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Role of EHD1 and EHBP1 in Perinuclear Sorting and Insulin-regulated GLUT4 Recycling in 3T3-L1 Adipocytes

2004; Elsevier BV; Volume: 279; Issue: 38 Linguagem: Inglês

10.1074/jbc.m401918200

ISSN

1083-351X

Autores

Adı́lson Guilherme, Neil A. Soriano, Paul S. Furcinitti, Michael Czech,

Tópico(s)

Pancreatic function and diabetes

Resumo

Insulin stimulates glucose transport in muscle and adipose tissues by recruiting intracellular membrane vesicles containing the glucose transporter GLUT4 to the plasma membrane. The mechanisms involved in the biogenesis of these vesicles and their translocation to the cell surface are poorly understood. Here, we report that an Eps15 homology (EH) domain-containing protein, EHD1, controls the normal perinuclear localization of GLUT4-containing membranes and is required for insulin-stimulated recycling of these membranes in cultured adipocytes. EHD1 is a member of a family of four closely related proteins (EHD1, EHD2, EHD3, and EHD4), which also contain a P-loop near the N terminus and a central coiled-coil domain. Analysis of cultured adipocytes stained with anti-GLUT4, anti-EHD1, and anti-EHD2 antibodies revealed that EHD1, but not EHD2, partially co-localizes with perinuclear GLUT4. Expression of a dominant-negative construct of EHD1 missing the EH domain (ΔEH-EHD1) markedly enlarged endosomes, dispersed perinuclear GLUT4-containing membranes throughout the cytoplasm, and inhibited GLUT4 translocation to the plasma membranes of 3T3-L1 adipocytes stimulated with insulin. Similarly, small interfering RNA-mediated depletion of endogenous EHD1 protein also markedly dispersed perinuclear GLUT4 in cultured adipocytes. Moreover, EHD1 is shown to interact through its EH domain with the protein EHBP1, which is also required for insulin-stimulated GLUT4 movements and hexose transport. In contrast, disruption of EHD2 function was without effect on GLUT4 localization or translocation to the plasma membrane. Taken together, these results show that EHD1 and EHBP1, but not EHD2, are required for perinuclear localization of GLUT4 and reveal that loss of EHBP1 disrupts insulin-regulated GLUT4 recycling in cultured adipocytes. Insulin stimulates glucose transport in muscle and adipose tissues by recruiting intracellular membrane vesicles containing the glucose transporter GLUT4 to the plasma membrane. The mechanisms involved in the biogenesis of these vesicles and their translocation to the cell surface are poorly understood. Here, we report that an Eps15 homology (EH) domain-containing protein, EHD1, controls the normal perinuclear localization of GLUT4-containing membranes and is required for insulin-stimulated recycling of these membranes in cultured adipocytes. EHD1 is a member of a family of four closely related proteins (EHD1, EHD2, EHD3, and EHD4), which also contain a P-loop near the N terminus and a central coiled-coil domain. Analysis of cultured adipocytes stained with anti-GLUT4, anti-EHD1, and anti-EHD2 antibodies revealed that EHD1, but not EHD2, partially co-localizes with perinuclear GLUT4. Expression of a dominant-negative construct of EHD1 missing the EH domain (ΔEH-EHD1) markedly enlarged endosomes, dispersed perinuclear GLUT4-containing membranes throughout the cytoplasm, and inhibited GLUT4 translocation to the plasma membranes of 3T3-L1 adipocytes stimulated with insulin. Similarly, small interfering RNA-mediated depletion of endogenous EHD1 protein also markedly dispersed perinuclear GLUT4 in cultured adipocytes. Moreover, EHD1 is shown to interact through its EH domain with the protein EHBP1, which is also required for insulin-stimulated GLUT4 movements and hexose transport. In contrast, disruption of EHD2 function was without effect on GLUT4 localization or translocation to the plasma membrane. Taken together, these results show that EHD1 and EHBP1, but not EHD2, are required for