EHD2 and the Novel EH Domain Binding Protein EHBP1 Couple Endocytosis to the Actin Cytoskeleton

Abstract

Here we identified two novel proteins denoted EH domain protein 2 (EHD2) and EHD2-binding protein 1 (EHBP1) that link clathrin-mediated endocytosis to the actin cytoskeleton. EHD2 contains an N-terminal P-loop and a C-terminal EH domain that interacts with NPF repeats in EHBP1. Disruption of EHD2 or EHBP1 function by small interfering RNA-mediated gene silencing inhibits endocytosis of transferrin into EEA1-positive endosomes as well as GLUT4 endocytosis into cultured adipocytes. EHD2 localizes with cortical actin filaments, whereas EHBP1 contains a putative actin-binding calponin homology domain. High expression of EHD2 or EHBP1 in intact cells mediates extensive actin reorganization. Thus EHD2 appears to connect endocytosis to the actin cytoskeleton through interactions of its N-terminal domain with membranes and its C-terminal EH domain with the novel EHBP1 protein. Here we identified two novel proteins denoted EH domain protein 2 (EHD2) and EHD2-binding protein 1 (EHBP1) that link clathrin-mediated endocytosis to the actin cytoskeleton. EHD2 contains an N-terminal P-loop and a C-terminal EH domain that interacts with NPF repeats in EHBP1. Disruption of EHD2 or EHBP1 function by small interfering RNA-mediated gene silencing inhibits endocytosis of transferrin into EEA1-positive endosomes as well as GLUT4 endocytosis into cultured adipocytes. EHD2 localizes with cortical actin filaments, whereas EHBP1 contains a putative actin-binding calponin homology domain. High expression of EHD2 or EHBP1 in intact cells mediates extensive actin reorganization. Thus EHD2 appears to connect endocytosis to the actin cytoskeleton through interactions of its N-terminal domain with membranes and its C-terminal EH domain with the novel EHBP1 protein. Components of the clathrin-dependent process whereby receptors and transporters are removed from the cell surface by endocytosis into intracellular vesicles have been well characterized (1Takei K. Haucke V. Trends Cell Biol. 2001; 11: 385-391Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). Following coating, invagination, and scission of internalized membrane, early endosomes containing the GTPase Rab5 and the endosomal protein EEA1 are formed, directing cargo to various destinations including the recycling endosome for transit back to the plasma membrane or to late endosomes and lysosomes where degradation of proteins occurs (2Woodman P.G. Traffic. 2000; 1: 695-701Crossref PubMed Scopus (84) Google Scholar). Although many of these steps have been studied in detail, a major gap in understanding endocytosis relates to the role of the actin cytoskeleton. In yeast, genetic analysis clearly shows a requirement of the actin cytoskeleton for endocytosis (3Geli M.I. Riezman H. J. Cell Sci. 1998; 111: 1031-1037Crossref PubMed Google Scholar, 4Munn A.L. Biochim. Biophys. Acta. 2001; 1535: 236-257Crossref PubMed Scopus (105) Google Scholar). This connection has been further strengthened by the identification of proteins in the Saccharomyces cerevisiae endocytic pathway that also bind actin or indirectly regulate actin. One of these proteins, Sla2p (5Yang S. Cope M.J. Drubin D.G. Mol. Biol. Cell. 1999; 10: 2265-2283Crossref PubMed Scopus (84) Google Scholar), binds actin directly through a talin-like domain, whereas two others, Pan1p (6Duncan M.C. Cope M.J. Goode B.L. Wendland B. Drubin D.G. Nat. Cell Biol. 2001; 3: 687-690Crossref PubMed Scopus (139) Google Scholar) and Abp1p (7Goode B.L. Rodal A.A. Barnes G. Drubin D.G. J. Cell Biol. 2001; 153: 627-634Crossref PubMed Scopus (164) Google Scholar), are activators of actin assembly nucleated by Arp2/3 complex. Genetic confirmation of the functional significance of these interactions provides strong support for the concept that actin polymerization can be coupled to the endocytic machinery, generating forces necessary for endocytosis. In mammalian cells the link between endocytosis and actin dynamics is less clear. Introducing actin-perturbing drugs or mutant forms of the Rho family of small GTPases disrupts endocytosis in some cells but not in all cases (8Gottlieb T.A. Ivanov I.E. Adesnik M. Sabatini D.D. J. Cell Biol. 1993; 120: 695-710Crossref PubMed Scopus (369) Google Scholar, 9Jackman M.R. Shurety W. Ellis J.A. Luzio J.P. J. Cell Sci. 1994; 107: 2547-2556Crossref PubMed Google Scholar, 10Lamaze C. Fujimoto L.M. Yin H.L. Schmid S.L. J. Biol. Chem. 1997; 272: 20332-20335Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 11Fujimoto L.M. Roth R. Heuser J.E. Schmid S.L. Traffic. 