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• W2000975813 abstract "The GLUT4 glucose transporter continuously recycles between the cell surface and an endosomal compartment in adipocytes. Insulin decreases the rate of GLUT4 endocytosis in addition to increasing its exocytosis. Endocytosis of the transporter is thought to occur at least in part via the clathrin-mediated endocytic system. The protein dynamin is involved in the final stages of clathrin-coated vesicle formation. Here we show that the dynamin II isoform is expressed in 3T3-L1 adipocytes and is present in isolated plasma membrane and low density microsomal fractions. Insulin reduced the levels of dynamin II associated with the plasma membrane by about half, raising the possibility that the hormone may reduce GLUT4 endocytosis by removing dynamin from the cell surface. A fusion protein containing the amphiphysin SH3 domain selectively bound dynamin II from 3T3-L1 adipocyte cell lysates. Microinjection of the fusion protein into these cells inhibited transferrin endocytosis and increased the levels of GLUT4 at the cell surface. GlutathioneS-transferase alone, the SH3 domains of spectrin and Crk, and a mutated amphiphysin SH3 domain unable to bind dynamin II did not affect GLUT4 distribution. However, a peptide containing the dynamin II sequence that binds amphiphysin increased the surface presence of GLUT4. Moreover, in cells first treated with insulin to externalize GLUT4, the dynamin peptide, but not an unrelated control peptide, inhibited GLUT4 internalization upon insulin removal. These results suggest that interactions of dynamin II with amphiphysin may play an important role in GLUT4 endocytosis. We hypothesize that insulin may reduce GLUT4 endocytosis by regulating the function of dynamin II at the cell surface, as part of the mechanism to increase glucose uptake. The GLUT4 glucose transporter continuously recycles between the cell surface and an endosomal compartment in adipocytes. Insulin decreases the rate of GLUT4 endocytosis in addition to increasing its exocytosis. Endocytosis of the transporter is thought to occur at least in part via the clathrin-mediated endocytic system. The protein dynamin is involved in the final stages of clathrin-coated vesicle formation. Here we show that the dynamin II isoform is expressed in 3T3-L1 adipocytes and is present in isolated plasma membrane and low density microsomal fractions. Insulin reduced the levels of dynamin II associated with the plasma membrane by about half, raising the possibility that the hormone may reduce GLUT4 endocytosis by removing dynamin from the cell surface. A fusion protein containing the amphiphysin SH3 domain selectively bound dynamin II from 3T3-L1 adipocyte cell lysates. Microinjection of the fusion protein into these cells inhibited transferrin endocytosis and increased the levels of GLUT4 at the cell surface. GlutathioneS-transferase alone, the SH3 domains of spectrin and Crk, and a mutated amphiphysin SH3 domain unable to bind dynamin II did not affect GLUT4 distribution. However, a peptide containing the dynamin II sequence that binds amphiphysin increased the surface presence of GLUT4. Moreover, in cells first treated with insulin to externalize GLUT4, the dynamin peptide, but not an unrelated control peptide, inhibited GLUT4 internalization upon insulin removal. These results suggest that interactions of dynamin II with amphiphysin may play an important role in GLUT4 endocytosis. We hypothesize that insulin may reduce GLUT4 endocytosis by regulating the function of dynamin II at the cell surface, as part of the mechanism to increase glucose uptake. The GLUT4 glucose transporter is the major insulin-responsive transporter of muscle and fat tissues. In the basal state, it is