EHD2 and the Novel EH Domain Binding Protein EHBP1 Couple Endocytosis to the Actin Cytoskeleton
2004; Elsevier BV; Volume: 279; Issue: 11 Linguagem: Inglês
10.1074/jbc.m307702200
ISSN1083-351X
AutoresAdı́lson Guilherme, Neil A. Soriano, Sahana Bose, John Holik, Avirup Bose, Darcy P. Pomerleau, Paul S. Furcinitti, John Leszyk, Silvia Corvera, Michael Czech,
Tópico(s)Axon Guidance and Neuronal Signaling
ResumoHere 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 at different stages of endocytosis and the structures of complexes at the interface of endocytic components and the actin cytoskeleton remain to be clarified. Using the recycling of GLUT4 glucose transporter proteins in cultured adipocytes as a model system, we identify here two novel proteins, EHD2 1The abbreviations used are: EHD2, EH domain protein 2; mEHD2, mouse EHD2; EHBP1, EH domain binding protein 1; CH domain, Calponin homology domain; GFP, green fluorescence protein; FITC, fluorescein isothiocyanate; PBS, phosphate buffered saline; HA, hemag-glutinin; EST, expressed sequence tag; aa, amino acid; EGFP, enhanced GFP; siRNA, small interfering RNA; GST, glutathione S-transferase.1The abbreviations used are: EHD2, EH domain protein 2; mEHD2, mouse EHD2; EHBP1, EH domain binding protein 1; CH domain, Calponin homology domain; GFP, green fluorescence protein; FITC, fluorescein isothiocyanate; PBS, phosphate buffered saline; HA, hemag-glutinin; EST, expressed sequence tag; aa, amino acid; EGFP, enhanced GFP; siRNA, small interfering RNA; GST, glutathione S-transferase. and EHBP1, that appear to connect actin dynamics with endocytosis. In this system, insertion of GLUT4 into the plasma membrane in response to insulin is accompanied by its rapid retrieval by the clathrin-dependent pathway (17Robinson L.J. Pang S. Harris D.S. Heuser J. James D.E. J. Cell Biol. 1992; 117: 1181-1196Crossref PubMed Scopus (257) Google Scholar, 18Kao A.W. Ceresa B.P. Santeler S.R. Pessin J.E. J. Biol. Chem. 1998; 273: 25450-25457Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 19Volchuk A. Narine S. Foster L.J. Grabs D. De Camilli P. Klip A. J. Biol. Chem. 1998; 273: 8169-8176Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). EHD2 is detected in membranes that contain some of the intracellular GLUT4. Through its EH domain, which is similar to those present in many proteins involved in endocytosis, EHD2 binds NPF motifs in EHBP1. Both EHD2 and EHBP1 also connect to the cytoskeleton through an acidic motif in the former and through a CH domain in the latter. High expression of either EHD2 or EHBP1 impairs endocytosis of GLUT4 in cultured adipocytes and transferrin in COS cells. Furthermore, loss of the endogenous EHD2 or EHBP1 mediated by siRNA gene silencing inhibits transferrin endocytosis, indicating that these interacting proteins function at an early stage of endocytosis near the plasma membrane by providing functional linkages to the actin cytoskeleton. Materials—Anti-EHD2 antibody was generated in rabbits using a GST-EHD2 fusion protein as an antigen and the IgG fraction isolated from rabbit serum. Anti-EHBP1 antibody was prepared by immunizing rabbits with a GST fusion protein containing residues 360-715 of EHBP1 sequence. Anti-EHBP1 was then affinity-purified from serum, using a cyanogen bromide-conjugated EHBP1 (GST-(360-715)) column. In order to test the specificity of anti-EHD2 and anti-EHBP1 antibodies, immunoblot analysis from lysates of cells expressing GFP-empty vector, GFP-EHD2, or GFP-EHBP1 were performed, using pre-immune or immune serums. Anti-EHD2 or anti-EHBP1, but not the pre-immune serum, recognizes specifically expressed GFP-EHD2 or GFP-EHBP1 proteins, respectively. Anti-EHD2-rhodamine red- and anti-EHBP1-Alexa488-conjugated antibodies were generated using protein labeling kits (Molecular Probes) following the manufacturer’s instructions. Goat anti-GLUT4 polyclonal antibody (C-20) and mouse anti-GLUT4 monoclonal antibody (clone IF8) used in whole mount and in freeze deep-etch EM analyses were purchased from Santa Cruz Biotechnology and Biogenesis, respectively. Rabbit anti-actin polyclonal antibody was a gift from Dr. Christine Chaponnier (University of Geneva, Switzerland). Mouse anti-Myc epitope (clone 9E10) monoclonal antibody was from NeoMarkers, Inc. Rabbit anti-HA polyclonal antibody was produced as described (20Bose A. Guilherme A. Robida S.I. Nicoloro S.M.C. Zhou Q.L. Jiang Z.Y. Pomerlau D.P. Czech M.P. Nature. 