2000; 1: 161-171Crossref PubMed Scopus (321) Google Scholar). However, analogous to studies in yeast, the case for a role of the actin cytoskeleton in mammalian endocytosis has been strengthened recently by identification of proteins that modulate actin dynamics and also interact with endocytic components (12Schafer D.A. Curr. Opin. Cell Biol. 2002; 14: 76-81Crossref PubMed Scopus (238) Google Scholar, 13Jeng R.L. Welch M.D. Curr. Biol. 2001; 11: R691-R694Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). These include mouse Abp1 (7Goode B.L. Rodal A.A. Barnes G. Drubin D.G. J. Cell Biol. 2001; 153: 627-634Crossref PubMed Scopus (164) Google Scholar) and mHip1R (14Engqvist-Goldstein A.E.Y. Kessels M.M. Chopra V.S. Hayden M.R. Drubin D.C. J. Cell Biol. 1999; 147: 1503-1518Crossref PubMed Scopus (177) Google Scholar), a protein closely related to the human Huntington interacting protein Hip1, and the yeast Sla2p, which bind actin directly. Two other proteins, intersectin (15Hussain N.K. Jenna S. Glogauer M. Quinn C.C. Wasiak S. Guipponi M. Antonarakis S.E. Kay B.K. Stossel T.P. Lamarche-Vane N. McPherson P.S. Nat. Cell Biol. 2001; 3: 927-932Crossref PubMed Scopus (300) Google Scholar) and syndapin (16Qualmann B. Kelly R.B. J. Cell Biol. 2000; 148: 1047-1062Crossref PubMed Scopus (251) Google Scholar), bind N-WASP, which in turn induces actin polymerization via Arp2/3. All of these proteins also bind components of the endocytic machinery such as clathrin-associated proteins or dynamin and thus could coordinately regulate actin assembly and trafficking events. However, potential roles played by actin filaments of endocytosis and the of the of endocytic components and the actin cytoskeleton to be the recycling of GLUT4 proteins in cultured as a we two novel proteins, EHD2 EH domain protein mouse EH domain protein domain, homology small interfering EH domain protein mouse EH domain protein domain, homology small interfering and that to connect actin dynamics with endocytosis. In of GLUT4 into the plasma membrane in to is by its by the clathrin-dependent pathway S. Heuser J. J. Cell Biol. PubMed Scopus Google Scholar, J.E. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). EHD2 is in membranes that some of the intracellular its EH domain, which is to in many proteins in EHD2 binds NPF in EHBP1. EHD2 and EHBP1 also connect to the cytoskeleton through an in the and through a domain in the High expression of EHD2 or EHBP1 endocytosis of GLUT4 in cultured and transferrin in of the EHD2 or EHBP1 by gene silencing inhibits transferrin that these interacting proteins function an early of endocytosis the plasma membrane by functional to the actin cytoskeleton. in a protein as an and the from by with a protein containing of EHBP1 from a EHBP1 In to the of and analysis from of cells or or or but not the or proteins, and protein the and mouse in and in from and a from of from as 2002; PubMed Scopus Google Scholar). and from from to and in analysis from mouse EHD2 from and and the to the in and by and the and with a or by and the and with a two and human EHBP1 from and from of of and and the two to the EHBP1 the EHBP1 by and the and with a or a to or and in or of EHD2 or EHBP1 as proteins in EHBP1 and the NPF from EHBP1 by generating and the and with a has been M. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). as D.C. J. W. K. R. S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). All the for to Cell and of to and as S.E. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). by as M. Sci. S. PubMed Scopus Google Scholar). cells and to for for and with the with of in with of or of cortical actin by expression of the in with of the the of EHD2 and from to the of of or of of the into as M. Sci. S. PubMed Scopus Google Scholar). from with in and in of the cells from with which to the cells by a of with a gene the of and a for we that with into cells and all cells of M. Sci. S. PubMed Scopus Google Scholar). the cells in and to for as of these cells for and actin by and cell a M. S. R. J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus (105) Google Scholar). the plasma membrane of by through by the for and the by the for from the a to the These have been characterized in J. PubMed Google Scholar). and of vesicles as M. S. R. J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus (105) Google Scholar). proteins by and by and the by of analysis as M. S. R. J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus (105) Google Scholar). the of EHD2 in as and of in and to EHD2 by in a to the of by but not not EHBP1 protein by with from and mouse as 2002; PubMed Scopus Google Scholar, S.E. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). of a and to a to to mouse by with and to the membrane an and transferrin cells the with or and the cells in 2 for 2 in the of for cells with and actin and with in and with and for with for 2 and with and for in cells with as Following with for with and for 1 with mouse cells with and the by the cells cells and with to cells the proteins by with and with an with and Cell cell cells with and cells for 2 and for with with by a with as D.C. J. W. K. R. S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). to a and the to a with a of of and to of by of in less 1 for in for from the of the cell by into which into a the cells and with and in to of and of early endosomes and of by not in the cell and and analysis as 2002; PubMed Scopus Google Scholar). with with as a with all and to and the cell and as 2002; PubMed Scopus Google Scholar). of and actin plasma membrane, from as S.E. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). membrane with and with by and with in for Scopus Google Scholar), which the of the for has been to the surface of the to the of or the for a and of proteins the to and the proteins by with and of EHBP1 proteins from as 1993; PubMed Scopus Google Scholar). other proteins from to the of EHD2 in from a of proteins that be in the trafficking of a membrane from to be in GLUT4 M. S. R. J. J. Biol. 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In to EHD2 a role in GLUT4 from the cell surface to the we the EHD2 and as These into cultured with a GLUT4 its to and with a in its M. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). In or and or or for of GLUT4 to the cell surface in to of the EHD2 could be the by of cell by the of These that the expression of EHD2 proteins not with GLUT4 in cultured adipocytes. In to plasma membrane back to the cells to with to and the of a of the cell with in the and with N. M. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In expression of the EHD2 a with of the cells the or in vesicles the cell surface These are with the that EHD2 function is for endocytic trafficking between plasma membrane and early and of an EHD2-binding EHD2 in endocytosis to proteins that interact with its EH domain. of with with or domain, and the proteins by and protein with a of with the domain but not with of by of two from and with from the human gene novel protein for EH domain protein the human gene by two with the of EHBP1 from and These two to the EHBP1 EHBP1 contains a protein of EHBP1 contains with to the two identified by EHBP1 contains a is its and in its N-terminal closely NPF NPF are to bind EH Kay B.K. M. Cell 1999; PubMed Scopus Google Scholar, M. Kay B.K. M. Nat. Biol. 2000; 11: Scopus Google Scholar) and have been identified in many proteins that a role in the endocytic process Kay B.K. M. Cell 1999; PubMed Scopus Google Scholar, S. 2000; Scopus Google Scholar). the potential of these NPF with the EH domain of of plasma membranes and with or with a protein containing the NPF of EHBP1 in EHD2 to bind to but not to This is by the EH domain of and but not bind NPF from EHBP1 These are with a of EHBP1 and EHD2 in by NPF and EH domain. of cells by that EHBP1 protein is with and plasma membrane with these EHD2 with EHBP1 in membranes the cell and EHD2 and EHBP1 to that are the of the cell of of EHD2 and EHBP1 occurs as the is of the between and in these cells with and cortical actin cytoskeleton in these membrane appears to be in with EHD2 and as in In an to the functional of to cortical actin of of of domain of EHD2 or of EHBP1 a of the cortical actin expression of or cortical actin in adipocytes. these that cortical assembly between these two novel proteins in adipocytes. we could not in transferrin in cells However, be that actin-perturbing drugs endocytosis in some cells but not in all cases (8Gottlieb T.A. Ivanov I.E. Adesnik M. Sabatini D.D. J. Cell Biol. 1993; 120: 695-710Crossref PubMed Scopus (369) Google Scholar, 9Jackman M.R. Shurety W. Ellis J.A. Luzio J.P. J. Cell Sci. 1994; 107: 2547-2556Crossref PubMed Google Scholar, 10Lamaze C. Fujimoto L.M. Yin H.L. Schmid S.L. J. Biol. Chem. 1997; 272: 20332-20335Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 11Fujimoto L.M. Roth R. Heuser J.E. Schmid S.L. Traffic. 2000; 1: 161-171Crossref PubMed Scopus (321) Google Scholar). of EHBP1 with EHD2 to EHBP1 also a role in endocytosis. to in to endocytosis and of transferrin to the of cells of EHBP1. and of the transferrin in the of these cells in whereas expression of these that of cells strong transferrin the whereas of cells of also to the expression of the of transferrin with in cells could be not the NPF in EHBP1 are a with to calponin homology which have been to bind and in proteins M. K. 2002; PubMed Scopus Google Scholar, M.J. J. 