perinuclear localization of GLUT4 and reveal that loss of EHBP1 disrupts insulin-regulated GLUT4 recycling in cultured adipocytes. Insulin stimulates glucose transport in skeletal muscle and adipose tissues by promoting the translocation of the glucose transporter GLUT4 from intracellular pools to the plasma membrane (1Czech M.P. Corvera S. J. Biol. Chem. 1999; 274: 1865-1868Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar, 2Pessin J.E. Thurmond D.C. Elmendorf J.S. Coker K.J. Okada S. J. Biol. Chem. 1999; 274: 2593-2596Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar, 3Simpson F. Whitehead J.P. James D.E. Traffic. 2001; 2: 2-11Crossref PubMed Scopus (84) Google Scholar, 4Watson R.T. Pessin J.E. Recent Prog. Horm. Res. 2001; 56: 175-193Crossref PubMed Scopus (188) Google Scholar, 5Rudich A. Klip A. Acta Physiol. Scand. 2003; 178: 297-308Crossref PubMed Scopus (61) Google Scholar, 6Holloszy J.O. Am. J. Physiol. 2003; 284: E453-E467Crossref PubMed Scopus (114) Google Scholar). Although this phenomenon was documented for the first time >20 years ago (7Cushman S.W. Wardzala L.J. 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J. 1994; 300: 631-635Crossref PubMed Scopus (330) Google Scholar) or depletion of Akt2 mediated by small interfering RNA (siRNA) 1The abbreviations used are: siRNA, small interfering RNA; ERC, endosomal recycling compartment; EH, Eps15 homology; HA, hemagglutinin; mEDH, murine EDH; YFP, yellow fluorescent protein; GST, glutathione S-transferase; CFP, cyan fluorescent protein; PBS, phosphate-buffered saline. 1The abbreviations used are: siRNA, small interfering RNA; ERC, endosomal recycling compartment; EH, Eps15 homology; HA, hemagglutinin; mEDH, murine EDH; YFP, yellow fluorescent protein; GST, glutathione S-transferase; CFP, cyan fluorescent protein; PBS, phosphate-buffered saline.-based gene silencing in 3T3-L1 adipocytes (15Jiang Z.Y. Zhou Q.L Coleman K.A. Chouinard M. Bose Q. Czech M.P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7569-7574Crossref PubMed Scopus (298) Google Scholar, 16Katome T. Obata T. Matsushima R. Masuyama N. Cantley L.C. Gotoh Y. Kishi K. Shiota H. Ebina Y. J. 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A number of fundamental questions about the membrane trafficking pathway of recycling GLUT4-containing membranes also remain unanswered. These include the following. 1) How does GLUT4 relate to the constitutive recycling pathway that is traversed by such proteins as the transferrin receptor? 2) What is the mechanism that retards movement of GLUT4 out of the perinuclear compartment in the basal state? 3) What are the site(s) in the GLUT4 recycling pathway regulated by insulin? In attempts to address these questions, several groups have reported that the trafficking of GLUT4 appears to partially overlap with the general endocytic recycling pathway, and several endosomal proteins have been identified in GLUT4 vesicles (19Kandror K.V. Coderre L. Pushkin A.V. Pilch P.F. Biochem. J. 1995; 307: 383-390Crossref PubMed Scopus (95) Google Scholar, 20Hashiramoto M. James D.E. Mol. Cell. Biol. 2000; 20: 416-427Crossref PubMed Scopus (91) Google Scholar, 21Guilherme A. Emoto M. Buxton J.M. Bose S. 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Evidence for a role of dynein during this latter step has been published recently, consistent with the hypothesis that the cytoskeleton plays a role in this process (10Patki V. Buxton J. Chawla A. Lifshitz L. Fogarty K. Carrington W. Tuft R. Corvera S. Mol. Biol. Cell. 2001; 12: 129-141Crossref PubMed Scopus (101) Google Scholar, 11Semiz S. Park J.G. Nicoloro S.M.C. Furcinitti P. Zhang C. Chawla A. Leszyk J. Czech M.P. EMBO J. 2003; 22: 2387-2399Crossref PubMed Scopus (128) Google Scholar, 21Guilherme 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 (104) Google Scholar, 25Huang J. Imamura T. Olefsky J.