2002; 420: 821-824Crossref PubMed Scopus (215) Google Scholar). Rhodamine-phalloidin, rhodamine-transferrin, goat anti-rabbit-Alexa350-conjugated and goat anti-mouse-Alexa594-conjugated antibodies were from Molecular Probes. Goat anti-rabbit FITC-conjugated antibody was from BIOSOURCE International (Camarillo, CA). The antibodies conjugated to 6- and 12-nm gold particles used in immunoelectron microscopy analysis were purchased from Jackson ImmunoResearch. Constructs—The murine EST encoding mouse EHD2 (GenBank™ accession number AI787872) was obtained from Genome Systems (St. Louis, MO). The plasmid DNA was isolated and sequenced, and the full-length mEHD2 was used to generate the different constructs used in this study. The plasmids expressing GFP-EHD2 (aa 1-543), GFP-ΔEH-EHD2 (aa 1-444), GFP-ΔEH-ΔA-EHD2 (aa 1-428), and GFP-EH (aa 444-543) were constructed by PCR amplification using primers creating XhoI and BamHI sites at the 5′ and 3′ ends, respectively. The PCR products were subcloned in-frame with a pEGFP-C2 vector. The plasmids expressing HA-EHD2, HA-ΔEH-EHD2, or HA-EH were similarly constructed by PCR amplification using primers creating KpnI and BamHI sites at the 5′ and 3′ ends, respectively. The PCR products were then subcloned in-frame with a 3XHA-pCMV5 vector. The two overlapping ESTs (aa 1-310; accession number BAA91391 and aa 235-1196; accession number BAA74926) encoding human EHBP1 were obtained from Kazusa DNA Research Institute and from Institute of Medical Science, University of Tokyo, Japan, respectively. The plasmid DNAs were isolated and sequenced, and the two ESTs were fused to generate the complete EHBP1 cDNA sequence. The plasmid expressing the full-length EHBP1 (aa 1-1196) was constructed by PCR amplification using primers creating KpnI and XmaI sites at the 5′ and 3′ ends, respectively. The PCR products were subcloned in-frame with a 3XHA-pCMV5 or a pEGFP-C3 vector to express HA-EHBP1 or GFP-EHBP1. For antibody production and in vitro pull-down assays, full-length or fragments of EHD2 or EHBP1 were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. EHD2, EHBP1 (aa 360-715), and the NPF motifs from EHBP1 (aa 227-407) were PCR-amplified by using primers generating BamHI and XhoI restriction sites at the 5′ and 3′ ends, respectively. The PCR products were subcloned in-frame with a pGEX5x3 vector. The Myc-GLUT4-EGFP construct has been described previously (21Jiang Z.Y. Chawla A. Bose A. Way M. Czech M.P. J. Biol. Chem. 2002; 277: 509-515Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). EGFP-EEA1 was generated as described (22Lawe D.C. Chawla A. Merithew E. Dumas J. Carrington W. Fogarty K. Lifshitz L. Tuft R. Lambright D. Corvera S. J. Biol. Chem. 2002; 277: 8611-8617Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). All the constructs were sequenced for verification prior to transfections. Cell Culture and Transfection of Differentiated 3T3-L1 Adipocytes— 3T3-L1 fibroblasts were grown to confluency and differentiated as described previously (23Bose A. Cherniack A.D. Langille S.E. Nicoloro S.M.C. Buxton J.M. Park J.G. Chawla A. Czech M.P. Mol. Cell. Biol. 2001; 21: 5262-5275Crossref PubMed Scopus (57) Google Scholar). Differentiated 3T3-L1 adipocytes were transfected by electroporation as described (24Jiang Z.Y. Zhou Q.L. Coleman K.A. Chouinard M. Boese Q. Czech M.P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7569-7574Crossref PubMed Scopus (303) Google Scholar). The