EHD1 and Eps15 Interact with Phosphatidylinositols via Their Eps15 Homology Domains
2007; Elsevier BV; Volume: 282; Issue: 22 Linguagem: Inglês
10.1074/jbc.m609493200
ISSN1083-351X
AutoresNaava Naslavsky, Juliati Rahajeng, Sylvie Chenavas, Paul L. Sorgen, Steve Caplan,
Tópico(s)CRISPR and Genetic Engineering
ResumoThe C-terminal Eps15 homology domain-containing protein, EHD1, regulates the recycling of receptors from the endocytic recycling compartment to the plasma membrane. In cells, EHD1 localizes to tubular and spherical recycling endosomes. To date, the mode by which EHD1 associates with endosomal membranes remains unknown, and it has not been determined whether this interaction is direct or via interacting proteins. Here, we provide evidence demonstrating that EHD1 has the ability to bind directly and preferentially to an array of phospholipids, preferring phosphatidylinositols with a phosphate at position 3. Previous studies have demonstrated that EH domains coordinate calcium binding and interact with proteins containing the tripeptide asparagine-proline-phenylalanine (NPF). Using two-dimensional nuclear magnetic resonance analysis, we now describe a new function for the Eps15 homology (EH) domain of EHD1 and show that it is capable of directly binding phosphatidylinositol moieties. Moreover, we have expanded our studies to include the C-terminal EH domain of EHD4 and the second of the three N-terminal EH domains of Eps15 and demonstrated that phosphatidylinositol binding may be a more general property shared by certain other EH domains. Further studies identified a positively charged lysine residue (Lys-483) localized within the third helix of the EH domain, on the opposite face of the NPF-binding pocket, as being critical for the interaction with the phosphatidylinositols. The C-terminal Eps15 homology domain-containing protein, EHD1, regulates the recycling of receptors from the endocytic recycling compartment to the plasma membrane. In cells, EHD1 localizes to tubular and spherical recycling endosomes. To date, the mode by which EHD1 associates with endosomal membranes remains unknown, and it has not been determined whether this interaction is direct or via interacting proteins. Here, we provide evidence demonstrating that EHD1 has the ability to bind directly and preferentially to an array of phospholipids, preferring phosphatidylinositols with a phosphate at position 3. Previous studies have demonstrated that EH domains coordinate calcium binding and interact with proteins containing the tripeptide asparagine-proline-phenylalanine (NPF). Using two-dimensional nuclear magnetic resonance analysis, we now describe a new function for the Eps15 homology (EH) domain of EHD1 and show that it is capable of directly binding phosphatidylinositol moieties. Moreover, we have expanded our studies to include the C-terminal EH domain of EHD4 and the second of the three N-terminal EH domains of Eps15 and demonstrated that phosphatidylinositol binding may be a more general property shared by certain other EH domains. Further studies identified a positively charged lysine residue (Lys-483) localized within the third helix of the EH domain, on the opposite face of the NPF-binding pocket, as being critical for the interaction with the phosphatidylinositols. Retraction: Factor XI binding to the platelet glycoprotein Ib-IX-V complex promotes factor XI activation by thrombin. VOLUME 277 (2002) PAGES 1662-1668Journal of Biological ChemistryVol. 282Issue 39PreviewRETRACTION Full-Text PDF Open Access The internalization of receptors is a critical process for eukaryotic cells. Receptors can be internalized from the plasma membrane by a variety of well described mechanisms, including via clathrin-coated pits, independently of clathrin, and through caveolae (1.Conner S.D. Schmid S.L. Nature. 