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13084-13089Crossref PubMed Scopus (108) Google Scholar). In the ERC, however, GLUT4 appears to be segregated away from the other recycling proteins that constitutively recycle back to the cell surface. 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James D.E. Mol. Biol. Cell. 2003; 14: 973-986Crossref PubMed Scopus (175) Google Scholar), although this appears to conflict with other data suggesting that a distinct compartment harbors insulin-sensitive GLUT4 (36Karylowski O. Zeigerer A. Cohen A. McGraw T.E. Mol. Biol. Cell. 2004; 15: 870-882Crossref PubMed Scopus (152) Google Scholar). Recently, results derived from several laboratories relating to a family of Eps15 homology (EH) domain-containing proteins, denoted EHD1-4 (37Guilherme A. Soriano N.P. Bose S. Holik J. Bose A. Pomerlau D.P. Furcinitti P. Leszyk J. Corvera S. Czech M.P. J. Biol. Chem. 2004; 279: 10593-10605Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 38Lin S.X. Grant B. Hirsh D. Maxfield F.R. Nat. Cell Biol. 2001; 3: 567-572Crossref PubMed Scopus (206) Google Scholar, 39Caplan 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 (239) Google Scholar, 40Picciano J.A. Ameen N. Grant B.D. Bradbury A.N. Am. J. Physiol. 2003; 285: C1009-C1018Crossref PubMed Scopus (76) Google Scholar, 41Rotem-Yehudar R. Galperin E. Horowitz M. J. Biol. Chem. 2001; 276: 33054-33060Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 42Shao 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 (141) Google Scholar, 43Galperin E. Benjamin S. Rapaport D. Rotem-Yehudar R. Tolchinsky S. Horowitz M. Traffic. 2002; 3: 557-589Crossref Scopus (67) Google Scholar), have converged to suggest a role for these proteins in regulated membrane recycling. Thus in Caenorhabditis elegans, a protein similar to EHD1, denoted RME-1, was found localized in perinuclear recycling membranes, and its disruption caused both redistribution of the endocytic recycling compartment and impaired transferrin recycling to the cell surface (38Lin S.X. Grant B. Hirsh D. Maxfield F.R. Nat. Cell Biol. 2001; 3: 567-572Crossref PubMed Scopus (206) Google Scholar). In mammalian cells, EHD1 was found to reside in a recycling membrane compartment related to exocytosis of major histocompatibility complex class I and the cystic fibrosis transmembrane conductance regulator (39Caplan 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 (239) Google Scholar, 40Picciano J.A. Ameen N. Grant B.D. Bradbury A.N. Am. J. Physiol. 2003; 285: C1009-C1018Crossref PubMed Scopus (76) Google Scholar) and to interact with EHD3 in a tubular membrane recycling compartment (43Galperin E. Benjamin S. Rapaport D. Rotem-Yehudar R. Tolchinsky S. Horowitz M. Traffic. 2002; 3: 557-589Crossref Scopus (67) Google Scholar). Findings in our laboratory showed that the related protein EHD2 localizes near the plasma membrane in actin-rich areas and is required for endocytosis of transferrin and the GLUT4 glucose transporter (37Guilherme A. Soriano N.P. Bose S. Holik J. Bose A. Pomerlau D.P. Furcinitti P. Leszyk J. Corvera S. Czech M.P. J. Biol. Chem. 2004; 279: 10593-10605Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). In this study, we therefore sought to determine whether EHD1 plays a role in GLUT4 recycling similar to its role in transferrin receptor trafficking and to compare the functions of EHD1 and EHD2 in cultured 3T3-L1 adipocytes. Here, we present evidence that EHD1 and its interacting protein EHBP1 (EH domain-binding protein-1), but not EHD2, are required for perinuclear sorting and insulin-regulated GLUT4 recycling in these cells. Loss of function of EHD1, mediated by expression of a dominant-negative construct or siRNA-based gene silencing, markedly enlarged the ERC, dispersed GLUT4 membranes, and impaired translocation and fusion of GLUT4 vesicles with the plasma membrane in response to insulin. Based on these results, we postulate that EHD1 is part of the cellular machinery necessary for normal