cells were then re-plated and allowed to recover for 24 h before serum starvation for 3 h and stimulation with insulin. For the myc-GLUT4-GFP internalization assays, adipocytes were cotransfected with 50 μg of myc-GLUT4-EGFP in combination with either 150 μg of pCMV5-3XHA-EHD2, 3XHAΔEH-EHD2, or 3XHA-EH. The effect of cortical actin rearrangement by expression of the GFP-EHD2 constructs was assayed in 3T3-L1 adipocyte electroporated with 50 μg of the plasmid encoding the indicated construct. siRNA-induced Degradation of EHD2 and EHBP1—The siRNA species purchased from Dharmacon were designed to target the following cDNA sequences: scrambled, 5′-CAGTCGCGTTTGCGACTGG-3′; EHD2-siRNA, 5′-AAGAAAGAGATGCCCACGGTGTT-3′; EHBP1-siRNA 1, AAGCTCTTGCCACCAGCAGCATT-3′; EHBP1-siRNA 2, AAGAGGAGAAGGCGGCAAAAATT-3′. Either 20 nmol of scrambled siRNA, 10 nmol of EHD2-siRNA, or 10 nmol of each of the EHBP1-siRNA species were electroporated into 3T3-L1 fibroblasts as described (24Jiang Z.Y. Zhou Q.L. Coleman K.A. Chouinard M. Boese Q. Czech M.P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7569-7574Crossref PubMed Scopus (303) Google Scholar). Briefly, fibroblasts were detached from culture dishes with 0.25% trypsin in PBS, washed twice, and resuspended in PBS. Half of the cells from one 150-mm dish were then mixed with siRNA, which was delivered to the cells by a pulse of electroporation with a Bio-Rad gene pulser II system at the setting of 0.18 kV and 950-microfarad capacitance. Using a Cy3-tagged siRNA for lamin A/C, we showed previously that Cy3-siRNA was introduced with virtually 100% efficiency into cells using this method, and nearly all cells showed loss of nuclear lamin A/C (24Jiang Z.Y. Zhou Q.L. Coleman K.A. Chouinard M. Boese Q. Czech M.P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7569-7574Crossref PubMed Scopus (303) Google Scholar). After electroporation the cells were reseeded in 12-well plates and allowed to rest for 48 h. Alexa594-transferrin uptake was then assayed as described below. A portion of these cells was analyzed for EHD2, EHBP1, and actin by Western blotting. Mass Spectral Analysis, Northern and Western Blotting—Rat fat cell subcellular fractions were prepared using a differential centrifugation procedure described previously (25Guilherme A. Emoto M. Buxton J.M. Bose S. Sabini R. Theurkauf W.E. Leszyk J. Czech M.P. J. Biol. Chem. 2000; 275: 38151-38159Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Briefly, the plasma membrane fraction was obtained after 20 min of centrifugation at 16,700 × g followed by centrifugation through sucrose. The high density microsomes were obtained by centrifuging the 16,700 × g supernatant at 38,700 × g for 20 min, and the low density microsomes were obtained by spinning the 38,700 × g supernatant at 150,000 × g for 90 min. The supernatant from the 150,000 × g centrifugation was concentrated using a Centriprep apparatus to obtain the cytosolic fraction. These fractions have been characterized in detail previously (52Piper R.C. Hess L.J. James D.E. Am. J. Physiol. 1991; 260: C570-C580Crossref PubMed Google Scholar). The isolation and fractionation of GLUT4-containing vesicles were carried out as described (25Guilherme A. Emoto M. Buxton J.M. Bose S. Sabini R. Theurkauf W.E. Leszyk J. Czech M.P. J. Biol. Chem. 2000; 275: 38151-38159Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The proteins were resolved by SDS-PAGE and visualized by silver staining (Bio-Rad), and the bands were excised. The peptide sequences were determined by matrix-assisted laser desorption ionization-time of flight-mass spectrometry analysis as described (25Guilherme A. Emoto M. Buxton J.M. Bose S. Sabini R. Theurkauf W.E. Leszyk J. Czech M.P. J. Biol. Chem. 2000; 275: 38151-38159Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). To examine the subcellular distribution of endogenous EHD2 in adipocytes, subcellular fractions were prepared as described before, and 50 μg of