FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1537397787> ?p ?o ?g. }

• W1537397787 endingPage "48465" @default.
• W1537397787 startingPage "48458" @default.
• W1537397787 abstract "Dynamin is a GTPase involved in endocytosis and other aspects of membrane trafficking. A critical function in the presynaptic compartment attributed to the brain-specific dynamin isoform, dynamin-1, is in synaptic vesicle recycling. We report that dynamin-2 specifically interacts with members of the Shank/ProSAP family of postsynaptic density scaffolding proteins and present evidence that dynamin-2 is specifically associated with the postsynaptic density. These data are consistent with a role for this otherwise broadly distributed form of dynamin in glutamate receptor down-regulation and other aspects of postsynaptic membrane turnover. Dynamin is a GTPase involved in endocytosis and other aspects of membrane trafficking. A critical function in the presynaptic compartment attributed to the brain-specific dynamin isoform, dynamin-1, is in synaptic vesicle recycling. We report that dynamin-2 specifically interacts with members of the Shank/ProSAP family of postsynaptic density scaffolding proteins and present evidence that dynamin-2 is specifically associated with the postsynaptic density. These data are consistent with a role for this otherwise broadly distributed form of dynamin in glutamate receptor down-regulation and other aspects of postsynaptic membrane turnover. Src homology 3 postsynaptic density amino acid(s) polymerase chain reaction glutathione S-transferase hemagglutinin Dynamin is a 100-kDa GTPase (1Obar R.A. Collins C.A. Hammarback J.A. Shpetner H.S. Vallee R.B. Nature. 1990; 347: 256-261Crossref PubMed Scopus (284) Google Scholar, 2Shpetner H.S. Vallee R.B. Nature. 1992; 355: 733-735Crossref PubMed Scopus (169) Google Scholar) that controls a variety of vesicular budding events including synaptic vesicle recycling, receptor-mediated endocytosis, caveolae internalization, phagocytosis, and secretory vesicle budding from the trans-Golgi network (3van 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, 4Herskovits J.S. Burgess C.C. Obar R.A. Vallee R.B. J. Cell Biol. 1993; 122: 565-578Crossref PubMed Scopus (394) Google Scholar, 5Gold E.S. Underhill D.M. Morrissette N.S. Guo J. McNiven M.A. Aderem A. J. Exp. Med. 1999; 190: 1849-1856Crossref PubMed Scopus (227) Google Scholar, 6Henley J.R. Krueger E.W.A. Oswalk B.J. McNiven M.A. J. Cell Biol. 1998; 141: 85-99Crossref PubMed Scopus (613) Google Scholar, 7Jones S.M. Howell K.E. Henley J.R. Cao H. McNiven M.A. Science. 1998; 279: 573-577Crossref PubMed Scopus (269) Google Scholar, 8Kreitzer G. Marmorstein A. Okamoto P. Vallee R. Rodriguez-Boulan E. Nat. Cell Biol. 2000; 2: 125-127Crossref PubMed Scopus (198) Google Scholar, 9van der Bliek A.M. Trends Cell Biol. 1999; 9: 96-102Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). It forms long spiral polymers around the necks of coated pits (10Takei K. McPherson P.S. Schmid S.L. De Camilli P. Nature. 1995; 374: 186-190Crossref PubMed Scopus (648) Google Scholar) and on lipid tubules (11Sweitzer S.M. Hinshaw J.E. Cell. 1998; 93: 1021-1029Abstract Full Text Full Text PDF PubMed Scopus (542) Google Scholar), suggesting that the protein may directly function in membrane scission. Alternatively, dynamin has also been postulated to act as a GTPase switch by recruiting other endocytic factors to the neck and then activating them to sever the coated vesicle (12Sever S. Muhlberg A.B. Schmid S.L. Nature. 