2003; 422: 37-44Crossref PubMed Scopus (3101) Google Scholar). Once internalized, the small vesicles containing the internalized cargo fuse with early endosomes (also known as sorting endosomes), and the receptors are then either sent to late endosomes and on to the lysosomal pathway for degradation or recycled back to the plasma membrane, where they may participate in additional rounds of internalization (2.Mellman I. Annu. Rev. Cell Dev. Biol. 1996; 12: 575-625Crossref PubMed Scopus (1346) Google Scholar). Receptor recycling occurs either directly from the sorting endosomes in a process known as "fast recycling" or indirectly in a process termed "slow" or "regulated" recycling (3.Maxfield F.R. McGraw T.E. Nat. Rev. Mol. Cell. Biol. 2004; 5: 121-132Crossref PubMed Scopus (1517) Google Scholar). Slow recycling has been better characterized and traverses a complex series of tubular and vesicular membrane structures that emerge from the microtubule-organizing center and is collectively known as the endocytic recycling compartment (3.Maxfield F.R. McGraw T.E. Nat. Rev. Mol. Cell. Biol. 2004; 5: 121-132Crossref PubMed Scopus (1517) Google Scholar, 4.Hopkins C.R. Trowbridge I.S. J. Cell Biol. 1983; 97: 508-521Crossref PubMed Scopus (448) Google Scholar). Despite advances in recent years, the process of recycling is not as well understood as internalization. Among the key regulatory proteins that control endocytic transport and recycling are the Rab family of GTP-binding proteins (5.Somsel Rodman J. Wandinger-Ness A. J. Cell Sci. 2000; 113: 183-192Crossref PubMed Google Scholar, 6.Pfeffer S.R. Trends Cell Biol. 2001; 11: 487-491Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar, 7.Zerial M. 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Cell Sci. 2004; 117: 6297-6311Crossref PubMed Scopus (124) Google Scholar) have been implicated at varying stages of sorting from the early endosome and recycling from the endocytic recycling compartment. In addition to Rab proteins and their direct effectors, SNARE 3The abbreviations used are: SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptors; PtdIns, phosphatidylinositol; PtdIns3P, phosphatidylinositol 3-phosphate; PtdIns(3,5)P2, phosphatidylinositol 3,5-bisphosphate; PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; LPPG, 1-palmitoyl-2-hydroxy-sn-glycero-3-(phospho-RAC-(1-glycerol)); BOG, n-octyl-β-d-glucopyranoside; EH, Eps15 homology; HSQC, heteronuclear single quantum correlation; GST, glutathione S-transferase; HRP, horseradish peroxidase; CMC, critical micelle concentration; PI3K, phosphatidylinositol 3-kinase; MHC, major histocompatibility complex; BSA, bovine serum albumin. proteins and other non-Rab proteins have also been implicated in the regulation of various stages of endocytosis. The Eps15 homology (EH) domain-containing family of proteins also has a well documented role in the regulation of endocytic transport and recycling. These proteins contain at least one copy of the EH domain, a highly conserved ∼100-amino acid stretch. The EH domain was originally identified in three copies at the N terminus of the protein Eps15 (17.Fazioli F. Minichiello L. Matoskova B. Wong W.T. Di Fiore P.P. Mol. Cell. Biol. 1993; 13: 5814-5828Crossref PubMed Scopus (238) Google Scholar, 18.Wong W.T. Schumacher C. Salcini A.E. Romano A. Castagnino P. Pelicci P.G. Di Fiore P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9530-9534Crossref PubMed Scopus (136) Google Scholar). Structurally, each EH domain is composed of two sets of helix-loophelix motifs (known as EF-hands) predicted to bind calcium and linked by a short antiparallel β-sheet (reviewed in Ref. 19.Confalonieri S. Di Fiore P.P. FEBS Lett. 2002; 513: 24-29Crossref PubMed Scopus (83) Google Scholar). In addition to binding calcium, it was discovered that EH domains bind to proteins containing the tripeptide asparagine-proline-phenylalanine (NPF) (20.Paoluzi S. Castagnoli L. Lauro I. Salcini A.E. Coda L. Fre S. Confalonieri S. Pelicci P.G. Di Fiore P.P. Cesareni G. EMBO J. 1998; 17: 6541-6550Crossref PubMed Scopus (101) Google Scholar, 21.Salcini A.E. Confalonieri S. Doria M. Santolini E. Tassi E. Minenkova O. Cesareni G. Pelicci P.G. Di Fiore P.P. Genes Dev. 1997; 11: 2239-2249Crossref PubMed Scopus (287) Google Scholar). Despite the wide variation of other functional domains found in EH domain-containing proteins, most of these proteins have a defined regulatory role in endocytic membrane transport (reviewed in Refs. 22.Miliaras N.B. Wendland B. Cell Biochem. Biophys. 