fractions indicated in Fig. 1D were loaded onto 10% polyacrylamide gels and transferred to nitrocellulose membranes. EHD2 was detected by immunoblotting, using anti-EHD2 antibody. As depicted in Fig. 1D, a 65-kDa band, corresponding to the molecular weight of EHD2, was detected by anti-EHD2 but not pre-immune serum (data not shown). The EHBP1 protein was detected by immunoblotting with affinity-purified anti-EHBP1 antibody. For Northern blot analysis, total RNA from 3T3-L1 fibroblasts, differentiated adipocytes, and different mouse tissues was isolated as described (20Bose A. Guilherme A. Robida S.I. Nicoloro S.M.C. Zhou Q.L. Jiang Z.Y. Pomerlau D.P. Czech M.P. Nature. 2002; 420: 821-824Crossref PubMed Scopus (215) Google Scholar, 23Bose A. Cherniack A.D. Langille S.E. Nicoloro S.M.C. Buxton J.M. Park J.G. Chawla A. Czech M.P. Mol. Cell. Biol. 2001; 21: 5262-5275Crossref PubMed Scopus (57) Google Scholar). 10 μg of total RNA was resolved on a 1.2% denaturing gel and was transferred to a Nytran membrane. DNA probe corresponding to nucleotides 1606-1881, unique to mouse EHD2, was prepared by PCR amplification, labeled with [α-32P]dCTP, and hybridized to the membrane at 42 °C. A phosphor screen was scanned using an Amersham Biosciences Storm 860 Scanner. Rhodamine-Transferrin Uptake and Immunofluorescence Microscopy—To assay transferrin uptake, COS-1 cells were transfected, using the calcium precipitation method, with either empty EGFP vector, GFP-EHD2, GFP-ΔEH-EHD2, or GFP-EH constructs, and after 24 h the cells were serum-starved in Krebs-Ringer/HEPES + 0.5% bovine serum albumin + 2 mm sodium pyruvate for 2 h. They were then incubated in the presence of 5 μg/ml transferrin-rhodamine for 5 min. The cells were then fixed with 4% formaldehyde and observed using confocal microscopy. To visualize endogenous actin and EHD2, 3T3-L1 adipocytes were fixed with 4% formaldehyde in phosphate-buffered saline (PBS), permeabilized, and blocked with 0.5% Triton X-100 and 1% fetal bovine serum for 20 min. Cells were incubated with primary antibodies for 2 h and with rhodamine-phalloidin and FITC-conjugated secondary antibody for 30 min. To analyze Myc-GLUT4-GFP internalization in adipocytes, cells were first cotransfected with plasmid DNA(s) as indicated. Following this, adipocytes were treated with 0.2 μm insulin for 45 min, washed with iced-cold PBS, and incubated on ice for 1 h with mouse anti-Myc monoclonal antibody. The cells were then washed with ice-cold PBS, and the myc-GLUT4-GFP internalization was initiated by warming the cells at 37 °C. At different time points, cells were fixed, permeabilized, and immunostained with Alexa594-labeled antimouse secondary antibody to visualize myc-GLUT4-GFP. The cells expressing the HA-tagged proteins were visualized by immunostaining with polyclonal anti-HA antibody and Alexa350-conjugated secondary antibody. Images were taken with an Olympus IX-70 microscope with CCD camera and then processed using Metamorph software. Live Cell Imaging—For live cell imaging COS-1 cells were transfected with GFP-tagged constructs, and after 24 h, cells were serum-starved for 2 h and then incubated for 7 min with Texas Red transferrin. Cells were washed with KHR buffer (25 mm HEPES, 125 mm NaCl, 5 mm KCl, 1.3 mm CaCl2, 1.2 mm MgSO4) followed by a 30-min chase with unlabeled transferrin. Cells were imaged using high speed, three-dimensional microscopy as described (22Lawe D.C. Chawla A. Merithew E. Dumas J. Carrington W. Fogarty K. Lifshitz L. Tuft R. Lambright D. Corvera S. J. Biol. Chem. 2002; 277: 8611-8617Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The microscope was configured to 133 nm/pixels using a ×60 objective, and the laser illumination was configured to provide a 488-nm excitation wavelength with a flux on specimen of ∼12 watts/cm2. Exposure times of 4 (GFP) and 15 ms (Texas Red) were used to acquire each of 21 optical sections, spaced by 250 nm. Each set of 21 optical sections