1999; 398: 481-486Crossref PubMed Scopus (312) Google Scholar). Dynamin contains an amino-terminal GTPase domain, followed by a central coiled-coil assembly domain (13Okamoto P.M. Tripet B. Litowski J. Hodges R.S. Vallee R.B. J. Biol. Chem. 1999; 274: 10277-10286Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), a pleckstrin homology domain, which binds to phosphoinositides and the βγ subunits of heterotrimeric GTPases (14Lin H.C. Gilman A.G. J. Biol. Chem. 1996; 271: 27979-27982Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 15Lin H.C. Barylko B. Achiriloaie M. Albanesi J.P. J. Biol. Chem. 1997; 272: 25999-26004Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), and a carboxyl-terminal coiled-coil region (also called the assembly or GTPase effector domain) that is involved in self-association (13Okamoto P.M. Tripet B. Litowski J. Hodges R.S. Vallee R.B. J. Biol. Chem. 1999; 274: 10277-10286Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 16Muhlberg A.B. Warnock D.E. Schmid S.L. EMBO J. 1997; 16: 6676-6683Crossref PubMed Scopus (197) Google Scholar, 17Smirnova E. Shurland D.-L. Newman-Smith E.D. Pishvaee B. van der Bliek A.M. J. Biol. Chem. 1999; 274: 14942-14947Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). At the extreme carboxyl terminus is a basic, proline-rich domain to which a number of Src homology 3 (SH3)1 domain-containing proteins, acidic phospholipids, and microtubules have been shown to bind (18Herskovits J.S. Shpetner H.S. Vallee R.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11468-11472Crossref PubMed Scopus (125) Google Scholar, 19Tuma P.L. Stachniak M.C. Collins C.A. J. Biol. Chem. 1993; 268: 17240-17246Abstract Full Text PDF PubMed Google Scholar, 20Schmid S.L. McNiven M. De Camilli P. Curr. Opin. Cell Biol. 1998; 10: 504-512Crossref PubMed Scopus (354) Google Scholar). Considerable insight into dynamin function at the synapse has come from genetic and morphological studies on the temperature-sensitive mutants of shibire, the dynamin ortholog in Drosophila (21Chen M.S. Obar R.A. Schroeder C.C. Austin T.W. Poodry C.A. Wadsworth S.C. Vallee R.B. Nature. 1991; 351: 583-586Crossref PubMed Scopus (433) Google Scholar, 22van der Bliek A.M. Meyerowitz E.M. Nature. 1991; 351: 411-414Crossref PubMed Scopus (587) Google Scholar). Single point mutations in the GTPase domain ofshibire cause paralysis at elevated temperatures, and ultrastructural analysis of nerve terminals under these conditions has revealed a depletion of synaptic vesicles, along with an accumulation of collared pits (23Koenig J.H. Saito K. Ikeda K. J. Cell Biol. 1983; 96: 1517-1522Crossref PubMed Scopus (85) Google Scholar, 24Koenig J.H. Ikeda K. J. Neurosci. 1989; 9: 3844-3860Crossref PubMed Google Scholar). In mammals, three closely related dynamin genes are expressed in a tissue-specific manner. Dynamin-1 is almost exclusively expressed in neurons (25Nakata T. Iwamoto A. Noda Y. Takemura R. Yoshikura H. Hirokawa N. Neuron. 1991; 7: 461-469Abstract Full Text PDF PubMed Scopus (73) Google Scholar). Dynamin-2 is found in the brain but is also widely expressed among other tissues (26Cook T.A. Urrutia R. McNiven M.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 644-648Crossref PubMed Scopus (160) Google Scholar, 27Sablin E.P. Kull F.J. Cooke R. Vale R.D. Fletterick R.J. Nature. 1996; 380: 555-559Crossref PubMed Scopus (321) Google Scholar, 28Sontag J. Fykse E.M. Yshkaryov Y. Liu J. Robinson P.J. Sudhof T.C. J. Biol. Chem. 1994; 269: 4547-4554Abstract Full Text PDF PubMed Google Scholar). Dynamin-3 was initially identified in testis (29Nakata T. Takemura R. Hirokawa N. J. Cell Sci. 1993; 105: 1-5Crossref PubMed Google Scholar) but is also found in brain, lung, and heart (30Cao H. Garcia F. McNiven M.A. Mol. Biol. Cell. 1998; 9: 2595-2609Crossref PubMed Scopus (338) Google Scholar). Differences in the subcellular distribution of the dynamin gene products and their alternative splice forms have been reported (30Cao H. Garcia F. McNiven M.A. Mol. Biol. Cell. 1998; 9: 2595-2609Crossref PubMed Scopus (338) Google Scholar). Because of its restriction to neurons, dynamin-1 has