2004; 41: 295-318Crossref PubMed Scopus (43) Google Scholar and 23.Polo S. Confalonieri S. Salcini A.E. Di Fiore P.P. Sci. STKE 2003. 2003; : re17Google Scholar). Among the best characterized EH domain-containing proteins is Eps15 itself (18.Wong W.T. Schumacher C. Salcini A.E. Romano A. Castagnino P. Pelicci P.G. Di Fiore P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9530-9534Crossref PubMed Scopus (136) Google Scholar). Eps15 and the related Eps15R are both found within assembly sites for clathrin-coated pits, serving as molecular scaffolds by associating with the AP-2 adaptor protein complex (24.Benmerah A. Gagnon J. Begue B. Megarbane B. Dautry-Varsat A. Cerf-Bensussan N. J. Cell Biol. 1995; 131: 1831-1838Crossref PubMed Scopus (151) Google Scholar) and the NPF-containing protein, epsin 1 (25.Chen H. Fre S. Slepnev V.I. Capua M.R. Takei K. Butler M.H. Di Fiore P.P. De Camilli P. Nature. 1998; 394: 793-797Crossref PubMed Scopus (272) Google Scholar). Recently, focus has turned to understanding the regulatory role of an atypical family of EH domain-containing proteins, the C-terminal EHD family (reviewed in Ref. 26.Naslavsky N. Caplan S. J. Cell Sci. 2005; 118: 4093-4101Crossref PubMed Scopus (89) Google Scholar). Unlike other mammalian EH domain-containing proteins, this subgroup of four homologous proteins has a single EH domain at its C terminus (27.Blume J.J. Halbach A. Behrendt D. Paulsson M. Plomann M. Exp. Cell Res. 2007; 313: 219-231Crossref PubMed Scopus (65) Google Scholar, 28.George M. Ying G. Rainey M.A. Solomon A. Parikh P.T. Gao Q. Band V. Band H. BMC Cell Biol. 2007; 8: 3Crossref PubMed Scopus (90) Google Scholar, 29.Mintz L. Galperin E. Pasmanik-Chor M. Tulzinsky S. Bromberg Y. Kozak C.A. Joyner A. Fein A. Horowitz M. Genomics. 1999; 59: 66-76Crossref PubMed Scopus (80) Google Scholar, 30.Pohl U. Smith J.S. Tachibana I. Ueki K. Lee H.K. Ramaswamy S. Wu Q. Mohrenweiser H.W. Jenkins R.B. Louis D.N. Genomics. 2000; 63: 255-262Crossref PubMed Scopus (83) Google Scholar), a central coiled-coil region involved in oligomerization (31.Galperin E. Benjamin S. Rapaport D. Rotem-Yehudar R. Tolchinsky S. Horowitz M. Traffic. 2002; 3: 575-589Crossref PubMed Scopus (69) Google Scholar, 32.Lee D.W. Zhao X. Scarselletta S. Schweinsberg P.J. Eisenberg E. Grant B.D. Greene L.E. J. Biol. Chem. 2005; 280: 280-290Google Scholar, 33.Naslavsky N. Rahajeng J. Sharma M. Jovic M. Caplan S. Mol. Biol. Cell. 2006; 17: 163-177Crossref PubMed Scopus (141) Google Scholar), and an N-terminal regulatory region that binds to nucleotides (32.Lee D.W. Zhao X. Scarselletta S. Schweinsberg P.J. Eisenberg E. Grant B.D. Greene L.E. J. Biol. Chem. 2005; 280: 280-290Google Scholar, 33.Naslavsky N. Rahajeng J. Sharma M. Jovic M. Caplan S. Mol. Biol. Cell. 2006; 17: 163-177Crossref PubMed Scopus (141) Google Scholar). Emerging evidence has implicated all four C-terminal EHD proteins in the process of endocytosis (26.Naslavsky N. Caplan S. J. Cell Sci. 2005; 118: 4093-4101Crossref PubMed Scopus (89) Google Scholar). EHD1, the best characterized C-terminal EHD protein thus far, has a well documented role in controlling recycling from the endocytic recycling compartment to the plasma membrane, a function similar to that attributed to Rab11 (31.Galperin E. Benjamin S. Rapaport D. Rotem-Yehudar R. Tolchinsky S. Horowitz M. Traffic. 