was acquired in less than 1 s, allowing 20 ms for each 250-nm shift in focus. Stacks were acquired every 20 s for 30 continuous minutes. The haze originating from light sources outside the in-focus plane of the cell was reduced by image restoration. Stacks were projected into single two-dimensional images, which were concatenated into a QuickTime video format. After imaging the cells were fixed, permeabilized, and stained with anti-HA and Alexa350-conjugated secondary antibodies in order to verify coexpression of GFP-EEA1 and HA-EHD2. Enlargement of early endosomes and increased cytosolic localization of GFP-EEA1 was induced by coexpressing HA-EHD2 (data not shown). The same phenotype was observed in the live cell coexpressing GFP-EEA1 and HA-EHD2 (Fig. 4, bottom panel). Electron Microscopy and Immunogold Labeling Analysis—Whole-mount electron microscopy analysis was performed as described (20Bose A. Guilherme A. Robida S.I. Nicoloro S.M.C. Zhou Q.L. Jiang Z.Y. Pomerlau D.P. Czech M.P. Nature. 2002; 420: 821-824Crossref PubMed Scopus (215) Google Scholar). Double immunogold labeling with different primary antibodies (rabbit anti-EHD2 with goat anti-GLUT4 or, as a negative control, rabbit non-immune IgG with goat non-immune IgG, all antibodies used at 10 μg/ml) and specific secondary antibodies conjugated to 6 and 12 nm gold particles were carried out on parafilm sheets at room temperature. After immunogold labeling, the cell preparations were fixed and processed as described (20Bose A. Guilherme A. Robida S.I. Nicoloro S.M.C. Zhou Q.L. Jiang Z.Y. Pomerlau D.P. Czech M.P. Nature. 2002; 420: 821-824Crossref PubMed Scopus (215) Google Scholar). For immunogold labeling of EHD2, GLUT4, and actin at plasma membrane, lawns from 3T3-L1 adipocytes were generated as described (23Bose A. Cherniack A.D. Langille S.E. Nicoloro S.M.C. Buxton J.M. Park J.G. Chawla A. Czech M.P. Mol. Cell. Biol. 2001; 21: 5262-5275Crossref PubMed Scopus (57) Google Scholar). Immediately after preparation, membrane lawns were fixed on coverslips with 3.7% formaldehyde and labeled with primary antibodies, followed by 6- and 12-nm gold-tagged secondary antibodies. Samples were fixed with 2.5% glutaraldehyde in PBS for 30 min. An alternate method (26Nation J.L. Stain Technol. 1983; 6: 347-351Crossref Scopus (615) Google Scholar), which employs the use of the transitional solvent hexamethyldisilazane for optimally drying soft tissues, has been used widely to preserve the fine surface details of soft tissue specimens without subjecting the specimens to the extreme pressures of Critical Point Drying or the prolonged periods at high vacuum needed for freeze-drying. Whole-mount grids were examined on a Philips CM 10 transmission electron microscope at 80 kV. Expression and Purification of Recombinant Proteins—The expressed fusion proteins were isolated using glutathione-agarose beads. To isolate recombinant protein, the fusion GST constructs were used to transform competent BL-21 cells, and the resulting fusion proteins were expressed by induction with isopropyl-β-thiogalactoside. EHD2, ΔEH-EHD2, and NPF-containing motifs of EHBP1 fusion proteins were isolated from bacterial lysates prepared as described (27Frangioni J.V. Neel B.G. Anal. Biochem. 1993; 210: 179-187Crossref PubMed Scopus (829) Google Scholar). The other GST fusion proteins were isolated from bacterial lysates prepared according to the standard procedures. Identification of EHD2 in Membrane Fractions from 3T3-L1 Adipocytes—As a means of identifying proteins that may be involved in regulating the trafficking of GLUT4-containing membranes, a purified membrane fraction from primary rat adipocytes shown to be highly enriched in GLUT4 (25Guilherme A. Emoto M. Buxton J.M. Bose S. Sabini R. Theurkauf W.E. Leszyk J. Czech M.P. J. Biol. Chem. 2000; 275: 38151-38159Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) was subjected to SDS-PAGE, and many of the silver-stained bands were