been assumed to be the synaptic isoform. The function of dynamin-2 is less well understood, and a role in neurons has not been identified. Overexpression of a dominant inhibitory mutant form of dynamin-2 in cultured hippocampal neurons was recently reported to inhibit glutamate-induced down-regulation of α-amino-3-5-methylisoxazole-4-propionic acid receptors (31Carroll R.C. Beattie E.C. Xia H., C, L.S. Altschuler Y. Nicoll R.A. Malenka R.C. von Zastrow M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14112-14117Crossref PubMed Scopus (343) Google Scholar). This result is of considerable current interest in view of evidence that up- and down-regulation of glutamate receptors plays a role in long term potentiation and depression (32Carroll R.C. Lissin D.V. von Zastrow M. Nicoll R.A. Malenka R.C. Nat. Neurosci. 1999; 2: 454-460Crossref PubMed Scopus (378) Google Scholar, 33Luscher C. Xia H. Beattie E.C. Carroll R.C. von Zastrow M. Malenka R.C. Nicoll R.A. Neuron. 1999; 24: 649-658Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar). Whether dynamin-2, in particular, functions postsynaptically is uncertain from these experiments, because of the interchangeability of dynamin isoforms when overexpressed (Ref. 13Okamoto P.M. Tripet B. Litowski J. Hodges R.S. Vallee R.B. J. Biol. Chem. 1999; 274: 10277-10286Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar; also see “Results”). To learn more about the specific functions of dynamin-2, we have used the yeast two-hybrid assay to identify dynamin-2-specific interacting proteins. We report here that dynamin-2 interacts with at least two members of the Shank/ProSAP family, which have recently been described as components of the postsynaptic density (PSD) (34Boeckers T.M. Winter C. Smalla K.H. Kreutz M.R. Bockmann J. Seidenbecher C. Garner C.C. Gundelfinger E.D. Biochem. Biophys. Res. Commun. 1999; 264: 247-252Crossref PubMed Scopus (145) Google Scholar, 35Naisbitt S. Kim E.K. Tu J.C. Xiao B. Sala C. Valtschanoff J. Weinberg R.J. Worley P.F. Sheng M. Neuron. 1999; 23: 569-582Abstract Full Text Full Text PDF PubMed Scopus (769) Google Scholar) Dynamin-2 exhibits a synaptic distribution in cultured hippocampal neurons and is specifically enriched relative to dynamin-1 in the postsynaptic density. Our findings indicate a close association between the endocytic machinery and the PSD and provide new insight into the dynamics of this structure. The entire dynamin-2bb cDNA was subcloned into the JK202 bait vector and used to screen an adult human brain cDNA library (Invitrogen, Inc.) for interactors in a LexA-based yeast two-hybrid assay utilizing the yeast strain, EGY191 (36Gyuris J. Golemis E. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1316) Google Scholar). Potential positives, as deemed by their ability to both grow in the absence of leucine and activate β-galactosidase expression, were further analyzed using BLAST and SMART searches. To qualitatively evaluate the β-galactosidase activities, highly sensitive filter assays were carried out as described in the Matchmaker LexA two-hybrid system user manual (CLONTECH Laboratories, Inc.). For the domain mapping studies and interaction specificity analyses, carboxyl-terminal deletion mutants of dynamin-2 in JK202 were generated using PCR. The dynamin-1 two-hybrid bait construct in JK202 has been described previously (13Okamoto P.M. Tripet B. Litowski J. Hodges R.S. Vallee R.B. J. Biol. Chem. 1999; 274: 10277-10286Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), and the light intermediate chain-2 (LIC2) bait construct was generously provided by Dr. Jorge Garces. The Shank1 cDNA was cloned from a rat brain cDNA library using reverse transcriptase-PCR with gene specific primers and LATaq polymerase (Takara Shuzo Ltd.). The library was generated using the Marathon cDNA amplification