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This domain of EHBP1 a function that the process of endocytosis through EHD2 as well as membranes via the with the actin cytoskeleton through its domain. with expression of in cells a cortical actin of the cells with membrane and the cell These are with the that EHBP1 with EHD2 to regulate endocytosis as well as actin in of EHD2 or EHBP1 by we or into cultured cells to degradation of EHD2 or EHBP1 and transferrin in these the EHD2 or EHBP1 protein in with to EHD2 or EHBP1 but not of the EHD2 or EHBP1 proteins in a of transferrin in of these shows that of cells with transferrin with whereas of the cells with of EHD2 or EHBP1 of the of the EHD2 or EHBP1 transferrin endocytosis also by transferrin cells or cells in which EHD2 or EHBP1 protein by gene silencing with transferrin for in and the by the of a in of EHD2 or EHBP1 proteins the of transferrin that in These that EHD2 and EHBP1 are for endocytic trafficking of of EHD2 the of the studies we that expression of EHD2 or in and cells induces a cortical as EHBP1 and to of cells with less as well as the cell cortical actin and in of cells In cells or cells EH domain These that the EHD2 protein of its EH domain is EHD2 in actin a role for the EH domain in cortical actin by EHD2 expression to the by which EHD2 induces of the of the protein family a of and a of the N-terminal of EH domain are also in proteins, and the yeast EH domain protein (6Duncan M.C. Cope M.J. Goode B.L. Wendland B. Drubin D.G. Nat. Cell Biol. 2001; 3: 687-690Crossref PubMed Scopus (139) Google Scholar, J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, J. C. R. Nat. Cell Biol. 2001; 3: PubMed Scopus Google Scholar). These are for of proteins with Arp2/3 a of J. Biol. 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M.C. Nat. Cell Biol. 2001; 3: PubMed Scopus Google Scholar, B. Nat. Cell Biol. 2001; 3: PubMed Scopus Google Scholar). a role of EHD2 in early endosome recycling back to the plasma membrane or transferrin early endosomes EHD2 is of EHD2 to membranes is through the N-terminal P-loop or EHD2 and mutant EHD2 of the EH domain to membrane whereas the EH domain not and Thus we that EHD2 to proteins in the plasma membrane through or these N-terminal and to through its C-terminal is that two are in the of EHD2 that link protein to the actin an and putative Arp2/3 and the EH domain which binds the novel protein EHBP1 and EHBP1 protein contains a domain to that of the 1 domain of which binds M. K. 2002; PubMed Scopus Google Scholar). in the that the actin cytoskeleton is in membrane and of endosomes to intracellular destinations in mammalian cells D.A. Curr. Opin. Cell Biol. 2002; 14: 76-81Crossref PubMed Scopus (238) Google Scholar, 13Jeng R.L. Welch M.D. Curr. Biol. 2001; 11: R691-R694Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, and B. Kessels M.M. Kelly R.B. J. Cell Biol. 2000; PubMed Scopus Google Scholar). have been actin function in the invagination, and scission steps of the clathrin-dependent endocytosis pathway but in some cell or of these steps for function (8Gottlieb T.A. Ivanov I.E. Adesnik M. Sabatini D.D. J. Cell Biol. 1993; 120: 695-710Crossref PubMed Scopus (369) Google Scholar, 10Lamaze C. Fujimoto L.M. Yin H.L. Schmid S.L. J. Biol. Chem. 1997; 272: 20332-20335Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 11Fujimoto L.M. Roth R. Heuser J.E. Schmid S.L. Traffic. 2000; 1: 161-171Crossref PubMed Scopus (321) Google Scholar). of internalized vesicles have been to actin filaments, including endocytic trafficking in cells (8Gottlieb T.A. Ivanov I.E. Adesnik M. Sabatini D.D. J. Cell Biol. 1993; 120: 695-710Crossref PubMed Scopus (369) Google Scholar, G. Traffic. 2001; PubMed Scopus Google Scholar). has been vesicles endocytosis is the M.R. G. S. M. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), and of endocytic vesicles through actin has been W. Nat. 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Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, R. J. Cell Biol. 2000; PubMed Scopus Google Scholar). be the role of in the of EHD2 and EHBP1 Arp2/3 to actin filaments, to polymerization of J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Biophys. 2000; PubMed Scopus Google Scholar), whereas the function of is to bind of actin filaments are in proteins M. K. 2002; PubMed Scopus Google Scholar). In of these the EHD2 and EHBP1 proteins be to of membranes with actin filaments, as in of steps in the endocytic pathway where actin roles include of the endocytic machinery (12Schafer D.A. Curr. Opin. Cell Biol. 2002; 14: 76-81Crossref PubMed Scopus (238) Google Scholar, B. Kessels M.M. Kelly R.B. J. Cell Biol. 2000; PubMed Scopus Google Scholar). that of actin is with of Nat. Cell Biol. 1999; 1: Scopus Google Scholar). or of the plasma membrane, the and from the plasma membrane also the actin cytoskeleton B. Kessels M.M. Kelly R.B. J. Cell Biol. 2000; PubMed Scopus Google Scholar, W. Nat. Cell Biol. 2002; PubMed Scopus Google Scholar). these we have that the that EHD2 and EHBP1 bind to membranes the membrane However, two components to the of the endocytic machinery in with the actin cytoskeleton. be to the or by which the of are with of the EHD2 and EHBP1 of for the the with we and from the of the of and the of support for the by of in the and of and by