largely sequestered in an intracellular compartment(s); however, several studies have shown that the transporter constitutively recycles between the cell surface and the intracellular loci (1Jhun B.H. Rampal A.L. Liu H. Lachaal M. Jung C.Y. J. Biol. Chem. 1992; 267: 17710-17715Abstract Full Text PDF PubMed Google Scholar, 2Yang J. Holman G.D. J. Biol. Chem. 1993; 268: 4600-4603Abstract Full Text PDF PubMed Google Scholar, 3Satoh S. Nishimura H. Clark A.E. Kozka I.J. Vannucci S.J. Simpson I.A. Quon M.J. Cushman S.W. Holman G.D. J. Biol. Chem. 1993; 268: 17820-17829Abstract Full Text PDF PubMed Google Scholar). There is ample evidence suggesting that the GLUT4 glucose transporter is internalized via clathrin-coated pits. GLUT4 has been localized to clathrin-coated regions by immunocytochemistry of plasma membrane lawns (4Robinson L.J. Pang S. Harris D.S. Heuser J. James D.E. J. Cell Biol. 1992; 117: 1181-1196Crossref PubMed Scopus (254) Google Scholar) and by immunoblotting of isolated clathrin-coated vesicles (5Chakrabarti R. Buxton J. Joly M. Corvera S. J. Biol. Chem. 1994; 269: 7926-7933Abstract Full Text PDF PubMed Google Scholar). Furthermore, lowering the intracellular K+ concentration, which disassembles clathrin lattices and prevents their reassembly, causes an accumulation of GLUT4 at the cell surface, presumably as a result of inhibiting the endocytic arm of its continuous recycling (6Nishimura H. Zarnowski M.J. Simpson I.A. J. Biol. Chem. 1993; 268: 19246-19253Abstract Full Text PDF PubMed Google Scholar). Within its N-terminal domain, GLUT4 contains a putative internalization motif (FQQI) that is similar to the internalization consensus sequence YXXΦ (where X represents any amino acid and Φ represents bulky hydrophobic residues) present in numerous recycling proteins (7Marks M.S. Ohno H. Kirchhausen T. Bonifacino J.S. Trends Cell Biol. 1997; 7: 124-128Abstract Full Text PDF PubMed Scopus (277) Google Scholar). GLUT4 also contains a dileucine motif near its C terminus. Both motifs have been shown to contribute to the internalization of GLUT4 (8Garippa R.J. Judge T.W. James D.E. McGraw T.E. J. Cell Biol. 1994; 124: 705-715Crossref PubMed Scopus (69) Google Scholar, 9Corvera S. Chawla A. Chakrabarti R. Joly M. Buxton J. Czech M.P. J. Cell Biol. 1994; 126: 979-989Crossref PubMed Scopus (103) Google Scholar, 10Haney P.M. Levy M.A. Strube M.S. Mueckler M. J. Cell Biol. 1995; 129: 641-658Crossref PubMed Scopus (86) Google Scholar, 11Marsh B.J. Alm R.A. McIntosh S.R. James D.E. J. Cell Biol. 1995; 130: 1081-1091Crossref PubMed Scopus (75) Google Scholar, 12Verhey K.J. Yeh J.-I. Birnbaum M.J. J. Cell Biol. 1995; 130: 1071-1079Crossref PubMed Scopus (114) Google Scholar, 13Garippa R.J. Johnson A. Park J. Petrush R.L. McGraw T.E. J. Biol. Chem. 1996; 271: 20660-20668Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Upon insulin stimulation, GLUT4-containing vesicles are mobilized to the cell surface (14James D.E. Strube M. Mueckler M. Nature. 1989; 338: 83-87Crossref PubMed Scopus (667) Google Scholar, 15Birnbaum M.J. Cell. 1989; 57: 305-315Abstract Full Text PDF PubMed Scopus (458) Google Scholar, 16Zorzano A. Wilkinson W. Kotliar N. Thoidis G. Wadzinkski B.E. Ruoho A.E. Pilch P.F. J. Biol. Chem. 1989; 264: 12358-12363Abstract Full Text PDF PubMed Google Scholar). In addition to increasing the rate of exocytosis of transporters, insulin also increases GLUT4 surface levels by reducing their endocytosis. Using an impermeant photoactivable glucose analog, it was observed that insulin lowered the rate of internalization of GLUT4 in rat adipocytes by 2.8-fold (1Jhun B.H. Rampal A.L. Liu H. Lachaal M. Jung C.Y. J. Biol. Chem. 1992; 267: 17710-17715Abstract Full Text PDF PubMed Google Scholar). A similar conclusion was reached