kit (CLONTECH Laboratories, Inc.) and kindly provided by Dr. Atsushi Mikami. The various PCR fragments were initially cloned into pCR2.1 using the TOPO-TA cloning system (Invitrogen, Inc.) and subsequently assembled into a full-length cDNA in the mammalian expression vector, pcDNA3.1+ vector (Invitrogen, Inc.). Three consecutive Myc tags, which were required for the detection of the epitope in overexpressed cells, were added to the amino terminus of Shank1 by PCR. All PCR-generated constructs were sequenced for accuracy. A Shank1 antibody was raised in rabbits against a His-tagged fusion protein consisting of the Shank1 region of a.a. 1792–2001. Carboxyl-terminal anti-dynamin-1 and -2 isoform-specific antibodies have been described previously (37Okamoto 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) as have a dynamin-2-specific antibody prepared against an internal sequence (Oncogene; Ref. 38Henley J.R. McNiven M.A. J. Cell Biol. 1996; 133: 761-775Crossref PubMed Scopus (107) Google Scholar) and antibodies against cortactin-binding protein, a generous gift from Dr. Tom Parsons (39Du Y. Weed S.A. Xiong W. Marshall T.D. Parsons J.T. Mol. Cell Biol. 1998; 18: 5838-5851Crossref PubMed Scopus (212) Google Scholar) and the Myc epitope (40Evan G.I. Lewis G.K. Ramsay G. Bishop J.M. Molec. Cell Biol. 1985; 5: 3610-3616Crossref PubMed Scopus (2151) Google Scholar, 41Gee M.A. Heuser J.E. Vallee R.B. Nature. 1997; 390: 636-639Crossref PubMed Scopus (261) Google Scholar). The other antibodies used in this study and their sources are anti-cortactin (p80/85 Src60 substrate, Santa Cruz Biotechnology, Inc.), anti-synaptophysin (Roche Molecular Biochemicals), anti-PSD95 (Calbiochem), anti-N-methyl-d-aspartic acid receptor 1 (PharMingen), anti-calmodulin kinase II (Calbiochem), and anti-HA (Covance). All of the secondary antibodies used were obtained from Jackson ImmunoResearch, Inc. For the immunoprecipitation assays in COS-7 cells, either dynamin-1 or -2 was co-transfected with Myc-tagged Shank2 or Shank1 using LipofectAMINE as suggested by the manufacturer (Invitrogen, Inc.), and the lysates were prepared in cold RIPA buffer as described previously (13Okamoto P.M. Tripet B. Litowski J. Hodges R.S. Vallee R.B. J. Biol. Chem. 1999; 274: 10277-10286Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Immunoprecipitation assays were carried out with either the dynamin-1 or -2 isoform-specific antibody on protein A beads for 4 h at 4 °C with end-over-end rotation, after which the beads were extensively washed with cold RIPA buffer. The samples were then resolved by SDS-polyacrylamide gel electrophoresis (7.5%), transferred to polyvinylidene difluoride, and blotted with Myc or the dynamin isoform-specific antibodies. For the immunoprecipitation assays from rat brain tissue, the cytosolic extract was prepared as described previously (37Okamoto 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), and the assays were carried out in the same manner as described for COS-7 cells (see above). The GST pull-down assays were performed essentially as described (37Okamoto 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). The bacterially expressed and purified recombinant GST fusion protein of a Shank2 fragment (a.a. 888–1180) that corresponds to the dynamin-2 binding region in Shank1 or the GST protein alone was immobilized on Sepharose 4B-glutathione beads (Amersham Pharmacia Biotech) in binding buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1% Nonidet 40, 1 mm EDTA, 1 mmphenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin) for 30 min at room temperature. The beads were then washed three times with binding buffer. Baculovirus expressed and purified recombinant full-length HA-tagged dynamin-2 was added, and binding was allowed to occur for an additional 1 h at room temperature with end-over-end