2002; 3: 575-589Crossref PubMed Scopus (69) Google Scholar, 33.Naslavsky N. Rahajeng J. Sharma M. Jovic M. Caplan S. Mol. Biol. Cell. 2006; 17: 163-177Crossref PubMed Scopus (141) Google Scholar, 34.Grant B. Zhang Y. Paupard M.C. Lin S.X. Hall D.H. Hirsh D. Nat. Cell Biol. 2001; 3: 573-579Crossref PubMed Scopus (218) Google Scholar, 35.Lin S.X. Grant B. Hirsh D. Maxfield F.R. Nat. Cell Biol. 2001; 3: 567-572Crossref PubMed Scopus (213) Google Scholar, 36.Caplan S. Naslavsky N. Hartnell L.M. Lodge R. Polishchuk R.S. Donaldson J.G. Bonifacino J.S. EMBO J. 2002; 21: 2557-2567Crossref PubMed Scopus (246) Google Scholar, 37.Naslavsky N. Boehm M. Backlund Jr., P.S. Caplan S. 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Ehlers M.D. Science. 2004; 305: 1972-1975Crossref PubMed Scopus (582) Google Scholar). Although no direct interaction has been demonstrated between EHD proteins and Rab proteins, recent studies have shown that EHD1 interacts directly with certain Rab effectors. For example, the divalent Rab4/Rab5 effector, Rabenosyn-5, interacts with the EH domain of EHD1 via several of its five NPF motifs (37.Naslavsky N. Boehm M. Backlund Jr., P.S. Caplan S. Mol. Biol. Cell. 2004; 15: 2410-2422Crossref PubMed Scopus (114) Google Scholar). Similarly, the Rab11 effector Rab11-FIP2, which contains three NPF motifs, interacts with EHD1 through NPF-EH domain interactions and plays a role in recycling from the endocytic recycling compartment (33.Naslavsky N. Rahajeng J. Sharma M. Jovic M. Caplan S. Mol. Biol. Cell. 2006; 17: 163-177Crossref PubMed Scopus (141) Google Scholar). One of the interesting features of EHD1 is that despite not having a hydrophobic transmembrane-spanning region, it localizes to an array of vesicular and tubular membrane structures. Membrane localization depends upon the ability of the EHD1 P-loop to bind nucleotides (35.Lin S.X. Grant B. Hirsh D. Maxfield F.R. Nat. Cell Biol. 2001; 3: 567-572Crossref PubMed Scopus (213) Google Scholar, 36.Caplan S. Naslavsky N. Hartnell L.M. Lodge R. Polishchuk R.S. Donaldson J.G. Bonifacino J.S. EMBO J. 2002; 21: 2557-2567Crossref PubMed Scopus (246) Google Scholar). In addition, EHD1 mutants incapable of oligomerizing become cytosolic in their subcellular localization, suggesting that oligomerization is a requirement for membrane association (32.Lee D.W. Zhao X. Scarselletta S. Schweinsberg P.J. Eisenberg E. Grant B.D. Greene L.E. J. Biol. Chem. 2005; 280: 280-290Google Scholar, 33.Naslavsky N. Rahajeng J. Sharma M. Jovic M. Caplan S. Mol. Biol. Cell. 2006; 17: 163-177Crossref PubMed Scopus (141) Google Scholar) and EHD1 function is severely compromised when these cytosolic mutants are expressed (34.Grant B. Zhang Y. Paupard M.C. Lin S.X. Hall D.H. Hirsh D. Nat. Cell Biol. 2001; 3: 573-579Crossref PubMed Scopus (218) Google Scholar, 35.Lin S.X. Grant B. Hirsh D. Maxfield F.R. Nat. Cell Biol. 2001; 3: 567-572Crossref PubMed Scopus (213) Google Scholar, 36.Caplan S. Naslavsky N. Hartnell L.M. Lodge R. Polishchuk R.S. Donaldson J.G. Bonifacino J.S. EMBO J. 2002; 21: 2557-2567Crossref PubMed Scopus (246) Google Scholar). Thus far, however, the mode by which EHD1 and other C-terminal EHD proteins associate with membranes has remained unknown. Indeed, until now, the question as to whether EHD1 associates indirectly with lipids through its various interaction partners or whether it directly binds to membranes has not been determined. In the study herein, we demonstrate that EHD1 is capable of directly interacting with membranes, by preferentially binding to phosphatidylinositols with a phosphate at position 3 via its EH domain. Our data lead to the conclusion that EH domains from other C-terminal EHD proteins, such as EHD4, as well as an N-terminal EH domain-containing protein (Eps15) are also capable of lipid binding. Moreover, we have identified an EHD1 residue (Lys-483) localized to the third helix