when the amount of GLUT4 at the cell surface was measured by taking advantage of its exofacial trypsin-sensitive site in 3T3-L1 adipocytes (17Czech M.P. Buxton J.M. J. Biol. Chem. 1993; 268: 9187-9190Abstract Full Text PDF PubMed Google Scholar). Moreover, insulin reduced the amount of GLUT4 associated with plasma membrane-derived clathrin-coated vesicles in the same cells (4Robinson L.J. Pang S. Harris D.S. Heuser J. James D.E. J. Cell Biol. 1992; 117: 1181-1196Crossref PubMed Scopus (254) Google Scholar, 5Chakrabarti R. Buxton J. Joly M. Corvera S. J. Biol. Chem. 1994; 269: 7926-7933Abstract Full Text PDF PubMed Google Scholar). The final event in the formation of the clathrin vesicles at the plasma membrane is the periplasmic fusion at the neck of the newly formed pit. This is thought to involve the 100-kDa GTPase dynamin since transfection of a dominant-negative mutant dynamin I, unable to bind and hydrolyze GTP, results in inhibition of clathrin-mediated endocytosis in mammalian cells (18Herskovits J.S. Burgess C.C. Obar R.A. Vallee R.B. J. Cell Biol. 1993; 122: 565-578Crossref PubMed Scopus (394) Google Scholar, 19van der Bliek A.M. Redelmeier T.E. Damke H. Tisdale E.J. Meyerowitz E.M. Schmid S.L. J. Cell Biol. 1993; 122: 553-563Crossref PubMed Scopus (585) Google Scholar, 20Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1031) Google Scholar, 21Damke H. Baba T. van der Bliek A.M. Schmid S.L. J. Cell Biol. 1995; 131: 69-80Crossref PubMed Scopus (342) Google Scholar). Furthermore, GTPγS 1The abbreviations used are: GTPγS, guanosine 5′-O-(3-thiotriphosphate); PM, plasma membrane(s); LDM, low density microsome(s); HDM, high density microsome(s); GST, glutathioneS-transferase; PBS, phosphate-buffered saline solution. 1The abbreviations used are: GTPγS, guanosine 5′-O-(3-thiotriphosphate); PM, plasma membrane(s); LDM, low density microsome(s); HDM, high density microsome(s); GST, glutathioneS-transferase; PBS, phosphate-buffered saline solution. was found to arrest synaptic vesicle endocytosis at the stage of invaginated clathrin-coated pits and to induce an accumulation of dynamin at the neck of these structures (22Takei K. McPherson P.S. Schmid S.L. De Camilli P. Nature. 1995; 374: 186-190Crossref PubMed Scopus (648) Google Scholar). In addition to a GTP-binding domain, dynamin I contains a pleckstrin homology domain and a proline-rich region at its C terminus that binds a specific subset of SH3 domain-containing proteins (23Gout I. Dhand R. Hiles I.D. Fry M.J. Panayotou G. Das P. Truong O. Totty N.F. Hsuan J. Booker G.W. Campbell I.D. Waterfield M.D. Cell. 1993; 75: 25-36Abstract Full Text PDF PubMed Scopus (482) Google Scholar, 24Miki H. Miura K. Matuoka K. Nakata T. Hirokawa N. Orita S. Kaibuchi K. Takai Y. Takenawa T. J. Biol. Chem. 1994; 269: 5489-5492Abstract Full Text PDF PubMed Google Scholar, 25Scaife R. Gout I. Waterfield M.D. Margolis R.L. EMBO J. 1994; 13: 2574-2582Crossref PubMed Scopus (77) Google Scholar, 26Seedorf K. Kostka G. Lammers R. Bashkin P. Daly R. Burgess W.H. van der Bliek A.M. Schlessinger J. Ullrich A. J. Biol. Chem. 1994; 269: 16009-16014Abstract Full Text PDF PubMed Google Scholar, 27David C. McPherson P.S. Mundigl O. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 331-335Crossref PubMed Scopus (349) Google Scholar, 28Okamoto P.M. Herskovits J.S. Vallee R.B. J. Biol. Chem. 1997; 272: 11629-11635Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 29Grabs D. Slepnev V.I. Songyang Z. David C. Lynch M. Cantley L.C. De Camilli P. J. Biol. Chem. 1997; 272: 13419-13425Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). One of the proteins that binds with great specificity to the proline-rich region of dynamin is amphiphysin (27David C. McPherson P.S. Mundigl O. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 331-335Crossref PubMed Scopus (349) Google Scholar). This interaction with amphiphysin is thought to play a role in the recruitment of dynamin to sites of endocytosis (27David C. McPherson P.S. Mundigl O. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 331-335Crossref PubMed Scopus (349) Google Scholar, 28Okamoto P.M. Herskovits J.S. Vallee R.B. J. Biol. Chem. 1997; 272: 11629-11635Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar,30Shupliakov O. Low P. Grabs D. Gad H. Chen H. David C. Takei K. De Camilli P. Brodin L. Science. 1997; 276: 259-263Crossref PubMed Scopus (396) Google Scholar). In this study, we have investigated the effect of disrupting SH3-mediated interactions of dynamin on the steady-state distribution of GLUT4 in 3T3-L1 adipocytes by microinjecting the SH3 domain of amphiphysin or a peptide of dynamin containing the amphiphysin-binding region. Mouse monoclonal anti-dynamin II-specific antibody was from Transduction Laboratories (Lexington, KY). Rabbit polyclonal anti-GLUT4 antibody was from Charles River Laboratories (Southbridge, MA). Monoclonal anti-clathrin heavy chain antibody was from Boehringer Mannheim (Laval, Quebec, Canada). Monoclonal anti-α1-Na/K-ATPase antibody 6H was a gift from Dr. M. Caplan (Yale University, New Haven, CT). Rhodamine-dextran (Mr 10,000) was from Molecular Probes, Inc. (Eugene, OR). 3T3-L1 fibroblasts were grown and induced to differentiate as described previously (31Volchuk A. Wang Q. Ewart H.S. Liu Z. He L. Bennett M.K. Klip A. Mol. Biol. Cell. 1996; 7: 1075-1082Crossref PubMed Scopus (126) Google Scholar). Four to six days after differentiation, cultures were deprived of serum for 2 h and stimulated with 100 nminsulin for 20 min at 37 °C, as indicated in the figure legends. Total membranes and subcellular fractions (PM, LDM, HDM, and cytosol) from control and insulin-stimulated cells were prepared as described previously (31Volchuk A. Wang Q. Ewart H.S. Liu Z. He L. Bennett M.K. Klip A. Mol. Biol. Cell. 1996; 7: 1075-1082Crossref PubMed Scopus (126) Google Scholar), except that cell breakage was performed with 10 cycles through a ball-bearing cell cracker (32Balch W.E. Dunphy W.G. Braell W.A. Rothman J.E. Cell. 1984; 39: 405-416Abstract Full Text PDF PubMed Scopus (477) Google Scholar) with a clearance of 0.0016 inches using 5 ml of homogenization buffer/10-cm dish. 3T3-L1 cell fractions and rat brain microsomes (33Nagamatsu S. Kornhauser J.M. Burant C.F. Seino S. Mayo K.E. Bell G.I. J. Biol. Chem. 1992; 267: 467-472Abstract Full Text PDF PubMed Google Scholar) were resolved by SDS-polyacrylamide gel electrophoresis and immunoblotted essentially as described earlier (34Volchuk A. Sargeant R. Sumitani S. Liu Z. He L. Klip A. J. Biol. Chem. 1995; 270: 8233-8240Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) using dynamin II, GLUT4, and clathrin heavy chain antibodies at 1:250, 1:1000, and 1:500 dilutions, respectively. A fusion protein comprising the SH3 domain of amphiphysin 1 linked to GST (construct V (residues 545–695), here called GST-AmphiSH3) was generated as described previously (27David C. McPherson P.S. Mundigl O. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 331-335Crossref PubMed Scopus (349) Google Scholar, 35David C. Solimena M. De Camilli P. FEBS Lett. 1994; 351: 73-79Crossref PubMed Scopus (128) Google Scholar). A fusion protein expressing two mutations (G684R and P687L) in the amphiphysin 1 SH3 domain (GST-AmphiSH3m) was generated as described (30Shupliakov O. Low P. Grabs D. Gad H. Chen H. David C. Takei K. De Camilli P. Brodin L. Science. 