rotation, after which the beads were washed four times with cold binding buffer. Bound HA-tagged dynamin-2 was eluted twice with elution buffer (50 mm reduced glutathione, 50 mm Tris-HCl, pH 8). The eluants were combined, resolved in 9% polyacrylamide-SDS gels, and processed for Western analysis. HA-dynamin-2 was assayed by immunoblotting with the anti-HA monoclonal antibody. Purification of the HA-tagged dynamin-2 protein was as reported previously (42Warnock D.E. Hinshaw J.E. Schmid S.L. J. Biol. Chem. 1996; 271: 22310-22314Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). For the blot overlay assays, cytosolic extracts of COS-7 cells transiently transfected with either Myc-tagged Shank1 or Myc-tagged dynein IC2C were prepared 48 h post-transfection in ice-cold RIPA buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 2 mm EDTA, 0.1% SDS, 1% sodium deoxycholate, 1% Triton X-100, 10 μg/ml each of aprotinin, leupeptin, pepstatin A, E-64, 16 μg/ml benzamidine, 1 mm4-(2-aminoethyl)-benzenesulfonyl fluoride). The extracts were spun 20 min at 13,000 rpm at 4 °C, after which the overexpressed Myc-tagged proteins were immunoprecipitated with an anti-Myc polyclonal antibody for 2 h at 4 °C followed by an incubation with protein A-Sepharose beads (Amersham Pharmacia Biotech) for 1 h at 4 °C. Immunocomplexes were washed once with 1m NaCl and 1% Nonidet P-40, twice with RIPA and 1m urea, and once with RIPA. The proteins were resolved on a 7.5% acrylamide/SDS gel, transferred to polyvinylidene difluoride, cut into strips, and allowed to renature in renaturation buffer (TBST with 5% nonfat dry milk) at 4 °C for 16 h. The strips were then incubated for 1 h at room temperature in overlay binding buffer (TBST, 0.1% bovine serum albumin, 2 mm MgSO4) in the presence or absence of 0.5 μm purified, recombinant HA-tagged dynamin-2 followed by two washes in wash buffer (TBST, 2 mm MgSO4) and then cross-linked to the membrane with 0.2% glutaraldehyde for 15 min at room temperature. After extensively washing the strips in wash buffer, they were then incubated for an hour with anti-HA monoclonal antibody in the overlay binding buffer. Enhanced chemiluminescence was used to process all of the above immunoblotting experiments. Rat hippocampal neuronal cultures were prepared from 18–19-day-old rat embryos as previously described (43Goslin G.B.A.K. Rat Hippocampal Neurons in Low-density Culture: Culturing Nerve Cells. MIT Press, Cambridge, MA1991: 339-370Google Scholar) and processed for immunofluorescence as essentially reported earlier (44Wu L. Wells D. Tay J. Mendis D. Abbott M.A. Barnitt A. Quinlan E. Heynen A. Fallon J.R. Richter J.D. Neuron. 1998; 21: 1129-1139Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar). Briefly, after 19–21 days in culture, the neurons were fixed in 4% paraformaldehyde for 20 min and then extracted with 0.05% saponin in phosphate-buffered saline for 5 min at room temperature. Incubation in anti-dynamin-2, anti-dynamin-1, or anti-synaptophysin primary antibody was overnight at 4 °C followed by incubation in either Cy3- or fluorescein isothiocyanate-conjugated species-specific secondary antibody for 1 h at room temperature. Immunofluorescence was visualized with a Zeiss Axiophot microscope. For the co-clustering experiments, COS7 cells were plated on coverslips that had been pretreated with poly-l-lysine and transiently transfected with HA-tagged dynamin-2, PSD95, Myc-tagged Shank1a, and/or GFP-tagged GKAP1a using LipofectAMINE Plus reagent (Invitrogen, Inc.). The cDNAs for PSD95 and GKAP1a were generously provided by Dr. Craig Garner (University of Alabama, Birmingham, AL). All of the constructs were made in pcDNA 3.1+ (Invitrogen, Inc.) with the exception of GFP-GKAP1a, which was subcloned into the pEGFP-N1 vector (CLONTECH, Inc.). After 24 h post-transfection, the