of PI(4,5)P2 is for the observed on transferrin The stable level of cellular PI(4,5)P2 is also with for expression of PIPKIγ661 in other J. I. T. J. T. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). as a of was also as an for a role for PIPKIγ661 in transferrin receptor an specific for the we expression in HeLa cells. These cells then used in the same transferrin assay as with the PIPKIγ661 stable was used as a for transferrin an we with an specific for this in and transferrin was on by in cells with effect was observed with or with specific for The observed for knockdown of via was also with in HeLa cells J. I. T. J. T. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). The level of knockdown for each protein via is in in the expression level of the transferrin receptor also to the observed of transferrin endocytosis upon knockdown of this we the transferrin receptor expression level these by in knockdown of PIPKIγ661 not the overall expression level of the transferrin that the observed on endocytosis be a direct of expression of PI(4,5)P2 is as a regulatory factor for clathrin-mediated endocytosis (11.Martin T.F. Curr. Opin. Cell Biol. 2001; 13: 493-499Crossref PubMed Scopus (328) Google Scholar). only it serve to modulate the of several proteins for clathrin coat assembly and but it also as an important targeting The AP2 complex is an example, as it two PI(4,5)P2 binding with site located on the and the other on the μ2-subunit (7.Collins B.M. McCoy A.J. Kent H.M. Evans P.R. Owen D.J. Cell. 2002; 109: 523-535Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar, J. Cell Biol. 2002; PubMed Scopus Google Scholar). the specific for of PI(4,5)P2 sites of endocytosis we have that PIPKIγ661 with the μ2-subunit of the AP2 adaptor complex both in and in This direct interaction is on a in the PIPKIγ661 that to a sorting We have that of of the within this motif in of the interaction with the μ2-subunit both in and in The direct interaction between AP2 and PIPKIγ661 a for targeting PIPKIγ661 to sites of endocytosis the plasma membrane this in of a highly of PI(4,5)P2 these The structure for the AP2 core to a for regulation of this In the AP2 crystal, the μ2-subunit is buried in a formed by the and (7.Collins B.M. McCoy A.J. Kent H.M. Evans P.R. Owen D.J. Cell. 2002; 109: 523-535Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar). In this conformation, the μ2-subunit docking site is from the membrane docking site of the complex. has been that of the linker domain of a that the subunit to of the pocket into an conformation and bind to cargo motifs the plasma membrane (7.Collins B.M. McCoy A.J. Kent H.M. Evans P.R. Owen D.J. Cell. 2002; 109: 523-535Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar, T. 2003; 4: PubMed Scopus Google Scholar, J. Cell Biol. 2002; PubMed Scopus Google Scholar, J. Cell Biol. 2002; PubMed Scopus Google Scholar). This for enhanced AP2 membrane via a direct interaction between and PI(4,5)P2, which is not in the conformation M. B. M. C. Owen D.J. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). Because PIPKIγ661 binding the cargo binding PIPKIγ661 bind to AP2 in this docking to the plasma membrane, PIPKIγ661 be displaced from by a in by of sorting motifs of This also be by of PIPKIγ661. Ohno H. M.C. Bonifacino J.S. J. Biol. Full Text Full Text PDF PubMed Scopus Google has that of the within the motif the interaction with in cargo binding H. M.C. Bonifacino J.S. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). We have that of PIPKIγ661 is by A.J. J. Cell Biol. 2003; PubMed Scopus Google Scholar). We have also that of PIPKIγ661 the with the μ2-subunit in several receptors upon binding to extracellular ligands J.S. Annu. Rev. Cell Dev. Biol. 13: PubMed Scopus Google Scholar). PIPKIγ661 and from AP2 upon targeting to receptors or to sites be of on PIPKIγ661 serve as an important regulatory for the interaction between PIPKIγ661 and AP2 the plasma membrane. This for targeting of PIPKIγ661 to endocytic sites is highly of the model by for targeting of PIPKIγ661 to targeting to via direct interaction with PIPKIγ661 be displaced from by A.J. J. Cell Biol. 2003; PubMed Scopus Google Scholar). both a is to the plasma membrane and then displaced from binding a highly of PI(4,5)P2 in a regulated targeting also PIPKIγ661 to endocytic sites in a The small factor has been to PIPKIγ661 endocytic sites and of AP2 to the plasma membrane M. M. P. J. Cell Biol. 2003; PubMed Scopus Google Scholar). factor has also been to with the AP2 complex in coimmunoprecipitation E. M. P. T. M. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). PIPKIγ661 serve as the factor factor to the AP2 complex, AP2 assembly via enhanced PI(4,5)P2 by PIPKIγ661. is not that several have various type I to clathrin-mediated J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google has in a that expression of wild type enhanced endocytosis of factor whereas expression of a kinase-dead had an inhibitory effect in cells. M. H. J. Cell Biol. 2003; PubMed Scopus Google recently that expression or knockdown of had a on transferrin endocytosis, whereas expression or knockdown of or had effect in and HeLa cells. The that these observed the of large of cellular PI(4,5)P2 upon modulation of for the of knockdown with this as we also observed in transferrin endocytosis these in HeLa cells. We also observed in PI(4,5)P2 in cells wild type or kinase-dead PIPKIγ661 or not with the by M. H. J. Cell Biol. 2003; PubMed Scopus Google Scholar), both for the endocytosis of transferrin receptor and cellular PI(4,5)P2 This be to the in the used or the that stable whereas M. H. J. Cell Biol. 2003; PubMed Scopus Google used a for a role for in transferrin receptor of via a of PI(4,5)P2 in the in the cells or which of the mediated by expression of the that the direct interaction between AP2 and PIPKIγ661 in modulation of highly of PI(4,5)P2, of the overall cellular level of PI(4,5)P2 in the of or knockdown protein expression we have discovered that the subunit of AP1B with PIPKIγ661 and this subunit binds specifically to PIPKIγ661 in not and The and both for sorting and this has been H. T. Bonifacino J.S. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). We have observed that of the motif within PIPKIγ661 to has on with the in not with these for have also been to have in both trafficking and M. P. PubMed Scopus Google Scholar, E. M. P. PubMed Scopus Google Scholar). Collectively, these that PIPKIγ661 have in both in endocytosis and via direct with AP2 and complexes. An of recently evidence has to the that AP2 in cargo as the for assembly Dev. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). is that this direct interaction with PIPKIγ661 only have on the of by AP2 transferrin the role for PIPKIγ661 in endocytosis be or the direct interaction between AP2 and PIPKIγ661 an for and of PI(4,5)P2 sites of AP2-mediated of this interaction is to a of clathrin-mediated endocytosis is regulated in cells. We and J. the of for and We also for in of several