analyzed by tryptic hydrolysis and mass spectrometry. Among a large number of proteins identified previously in GLUT4-containing membranes (25Guilherme A. Emoto M. Buxton J.M. Bose S. Sabini R. Theurkauf W.E. Leszyk J. Czech M.P. J. Biol. Chem. 2000; 275: 38151-38159Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), a protein band of about 65 kDa was found to contain peptides identical to a human isoform of the EHD2 protein (Fig. 1A). DNA sequences encoding this isoform, EHD2, were reported previously in the context of a genomics study without further characterization (28Pohl 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 (81) Google Scholar), and the murine homolog has not been identified previously. Mouse EHD2 belongs to a group of four closely related proteins expressed in human cells that present a P-loop near the N terminus, followed by a coiled-coil region and an EH domain at the C terminus (28Pohl 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 (81) Google Scholar). This family of proteins has been suggested to function in endocytosis and membrane trafficking through binding of their EH domains to partner proteins with NPF motifs (29Santolini E. Salcini A.E. Kay B.K. Yamabhai M. Di Fiore P.P. Exp. Cell Res. 1999; 253: 186-209Crossref PubMed Scopus (115) Google Scholar, 30Confalonieri S. Di Fiore P.P. FEBS Lett. 2000; 513: 24-29Crossref Scopus (81) Google Scholar, 31de Beer T. Hoofnagle A.N. Enmon J.L. Bowers R.C. Yamabhai M. Kay B.K. Overduin M. Nat. Struct. Biol. 2000; 11: 1018-1022Crossref Scopus (89) Google Scholar). Sequence analysis of the full-length mEHD2 cDNA, derived from an EST clone obtained from Genome Systems, revealed the expected deduced domain structure for this protein as depicted in Fig. 1B. The expression profile of mEHD2 reveals high abundance in lung, fat, and skeletal and heart muscle (Fig. 1C). The latter three tissues are insulin-sensitive and uniquely express GLUT4 glucose transporters. Also, mEHD2 mRNA is greatly elevated upon differentiation of mouse 3T3-L1 fibroblasts to adipocytes in culture (Fig. 1C). These expression data suggest a functional significance of EHD2 expression in the context of GLUT4 trafficking. Consistent with this possibility, recent studies with EHD1 and its Caenorhabditis elegans homolog RME-1 suggest that these proteins are part of the molecular machinery responsible for recycling of receptors to the plasma membrane (32Grant B. Zhang Y. Paupard M.C. Lin S.X. Hall D.H. Hirsh D. Nat. Cell Biol. 2001; 3: 573-579Crossref PubMed Scopus (214) Google Scholar, 33Lin S.X. Grant B. Hirsh D. Maxfield F.R. Nat. Cell Biol. 2001; 3: 567-572Crossref PubMed Scopus (209) Google Scholar, 34Caplan 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 (241) Google Scholar). Furthermore, connections between EHD1 and endocytosis of IGF-1 receptor in Chinese hamster ovary cells has been described (35Rotem-Yehudar R. Galperin E. Horowitz M. J. Biol. Chem. 2001; 276: 33054-33060Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). A role for an EHD4-related protein, Pincher, in mediating endocytosis and trafficking of the nerve growth factor receptor TrkA in PC12 cells has also been reported recently (36Shao Y. Akmentin W. Toledo-Aral J.J. Rosenbaum J. Valdez G. Cabot J.B. Hilbush B.S. Halegoua S. J. Cell Biol. 2002; 157: 679-691Crossref PubMed Scopus (142) Google Scholar). Thus, these recent reports combined with the findings in Fig. 1 suggest the possibility that EHD2 is a novel mouse protein that functions in some aspect of the trafficking of membranes containing GLUT4 and other recycling proteins. EHD2 and GLUT4-containing Vesicles Localize Near the Plasma Membrane—The subcellular localization of EHD2 was determined using a polyclonal antibody against GST-EHD2 in biochemical and morphological approaches. As shown in Fig. 1D (left), subcellular fractionation of adipocytes followed by immunoblot
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