cells were fixed in 4% paraformaldehyde for 20 min at room temperature and then extracted with ice-cold methanol for 5 min at −20 °C. HA, Myc, and PSD primary antibodies were applied to the cells for 1 h at 37 °C, after which they were briefly washed and then incubated with Cy3-, Cy5-, or Alexa Green-conjugated, species-specific secondary antibodies for an additional 1 h at 37 °C. All of the antibodies were diluted in 1% bovine serum albumin. The images were gathered and processed with a Leica DMIRBE immunofluorescent microscope interfaced to a CCD camera using the Metamorph software. Rat brain homogenate and synaptosomal and postsynaptic density fractions were obtained by differential centrifugation following previously published protocol (45Abbott M.A. Wells D.G. Fallon J.R. J. Neurosci. 1999; 19: 7300-7308Crossref PubMed Google Scholar, 46Carlin R.K. Grab D.J. Cohen R.S. Siekevitz P. J. Cell Biol. 1980; 86: 831-845Crossref PubMed Scopus (597) Google Scholar) with the exception that 25 unstripped frozen whole rat brains (Pel-Freeze) were used. Twenty micrograms of each fraction was resolved by SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride, and blotted for various synaptic proteins at the following dilutions: anti-synaptophysin, 1:200; anti-dynamin-2, 1:1000; anti-dynamin-1b, 1:6000; anti-dynamin-1a, 1:5000; anti-Shank1, 1:2000; anti-PSD95, 1:200; anti-calmodulin kinase II, 1:200; and anti-NMDAR1, 1:1000. All blots were processed for imaging using enhanced chemiluminescence. To identify proteins that interact with dynamin-2, we used a full-length dynamin-2 construct as bait in a yeast two-hybrid screen of a human brain cDNA library. Of ∼3 × 107 transformants that were screened, 24 grew on Leu− medium and activated β-galactosidase. Two of the prey sequences coded for a 188-a.a. fragment of Shank1 (also known as ProSAP2 and synamon), a member of the recently discovered Shank/ProSAP PDZ domain-containing protein family that has been reported to act as scaffolding components of the PSD (35Naisbitt S. Kim E.K. Tu J.C. Xiao B. Sala C. Valtschanoff J. Weinberg R.J. Worley P.F. Sheng M. Neuron. 1999; 23: 569-582Abstract Full Text Full Text PDF PubMed Scopus (769) Google Scholar). Shank1 was initially identified on the basis of its interaction with GKAP/SAPAP/DAP1 (34Boeckers T.M. Winter C. Smalla K.H. Kreutz M.R. Bockmann J. Seidenbecher C. Garner C.C. Gundelfinger E.D. Biochem. Biophys. Res. Commun. 1999; 264: 247-252Crossref PubMed Scopus (145) Google Scholar, 35Naisbitt S. Kim E.K. Tu J.C. Xiao B. Sala C. Valtschanoff J. Weinberg R.J. Worley P.F. Sheng M. Neuron. 1999; 23: 569-582Abstract Full Text Full Text PDF PubMed Scopus (769) Google Scholar, 47Yao I. Hata Y. Hirao K. Deguchi M. Ide N. Takeuchi M. Takai Y. J. Biol. Chem. 1999; 274: 27463-27466Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), which in turn interacts with the well characterized PSD component PSD95/SAP90 (35,48,49; Fig.1 a). Besides its PDZ domain, Shank1 also contains an ankyrin repeat region, an SH3 domain, a polyhistidine segment, and a sterile α motif domain (Fig.1 a). Our 188-a.a. fragment mapped to a proline-rich region (a.a. 1603–1790) located between the latter two domains (Fig. 1,a and b). We also tested for an interaction between dynamin-2 and Shank2. Shank2 (50Lim S. Naisbitt S. Yoon J. Hwang J.I. Suh P.G. Sheng M. Kim E. J. Biol. Chem. 1999; 274: 29510-29518Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar), also known as ProSAP1 (51Boeckers T.M. Kreutz M.R. Winter C. Zuscharatter W. Smalla K.H. Sanmarti-Vila L. Wex H. Langnaese K. Bockmann J. Garner C.C. Gundelfinger E.D. J. Neurosci. 