sequestration and regulation of GLUT4-containing vesicles. Material and Chemicals—Anti-EHD1 antibody was a gift from Dr. Mia Horowitz (Tel Aviv University, Tel Aviv-Jaffa, Israel). Rabbit anti-EHD2, affinity-purified anti-EHBP1, and anti-hemagglutinin (HA) polyclonal antibodies were generated as described (37Guilherme A. Soriano N.P. Bose S. Holik J. Bose A. Pomerlau D.P. Furcinitti P. Leszyk J. Corvera S. Czech M.P. J. Biol. Chem. 2004; 279: 10593-10605Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Rabbit anti-Arp3 and mouse anti-phosphotyrosine (clone 4G10) antibodies were from Upstate Biotechnology, Inc. Goat anti-GLUT4 polyclonal (C-20) and mouse anti-insulin receptor monoclonal antibodies were from Santa Cruz Biotechnology, Inc. Mouse anti-HA monoclonal antibody was from Covance. Anti-phospho-Ser473 Akt and anti-Akt antibodies were from Cell Signaling Technology. Rabbit polyclonal antibody against Acrp30 (adipocyte complement-related protein of 30 kDa) was from Affinity BioReagents. Mouse anti-Myc epitope monoclonal antibody (clone 9E10) was conjugated to rhodamine red with a protein labeling kit (Molecular Probes, Inc.) following the manufacturer's specifications. Rhodamine-conjugated phalloidin and rhodamine-conjugated transferrin were from Molecular Probes, Inc. Cy3-conjugated mouse anti-goat and fluorescein isothiocyanate-conjugated mouse anti-rabbit secondary antibodies were from Jackson ImmunoResearch Laboratories, Inc. Anti-α-tubulin monoclonal antibody was from Amersham Biosciences. DNA Constructs—The murine expressed sequence tag encoding mouse EHD1 (GenBank™/EBI accession number BC037094) was obtained from American Type Culture Collection. The plasmid DNA was isolated and sequenced, and full-length mEHD1 was used to generate the different constructs in this study. The plasmid expressing yellow fluorescent protein (YFP)-tagged ΔEH-EHD1 (amino acids 1-442) was constructed by PCR amplification using primers creating XhoI and HindIII sites at the 5′- and 3′-ends, respectively. The PCR product was subcloned in-frame with a pEYFP-C3 vector. The plasmid expressing full-length HA-EHD1 was constructed using a linker (5′-ATAAGCTTGATGTTCAGCTGGGTGAGCAAGGATGCCCGCCGCAAGAAGGAGCCGGAGCTC-3′) with HindIII and SacI sites at the 5′- and 3′-ends, respectively, designed to place Ehd1 cDNA in-frame with the 3XHA vector. Full-length EHD1 in pCMV-sport6.0 was digested with SacI and XbaI, and this fragment was ligated with the linker into the HA vector to yield full-length HA-EHD1. The plasmid expressing HA-ΔEH-EHD1 was constructed by excising the ΔEH-EHD1 fragment (amino acids 1-442) from the YFP-ΔEH-EHD1 vector described above and subcloned in-frame with the 3XHA vector using the linker described above. The plasmids expressing YFP-ΔEH-EHD2, HA-EHD2, and HA-ΔEH-EHD2 were constructed as described (37Guilherme A. Soriano N.P. Bose S. Holik J. Bose A. Pomerlau D.P. Furcinitti P. Leszyk J. Corvera S. Czech M.P. J. Biol. Chem. 2004; 279: 10593-10605Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). For in vitro pull-down assays, full-length EHD1 or EHD2 or fragments were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli as described (37Guilherme A. Soriano N.P. Bose S. Holik J. Bose A. Pomerlau D.P. Furcinitti P. Leszyk J. Corvera S. Czech M.P. J. Biol. Chem. 2004; 279: 10593-10605Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). EHD2 (amino acids 1-543), ΔEH-EHD2 (amino acids 1-444), ΔEHΔA-EHD2 (amino acids 1-428), and the EH domain (amino acids 444-543) were PCR-amplified using primers generating BamHI and XhoI restriction sites at the 5′- and 3′-ends, respectively. ΔEH-EHD1 (amino acids 1-442) constructs were PCR-amplified using primers generating SalI and NotI restriction sites at the 5′- and 3′-ends, respectively. The PCR products were subcloned in-frame with a pGEX5×3 vector. The GST-VCA-expressing plasmid was similarly constructed by amplifying the region encoding amino acids 398-501 in rat neural Wiskott-Aldrich syndrome protein. All constructs were sequenced for verification prior to transfection. Cell Culture, Transfections, Myc-GLUT4-CFP Translocation, and Transferrin Uptake Assay—CHO-T cells expressing the human insulin receptor were grown in nutrient mixture F-12 supplemented with 10% fetal bovine serum. 