1997; 276: 259-263Crossref PubMed Scopus (396) Google Scholar). Two GST fusion proteins encoding the SH3 domains of spectrin and Crk were generous gifts from Dr. A. Pawson (Mount Sinai Hospital, Toronto) (23Gout I. Dhand R. Hiles I.D. Fry M.J. Panayotou G. Das P. Truong O. Totty N.F. Hsuan J. Booker G.W. Campbell I.D. Waterfield M.D. Cell. 1993; 75: 25-36Abstract Full Text PDF PubMed Scopus (482) Google Scholar). A 15-oligomer peptide (PPPQVPSRPNRAPPG) representing amino acids 828–842 of dynamin Iaa containing the amphiphysin SH3 domain-binding site was synthesized (30Shupliakov O. Low P. Grabs D. Gad H. Chen H. David C. Takei K. De Camilli P. Brodin L. Science. 1997; 276: 259-263Crossref PubMed Scopus (396) Google Scholar). This sequence is highly conserved in dynamin II, which has recently been shown to interact with amphiphysin (28Okamoto P.M. Herskovits J.S. Vallee R.B. J. Biol. Chem. 1997; 272: 11629-11635Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). A 19-oligomer peptide (CVRRASEPGNRKGRLGNEK) (generous gift from Dr. S. Grinstein, Hospital for Sick Children, Toronto) was used as an unrelated control peptide. 3T3-L1 adipocytes were washed twice in PBS and lysed with 1.2 ml of lysis buffer/10-cm dish (lysis buffer = 20 mm HEPES, 100 mm KCl, 1% Triton X-100, 1 mm dithiothreitol, 2 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride, 1 μmpepstatin A, pH 7.4). Cells were placed on a shaker at 4 °C for 30 min, scraped, passed 10 times through a 25-gauge syringe, and centrifuged for 10 min at 4 °C at 12,000 rpm in a microcentrifuge. The supernatant (lysate) was removed, and the protein concentration was measured by the BCA method (Pierce). Forty micrograms of GST, GST-AmphiSH3, or GST-AmphiSH3m were linked to glutathione-agarose beads by incubation at 4 °C for 3 h with gentle tumbling, followed by three washes with lysis buffer. To the beads was added 1 mg of adipocyte lysate, and incubation was continued with tumbling for 3 h at 4 °C. In competition experiments, the dynamin peptide was added together with the lysate at a final concentration of 300 μm. Beads were washed four times in lysis buffer, and bound material was eluted with 25 μl of 2× concentrated sample buffer and subjected to electrophoresis. 3T3-L1 cells were grown and differentiated on 25-mm diameter coverslips placed in 6-well dishes. Coverslips were placed in coverslip chambers (Medical Systems Corp., Greenvale, NY) containing 1 ml of RPMI 1640 medium (R 4130 Sigma) supplemented with 20 mm HEPES and 4% fetal bovine serum. A region of ∼1 mm2 on the bottom of the coverslip was marked for subsequent microinjection on the stage of a Nikon fluorescence microscope resting on a Newport BenchTop vibration isolation system fitted with an Eppendorf microinjection unit (Micromanipulator 5171 and Transjector 5246). Borosilicate microinjection pipettes (World Precision Instruments, Inc.) were pulled using a Sutter Instrument Flaming/Brown micropipette puller (Model p-97). The majority (∼90%) of the cells in the marked area (typically 100–150 cells) were microinjected with a solution containing 2.0 mg/ml GST, GST-AmphiSH3, GST-AmphiSH3m, GST-spectrinSH3, or GST-CrkSH3, plus rhodamine-dextran (1.1 mg/ml) in microinjection buffer (110 mm potassium acetate, pH 7.2, 10 mmHEPES, 1 mm EDTA). The molar concentrations of these proteins in the microinjection solution were 75, 42, 41, 61, and 61 μm, respectively. When the dynamin peptide or the unrelated peptide was microinjected, the concentration of the peptide in this buffer was 17 mm. The medium was then changed to Dulbecco's minimal essential medium containing 10% fetal bovine serum, and cells were incubated at 37 °C for 30 min, followed by a 2-h incubation in Dulbecco's minimal essential medium alone. Cells in the injected and non-injected areas of the same coverslip were photographed 1.5 h after microinjection with phase-contrast and fluorescence optics under a 