of the EH domain as being critical for the interaction with the phosphatidylinositols. Overall, we describe phosphoinositide-binding as a new function for EH domains, which may in part explain the association of EHD proteins with membranes. Recombinant DNA Constructs−Full-length EHD1 and EHD3, the second EH domain of Eps15, and the EH domains of EHD4 and EHD1 were subcloned into the GST fusion bacterial expression vector pGEX-6P-2 (GE Life Sciences). Wild-type Myc-EHD1 and Myc-EHD1 ΔEH have been previously described (36.Caplan S. Naslavsky N. Hartnell L.M. Lodge R. Polishchuk R.S. Donaldson J.G. Bonifacino J.S. EMBO J. 2002; 21: 2557-2567Crossref PubMed Scopus (246) Google Scholar). Myc-EHD1 K483E was prepared by site-directed mutagenesis with the QuikChange kit (Stratagene, La Jolla, CA). Phosphatidylinositols and Other Lipids−18:1 Phosphatidylinositol (PtdIns), 8:0 1,2-dioctanoyl-sn-glycero-3-(phosphoinositol 3,4,5-trisphosphate) (PtdIns(3,4,5)P3), 8:0 1,2-dioctanoyl-sn-glycero-3-(phosphoinositol 3,5-bisphosphate) (PtdIns(3,5)P2), 8:0 phosphatidic acid, and 16:0 1-palmitoyl-2-hydroxy-sn-glycero-3-(phospho-RAC-(1-glycerol)) (LPPG) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). 8:0 octyl-β-d-glucopyranoside (BOG) was obtained from Calbiochem. Each lipid was received as a lyophilized powder and all were completely soluble in phosphate-buffered saline (pH 7.4) buffer. Critical micelle concentration (CMC) measurements are not available for eight-carbon phosphatidylinositol phosphates, and LPPG and would be cost-prohibitive to determine (44.Campbell R.B. Liu F. Ross A.H. J. Biol. Chem. 2003; 278: 33617-33620Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). However, the CMC for PtdIns is 60 μm, and the addition of phosphates to the inositol ring is expected to increase the head group size, charge, and therefore the CMC (44.Campbell R.B. Liu F. Ross A.H. J. Biol. Chem. 2003; 278: 33617-33620Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). For example, the CMC of di-C8-phosphatidylserine has been measured at 2.28 mm (45.Kleinschmidt J.H. Tamm L.K. Biophys. J. 2002; 83: 994-1003Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Accordingly, the 0.75 mm concentration utilized of PtdIns(3,4,5)P3, PtdIns(3,5)P2, and BOG (CMC 20-25 mm) is expected to render them monodispersed and unable to form micelles or membranes. Antibodies−Peptide-specific affinity-purified polyclonal anti-EHD1 antibodies have been previously described (33.Naslavsky N. Rahajeng J. Sharma M. Jovic M. Caplan S. Mol. Biol. Cell. 2006; 17: 163-177Crossref PubMed Scopus (141) Google Scholar), goat anti-GST-horseradish peroxidase (HRP) was purchased from GE Life Sciences, and anti-PtdIns(3,4,5)P3 (46.Chen R. Kang V.H. Chen J. Shope J.C. Torabinejad J. DeWald D.B. Prestwich G.D. J. Histochem. Cytochem. 2002; 50: 697-708Crossref PubMed Scopus (65) Google Scholar) and Alexa fluorochromes conjugated to secondary antibodies were obtained from Molecular Probes, Inc. (Carlsbad, CA). Protein Purification−GST fusion proteins were generated by standard methods. For NMR studies, 15N labeling was done as previously described (47.Duffy H.S. Sorgen P.L. Girvin M.E. O'Donnell P. Coombs W. Taffet S.M. Delmar M. Spray D.C. J. Biol. Chem. 2002; 277: 36706-36714Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), and the GST was cleaved from the EH domains with PreScission Protease (GE Life Sciences) and concentrated as previously described (47.Duffy H.S. Sorgen P.L. Girvin M.E. O'Donnell P. Coombs W. Taffet S.M. Delmar M. Spray D.C. J. Biol. Chem. 2002; 277: 36706-36714Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Protein-Lipid Binding Assay−PIP (phosphatidylinositol) strips, PIP (phosphatidylinositol) arrays, and SphingoStrips (Echelon Biosciences, Salt Lake City, UT) were blocked in TBST-BSA (10 mm Tris, pH 8, 150 mm NaCl, 0.1% Tween 20 (v/v)) supplemented with 3% fatty acid-free BSA (Sigma) for 1 h at room temperature and then incubated with 1 μg/ml GST fusion protein diluted in TBST-BSA overnight at 4 °C. After washes with TBST-BSA, the membranes were blotted with goat anti-GST conjugated to HRP and visualized by enhanced chemiluminescence. Densitometric analysis was applied to determine the relative affinity of EHD1 binding to the various phosphatidylinositols. Numerical densitometric values were attributed to each of the five concentrations measured after subtracting backgrounds, and the total additive value for all five measurements was used as an indication of affinity. The highest value, for EHD1 binding to PtdIns(3,5)P2, was arbitrarily assigned "100% binding," and all other phosphatidylinositols were normalized compared with PtdIns(3,5)P2. Protein-Liposome Binding Assay−PolyPIPosomes (Echelon) contain phosphatidylcholine (65 mol %), PE (29 mol %), biotin-PE (1 mol %), and phosphatidylinositol (5 mol %). The phosphatidylinositols used in this assay were PtdIns, PtdIns(3,5)P2, and PtdIns(3,4,5)P3.15 μmol of PolyPIPosomes were diluted in 100 μl of binding buffer (50 mm Tris, pH 7.8, 150 mm NaCl, 0.05% Nonidet P-40 (v/v), 20 mm iodoacetamide) and incubated 15 min at room temperature with 10 μg of GST fusion protein. Liposome-protein complexes were recovered by 10 min centrifugation at room temperature (11,356 × g), and pellets were resuspended in 500 μl of binding buffer and further incubated with streptavidin-agarose (Invitrogen) for 1 h at room temperature. Biotinylated liposomes bound to GST proteins were washed with immunoprecipitation buffer (50 mm Tris, pH 7.4, 150 mm NaCl, 0.5% Triton X-100 (v/v), 20 mm iodoacetamide) and eluted with Laemmli sample buffer containing 5% 2-mercaptoethanol, separated by SDS-PAGE, and immunoblotted with anti-GST-HRP (as described under "Protein-Lipid Binding Assay"). To negate liposome-independent pull-down of GST proteins, a control sample comprising the streptavidin-agarose and GST proteins (without liposomes) was also included. Protein-Lipid Bead Binding Assays−Bead Binding assays were done essentially as described by Rao and co-workers (48.Rao V.R. Corradetti M.N. Chen J. Peng J. Yuan J. Prestwich G.D. Brugge J.S. J. Biol. Chem. 1999; 274: 37893-37900Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Briefly, streptavidin-agarose beads bound to biotinylated DiC6-PtdIns or -PtdIns(3,4,5)P3 (Echelon) were used to bind and isolate full-length GST fusion proteins. A 10-μl slurry of beads was washed once in 500 μl of binding buffer (10 mm HEPES, pH 7.4, 150 mm NaCl, 0.5% (v/v) Nonidet P-40, 5 mm dithiothreitol) and resuspended in the same buffer, supplemented with 5 μg of full-length GST fusion protein, rotating for 2 h at 4 °C. After extensive washing with binding buffer, beads were eluted with Laemmli sample buffer containing 5% 2-mercaptoethanol, separated by SDS-PAGE, and immunoblotted with anti-GST-HRP. Two-dimensional Heteronuclear Single Quantum Correlation (HSQC) Analysis−NMR data were acquired at 25 °C using a 600-MHz Varian INOVA NMR spectrometer fitted with a cryoprobe at the University of Nebraska Medical Center NMR Shared Resource Facility. Gradient-enhanced two-dimensional 15N HSQC experiments (49.Kay L.E. Keifer P. Saarinen T. J. Am. Chem. Soc. 1992; 114: 10663-10665Crossref Scopus (2439) Google Scholar) were used to observe all backbone amide resonances in 15N-labeled EH domains. Data were acquired with 1024 complex points in the direct dimension and 128 complex points in the indirect dimension. Sweep widths were 10,000 Hz in the proton dimension and 2500 Hz in the nitrogen dimension. NMR spectra were processed using NMRPipe (50.Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11638) Google Scholar) and analyzed using NMRView (51.Johnson B.A. Bevins R.A. J. Biomol. NMR. 1994; 4: 603-614Crossref PubMed Scopus (2686) Google Scholar). EHD1 Associates Directly with Phosphatidylinositol Lipids−To determine whether EHD1 is capable of directly interacting with lipids, we first utilized an assay in which a wide variety of lipids, immobilized on nitrocellulose, were probed with GST as a control (data not shown) or with a GST-EHD1 fusion protein. As demonstrated in Fig. 1, A and B, GST-EHD1 bound to a series of phosphatidylinositol moieties, including phosphatidic acid, phosphatidylinositol-3-phosphate (PtdIns3P), phosphatidylinositol 4-phosphate, phosphatidylinositol 5-phosphate, PtdIns(3,5)P2, and PtdIns(3,4,5)P3, but showed no binding to various other lipids, including phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingosine, cholesterol, ceramide, and others (Fig. 1, A and B). To quantitatively assess the binding specificity of these lipids for EHD1, GST-EHD1 was used to probe an array of phosphatidylinositol moieties immobilized on nitrocellulose membranes in decreasing concentrations from 100 to 6.2 pmol (Fig. 1C) and quantified by densitometry (Fig. 1D). As demonstrated, phosphatidylinositol moieties with a phosphate at position 3 of the inositol ring, including PtdIns(3,5)P2 and PtdIns(3,4,5)P3, showed binding even at 25 and 12.5 pmol, respectively. These data indicate that EHD1 can directly bind to phosphatidylinositols with phosphates on the inositol ring. To further validate our findings that EHD1 can interact directly with phospholipids, we used a pull-down assay, whereby streptavidin beads coated with either PtdIns(3,4,5)P3 or PtdIns were then incubated with GST-EHD1, GST-EHD3 (the closest mammalian homolog to EHD1 at 85% identity), or GST alone. By this in vitro assay, we again demonstrated that EHD1 as well as EHD3 bind to PtdIns(3,4,5)P3 and PtdIns (Fig. 2A). To determine whether EHD1 interacts directly with phospholipids in the context of a monolayer more closely resembling physiological membrane composition, we utilized liposomes composed of 1% biotinylated PE, 29% PE, and 65% phosphatidylcholine with either 5% PtdIns(3,4,5)P3, PtdIns(3,5)P2, or PtdIns (PolyPIPosomes; Echelon). The liposomes were incubated with GST-EHD1, GST-EHD3, or GST alone as a control. Isolated complexes of biotinylated liposomes bound to GST proteins were further purified by precipitation with streptavidin-coated Sepharose beads (Fig. 2B). Approximately 10-20% of both GST-EHD1 and GST-EHD3 that was incubated with the liposomes precipitated with liposomes containing PtdIns(3,4,5)P3 and PtdIns(3,5)P2, whereas less than 3% precipitated with PtdIns. The GST-only control did not bind to any of the liposome compositions. Additional control experiments with streptavidin beads but without liposomes demonstrated that the efficient pull-down of GST-EHD1 and GST-EHD3 was entirely dependent on the presence of the liposomes. Moreover, the preferential binding to PtdIns(3,5)P2 and PtdIns(3,4,5)P3 as compared with PtdIns is consistent with our findings in Fig. 1. Taken together, our data support a direct interaction between EHD1 and phosphoinositol lipids. Inhibition of Phosphatidylinositol 3-Kinase (PI3K) Activity Affects the Subcellular Distribution of EHD1−Within cells, EHD1 resides on tubular and vesicular membrane-bound organelles. Although efficient/specific antibodies are not available for many of the phospholipids, commercial antibodies suitable for immune fluorescence are available for PtdIns(3,4,5)P3 (46.Chen R. Kang V.H. Chen J. Shope J.C. Torabinejad J. DeWald D.B. Prestwich G.D. J. Histochem. Cytochem. 2002; 50: 697-708Crossref PubMed Scopus (65) Google Scholar), one of the phospholipids to which EHD1 displayed relatively high affinity (Figs. 1 and 2). To determine whether EHD1 localizes in vivo to PtdIns(3,4,5)P3-containing membranes, we fixed and incubated HeLa cells with antibodies directed against endogenous EHD1 and PtdIns(3,4,5)P3 (Fig. 3). As shown, EHD1 localizes to an array of
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