3T3-L1 fibroblasts were differentiated into adipocytes as described (37Guilherme A. Soriano N.P. Bose S. Holik J. Bose A. Pomerlau D.P. Furcinitti P. Leszyk J. Corvera S. Czech M.P. J. Biol. Chem. 2004; 279: 10593-10605Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Transfection of 3T3-L1 adipocytes, COS-1 cells, and CHO-T cells with ΔEH-EHD1 or ΔEH-EHD2 was performed as described (37Guilherme A. Soriano N.P. Bose S. Holik J. Bose A. Pomerlau D.P. Furcinitti P. Leszyk J. Corvera S. Czech M.P. J. Biol. Chem. 2004; 279: 10593-10605Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Myc-GLUT4-CFP translocation assay and rhodamine-conjugated transferrin uptake assay have been described previously (37Guilherme A. Soriano N.P. Bose S. Holik J. Bose A. Pomerlau D.P. Furcinitti P. Leszyk J. Corvera S. Czech M.P. J. Biol. Chem. 2004; 279: 10593-10605Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). siRNA-induced Degradation of EHD1 and EHBP1—The siRNA species purchased from Dharmacon were designed to target the following cDNA sequences: scrambled, 5′-CAGTCGCGTTTGCGACTGG-3′; and EHD1 siRNA, 5′-CAGCCGAGGTTATGACTTT-3′; EHBP1 siRNA1, 5′-AAGCTCTTGCCACCAGCAGCATT-3′; EHBP1 siRNA2, 5′-AAGAGGAGAAGGCGGCAAAAATT-3′. 20 nmol of scrambled siRNA, 20 nmol of EHD1 siRNA, or 10 nmol of each of the EHBP1 siRNA species were electroporated into 3T3-L1 adipocytes as described (37Guilherme A. Soriano N.P. Bose S. Holik J. Bose A. Pomerlau D.P. Furcinitti P. Leszyk J. Corvera S. Czech M.P. J. Biol. Chem. 2004; 279: 10593-10605Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Briefly, adipocytes were detached from culture dishes with 0.25% trypsin in phosphate-buffered saline (PBS), washed twice, and resuspended in PBS. Half of the cells from one 150-mm dish were mixed with siRNA and then delivered to the cells by a pulse of electroporation with a Bio-Rad Gene Pulser II system at a setting of 0.18 kV and 950-microfarad capacitance. Using a Cy3-tagged siRNA for lamin A/C, we showed previously that Cy3-tagged siRNA was introduced with virtually 100% efficiency into cells using this method and that nearly all cells showed loss of nuclear lamin A/C (15Jiang Z.Y. Zhou Q.L Coleman K.A. Chouinard M. Bose Q. Czech M.P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7569-7574Crossref PubMed Scopus (298) Google Scholar). After 72 h, cells were harvested, and equal amounts of protein from different lysates were resolved by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. A portion of these cells were analyzed by immunofluorescence microscopy. Immunofluorescence Microscopy Analysis—For immunofluorescence microscopy analysis, differentiated 3T3-L1 adipocytes were transfected by electroporation as described (37Guilherme A. Soriano N.P. Bose S. Holik J. Bose A. Pomerlau D.P. Furcinitti P. Leszyk J. Corvera S. Czech M.P. J. Biol. Chem. 2004; 279: 10593-10605Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). The cells were then replated and allowed to recover for at least 24 h. To visualize endogenous EHD1, GLUT4, actin, and microtubules, 3T3-L1 adipocytes were fixed with 4% formaldehyde in PBS; permeabilized; and blocked with PBS containing 0.05% Triton X-100, 0.05% Tween 20, and 0.1% bovine serum albumin. Cells were incubated with the indicated primary antibody followed by a secondary antibody or rhodamine-conjugated phalloidin to detect F-actin. For analysis of co-localization of endogenous GLUT4 and EHD1, z-series stacks of images were deconvoluted and reconstituted using specialized software as described (37Guilherme A. Soriano N.P. Bose S. Holik