40× objective. Plasma membrane lawns (sheets) were prepared by a modification of the procedure of Robinson et al. (4Robinson L.J. Pang S. Harris D.S. Heuser J. James D.E. J. Cell Biol. 1992; 117: 1181-1196Crossref PubMed Scopus (254) Google Scholar). Following the various treatments, cells were placed on ice and washed twice in ice-cold PBS. Hypotonic swelling buffer (23 mm KCl, 10 mmHEPES, 2 mm MgCl2, 1 mm EGTA, pH 7.5) was added in three quick rinses. Five milliliters of breaking buffer (70 mm KCl, 30 mm HEPES, 5 mm MgCl2, 3 mm EGTA, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 1 μm pepstatin A, pH 7.5) were added, and the solution was aspirated up and down using a 1.0-ml pipettor to promote cell breakage. The adhered plasma membrane lawns were washed three times in breaking buffer and incubated with cold 3% paraformaldehyde in breaking buffer for 10 min on ice. The coverslips were washed three times in PBS, and excess fixative was quenched with 50 mmNH4Cl/PBS for 5 min, followed by three washes with PBS at room temperature. The lawns were subsequently blocked by a 1-h incubation in 5% goat serum in PBS at room temperature. Labeling with rabbit anti-GLUT4 antiserum (1:150) for 30 min at room temperature ensued, followed by three washes with PBS and labeling with fluorescein isothiocyanate-conjugated donkey anti-rabbit antiserum (1:50) for 30 min. Immunolabeled lawns were rinsed four times with PBS and mounted with ProLong Antifade mounting solution (Molecular Probes, Inc.). Confocal images were obtained using a Leica TCS 4D laser confocal fluorescence microscope with a 63× objective. All images were collected under identical gain settings established in preliminary experiments. Confocal images were processed with Adobe Photoshop software for figure production or used to quantitate fluorescence intensity using NIH Imaging software. For quantitation, the fluorescence/unit area of each lawn was measured in two to four fields of microinjected and non-injected cells (each field contained 8–15 lawns). The results were collected in arbitrary units, and the S.E. for each experimental condition was calculated. Because each experimental condition had its own control (basal non-injected cells), once the S.E. values were calculated for the fluorescence intensity/unit area, the results were normalized to that control. Statistical analysis was by analysis of variance. Monolayers of 3T3-L1 fibroblasts or adipocytes on glass coverslips were microinjected with GST-AmphiSH3 or GST-AmphiSH3m as described above, except that detection of injection was achieved by co-injection of lucifer yellow (0.5 mg/ml). Following microinjection, cells were serum-deprived for 1 h and then exposed to 0.25 μg/ml rhodamine-labeled transferrin for 1 h at 37 °C (18Herskovits J.S. Burgess C.C. Obar R.A. Vallee R.B. J. Cell Biol. 1993; 122: 565-578Crossref PubMed Scopus (394) Google Scholar, 36Wigge P. Vallis Y. McMahon H.T. Curr. Biol. 1997; 7: 554-560Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Control experiments were performed at 4 °C. Coverslips were washed three times in PBS, fixed in 3% paraformaldehyde/PBS for 1 h, and mounted as described above. Confocal images were obtained as above using a 63× objective. Three mammalian dynamin genes have been cloned, each with several alternatively spliced forms (37Liu J.-P. Robinson P.J. Endocr. Rev. 1995; 16: 590-607PubMed Google Scholar, 38Urrutia R. Henley J.R. Cook T. McNiven M.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 377-384Crossref PubMed Scopus (253) Google Scholar). Dynamin I is expressed exclusively in brain (39Robinson P.J. Sontag J.-M. Liu J.-P. Fykse E.M. Slaughter C. McMahon H. Sudhof T.C. Nature. 1993; 365: 163-166Crossref PubMed Scopus (235) Google Scholar); dynamin III is present abundantly in testis, with lower amounts present in brain and lung (40Nakata T. Takemura R. Hirokawa N. J. Cell Sci. 1993; 105: 1-5Crossref PubMed Google Scholar, 41Cook T. Mesa K. Urrutia R. J. Neurochem. 1996; 67: 927-931Crossref PubMed Scopus (75) Google Scholar); and dynamin II was found to varying degrees in all tissues tested (42Sontag J.-M. Fykse E.M. Ushkaryov Y. Liu J.-P. Robinson P.J. Sudhof T.C. J. Biol. Chem. 1994; 269: 4547-4554Abstract Full Text PDF PubMed Google Scholar, 43Cook T.A. Urrutia R. McNiven M.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 644-648Crossref PubMed Scopus (160) Google Scholar). Dynamin I could not be detected in 3T3-L1 adipocytes using an isoform-specific antibody (data not shown). Using an antibody specific to dynamin II, we examined whether this protein is present in these cells. In Fig.1 A, the content of dynamin II is compared in total membranes from undifferentiated 3T3-L1 fibroblasts and differentiated 3T3-L1 adipocytes. The protein was readily detected at both stages of differentiation to comparable extents. By comparison, relatively little dynamin II was detected in total brain microsomes. Dynamin II localization in 3T3-L1 adipocytes was further analyzed by subcellular fractionation. Fig. 1 B shows the presence of different proteins in subcellular fractions isolated from unstimulated (basal) and insulin-stimulated 3T3-L1 adipocytes. Clathrin was found largely in the cytosol and to varying degrees in the membrane fractions. Its distribution was not affected by insulin stimulation. As expected, the cell-surface marker α1-Na/K-ATPase was found almost exclusively in the PM, and its distribution was also not affected by insulin stimulation. Dynamin II was detected in all membrane fractions, but was scarce in the cytosol. The concentration of dynamin II/unit protein was similar in the PM, LDM, and HDM fractions from unstimulated cells. Interestingly, insulin stimulation resulted in a decrease in dynamin II content in the PM of 44 ± 10% (n = 4), with small gains in both the LDM and HDM. In contrast, and confirming previous reports, the GLUT4 glucose transporter was most abundant in the LDM fraction in unstimulated cells, and its concentration dropped in this fraction and increased in the PM as a result of insulin stimulation. To assess whether dynamin plays a role in GLUT4 traffic, we attempted to interfere with its action by using the fusion protein GST-AmphiSH3, a construct that was recently shown to block synaptic vesicle endocytosis in lamprey axons (30Shupliakov O. Low P. Grabs D. Gad H. Chen H. David C. Takei K. De Camilli P. Brodin L. Science. 1997; 276: 259-263Crossref PubMed Scopus (396) Google Scholar). To this effect, it was important to establish that this fusion protein, which encompasses the SH3 domain of the human amphiphysin 1 cDNA, can indeed bind the dynamin isoform present in 3T3-L1 adipocytes. Therefore, Triton X-100 lysates of 3T3-L1 adipocytes were incubated with glutathione-containing agarose beads bound to either GST or GST-AmphiSH3. Fig.2 A shows the protein profile of the cell lysate and of the material bound to both types of beads. The GST pellet showed the presence of a few proteins, which are considered to bind nonspecifically to GST-glutathione-agarose. The GST-AmphiSH3 pellet showed three additional proteins of ∼100, 45, and 30 kDa. The 100-kDa protein reacted with the anti-dynamin II-specific antibody (Fig. 2 B). The 45- and 30-kDa bands reacted with the anti-GST antibody and are therefore likely degradation products of the fusion protein. The experiment was repeated using GST-AmphiSH3m (containing the point mutations G684R and P687L). No dynamin was sedimented by this construct (Fig. 2 C), demonstrating the specificity of GST-AmphiSH3 to bind this