J. Bose A. Pomerlau D.P. Furcinitti P. Leszyk J. Corvera S. Czech M.P. J. Biol. Chem. 2004; 279: 10593-10605Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). The co-localization of endogenous EHD1 with perinuclear GLUT4 (see Fig. 6A) and that of ΔEH-EHD1 with GLUT4 (see Fig. 7A) were measured using the Metamorph software package. A rectangular region of interest in a single z-plane of the deconvolved images of cells costained with anti-EHD1 and anti-GLUT4 antibodies (Figs. 6A and 7A) was drawn. An intensity threshold was set in each image, and the percentages of EHD1 and truncated EHD1 that overlapped with GLUT4 were measured in the boxed region. The percentages of GLUT4 that overlapped with EHD1 and truncated EHD1 were also similarly measured.Fig. 7Expression of ΔEH-EHD1, but not ΔEH-EHD2, disrupts perinuclear localization of GLUT4 in 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were transfected with YFP-ΔEH-EHD1 (A) or full-length EHD1 or YFP-ΔEH-EHD2 (B). After 24 h, cells were fixed, permeabilized, and stained with anti-GLUT4 antibody to visualize endogenous GLUT4. GLUT4-containing membranes were quite dispersed in cells expressing truncated EHD1 (A), but not in untransfected cells (arrows) or in cells expressing truncated EHD2 or full-length EHD1 (B). Overlay images (Merge) depict GLUT4 (red) in cells transfected with YFP constructs (green). In cells expressing YFP-ΔEH-EHD1, ring-shaped and tubular compartments containing both truncated EHD1 and GLUT4 at the cell periphery were observed. 80-90% of the GLUT4 protein in boxed regions a and b co-localized with YFP-ΔEH-EHD1. The number at the top right of each panel indicates the distance (in micrometers) from the bottom. Enlargements of the areas containing these structures in the merged images are depicted in panels a and b.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Analysis of Interaction of the Arp2/3 Complex and EHD Protein Variants—GST or GST-tagged EHD variants (25 μg) immobilized on glutathione beads were mixed with 0.3 mg of cytosolic fractions from 3T3-L1 adipocytes in buffer A (PBS containing 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, and 5 μg/ml aprotinin). The samples were incubated at 4 °C end-over-end, and the beads were washed five times with buffer A and then boiled in SDS sample buffer. The Arp2/3 complex was detected by immunoblotting using anti-Arp3 polyclonal antibody after resolution by SDS-PAGE. 2-Deoxyglucose Uptake Assay—Insulin-stimulated glucose transport in 3T3-L1 adipocytes was estimated by measuring 2-deoxyglucose uptake as described (9Bose 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 (213) Google Scholar, 15Jiang Z.Y. Zhou Q.L Coleman K.A. Chouinard M. Bose Q. Czech M.P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7569-7574Crossref PubMed Scopus (298) Google Scholar). Briefly, siRNA-transfected cells were reseeded on 24-well plates, cultured for 72 h, washed twice, and starved for 2 h with Krebs-Ringer Hepes buffer (130 mm NaCl, 5 mm KCl, 1.3 mm CaCl2, 1.3 mm MgSO4, and 25 mm Hepes, pH 7.4) supplemented with 0.5% bovine serum albumin and 2 mm sodium pyruvate. Cells were then stimulated with insulin for 30 min at 37 °C. Glucose uptake was initiated by addition of 2-[1,2-3H]deoxy-d-glucose to a final assay concentration of 100 μm for 5 min at 37 °C. Assays were terminated by three washes with ice-cold Krebs-Ringer Hepes buffer; the cells were solubilized with 0.4 ml of 1% SDS; and 3H was determined by scintillation counting. Nonspecific deoxyglucose uptake was measured in the presence of 20 μm cytochalasin B and subtracted from each determination to obtain specific uptake. Distinct Functions of EHD1 Versus EHD2 in Recycling Endosomes—To compare the cellular localization of EHD1 and EHD2 proteins, HA-tagged constructs of these proteins were expressed at low levels in COS-1 cells (Fig. 1A). Detectio

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