protein. To verify that GST-AmphiSH3 interferes with endocytosis of proteins that internalize via clathrin-coated pits, we incubated 3T3-L1 adipocytes with fluorescently labeled transferrin. The presence of intracellular transferrin was used as a measure of its endocytosis. Under the conditions of this assay, very little transferrin is left at the cell surface upon washing prior to fixation (18Herskovits J.S. Burgess C.C. Obar R.A. Vallee R.B. J. Cell Biol. 1993; 122: 565-578Crossref PubMed Scopus (394) Google Scholar, 36Wigge P. Vallis Y. McMahon H.T. Curr. Biol. 1997; 7: 554-560Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). The internalized fluorescence was examined in cells that were microinjected with GST-AmphiSH3 as well as in the non-injected cells on the same coverslips (Fig. 3). On coverslips incubated at 37 °C, the distribution of transferrin in the non-injected cells was punctate within the cytoplasm and concentrated in the perinuclear region. On the same coverslips, cells microinjected with GST-AmphiSH3 showed a marked reduction in intracellular transferrin; the perinuclear staining was no longer observed; and the cytoplasmic staining was equal to that observed in non-injected cells incubated at 4 °C. Control experiments with 3T3-L1 fibroblasts confirmed that GST-AmphiSH3 prevented transferrin endocytosis and that this effect was not reproduced by GST-AmphiSH3m (data not shown). GST-AmphiSH3 was then microinjected into 3T3-L1 adipocytes to assess its effects on cell-surface GLUT4 levels. The microinjected 3T3-L1 adipocytes were allowed to recover for 30 min and deprived of serum for 2 h prior to generation of plasma membrane lawns. Fig.4 A illustrates one experiment representative of three. As anticipated, in a non-injected region of the coverslip (first column), the level of GLUT4 immunofluorescence on plasma membrane lawns was low (bottom panel), indicating that only a small amount of GLUT4 is present at the surface of unstimulated cells. Approximately 80% of the microinjected cells in the 1-mm2 marked region of the coverslip clearly retained the microinjected rhodamine-dextran marker and were thus considered to be intact and retaining microinjected material (second column, middle panel). In the plasma membrane lawns of unstimulated cells that were microinjected with GST-AmphiSH3, the GLUT4 immunofluorescence was higher in many of the lawns compared with those of non-injected cells (second column, bottom panel). This suggests that the fusion protein elevated the surface content of GLUT4. Microinjection of GST did not affect the GLUT4 immunofluorescence compared with non-injected cells (third and fourth columns, bottom panels). Fig. 4 B shows one experiment representative of three in which GLUT4 immunofluorescence was detected in the lawn of cells microinjected with GST-AmphiSH3m. As with GST alone, the signal was not different from that in control cells. By comparison, insulin stimulation of cells resulted in a marked gain in surface GLUT4. The quantitated results of all experiments are shown in Fig. 4 C. Microinjection of GST-AmphiSH3 significantly doubled the amount of GLUT4 at the plasma membrane (p < 0.005). In contrast, neither GST nor GST-AmphiSH3m significantly affected the amount of surface GLUT4. Insulin stimulation of intact cells increased the amount of GLUT4 at the cell surface by ∼3.5-" @default.
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• W2000975813 title "Perturbation of Dynamin II with an Amphiphysin SH3 Domain Increases GLUT4 Glucose Transporters at the Plasma Membrane in 3T3-L1 Adipocytes" @default.
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• W2000975813 doi "https://doi.org/10.1074/jbc.273.14.8169" @default.
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