1999; 19: 6506-6518Crossref PubMed Google Scholar), and cortactin-binding protein 1, or CortBP1 (39Du Y. Weed S.A. Xiong W. Marshall T.D. Parsons J.T. Mol. Cell Biol. 1998; 18: 5838-5851Crossref PubMed Scopus (212) Google Scholar), has been shown to be present in PSDs, neuronal growth cones, and other actin-containing cytoskeletal structures (39Du Y. Weed S.A. Xiong W. Marshall T.D. Parsons J.T. Mol. Cell Biol. 1998; 18: 5838-5851Crossref PubMed Scopus (212) Google Scholar) and exhibits 32% identity (53% similarity) to Shank1 within the dynamin-2 binding region we identified. As shown in Fig. 1 b, clear evidence for an interaction with full-length Shank2 was observed. We also tested for dynamin isoform specificity in a directed two-hybrid assay. No interaction could be detected with dynamin-1 as bait (Fig.1 b). Taken together, these results reveal the identification of a novel class of isoform-specific dynamin-binding partners. Because the dynamin-1 and -2 sequences differ most prominently within the carboxyl-terminal proline-rich domain, we reasoned that the binding site for the Shank/ProSAP proteins might lie within this region. To test this possibility, we used carboxyl-terminal dynamin-2 deletion mutants in a directed two-hybrid assay. Binding of both Shank1 and Shank2 persisted in constructs lacking up to 31 a.a. from the carboxyl terminus (Dyn-2 C837–C868; Fig. 1 c). Removal of an additional 10 amino acids (Dyn-2 C824), however, completely abolished the interaction (Fig. 1 c). At least two Shank family members (Shank2/CortBP1 and Shank3a) have been found to interact with the actin-binding protein, cortactin, which has recently also been reported to interact with dynamin-2 (35Naisbitt S. Kim E.K. Tu J.C. Xiao B. Sala C. Valtschanoff J. Weinberg R.J. Worley P.F. Sheng M. Neuron. 1999; 23: 569-582Abstract Full Text Full Text PDF PubMed Scopus (769) Google Scholar, 39Du Y. Weed S.A. Xiong W. Marshall T.D. Parsons J.T. Mol. Cell Biol. 1998; 18: 5838-5851Crossref PubMed Scopus (212) Google Scholar, 52McNiven M.A. Kim L. Krueger E.W. Orth J.D. Cao H. Wong T.W. J. Cell Biol. 2000; 151: 187-198Crossref PubMed Scopus (340) Google Scholar). We found that cortactin interacts with the carboxyl-terminal portion of dynamin-2 in our two-hybrid assay, consistent with previous results (52McNiven M.A. Kim L. Krueger E.W. Orth J.D. Cao H. Wong T.W. J. Cell Biol. 2000; 151: 187-198Crossref PubMed Scopus (340) Google Scholar). However, binding was completely abolished in three dynamin-2 constructs that still exhibited a strong interaction with Shank1 and Shank 2 (Dyn-2 C837–C844; Fig. 1 c). Using the yeast two-hybrid assay, we also defined the region within the original Shank1 prey fragment responsible for dynamin-2 binding. Binding was abolished upon deletion of the amino-terminal 29 a.a. from the Shank1 188-a.a. prey fragment (Fig. 1 c). This region is among the more highly conserved parts of the prey fragment (72% identity; 86% similar; Fig. 1 c), consistent with a conserved functional role. To test further for an association between dynamin-2 and members of the Shank/ProSAP family, we overexpressed either Myc-tagged dynamin-1 or dynamin-2 with Shank2 in COS-7 cells. Full-length untagged Shank2 co-immunoprecipitated with either Myc-dynamin-1 or -2 (Fig.2 a). This result supports the two-hybrid data, but the co-immunoprecipitation of both dynamin isoforms with Shank2 appears to contradict the dynamin isoform specificity revealed by the two-hybrid analysis. However, we believe that co-immunoprecipitation of overexpressed dynamin-1 with Shank2 occurs through an interaction between dynamin-1 and dynamin-2, which is the predominant isoform expressed in COS7 and other non-neuronal cultured cell lines (data not shown). Direct evidence for the free interaction between dynamin isoforms can be observed in Fig.2 c (see below) and in our previous results (13Okamoto P.M. Tripet B. Litowski J. Hodges R.S. Vallee R.B. J. Biol. Chem. 1999; 274: 10277-10286Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), and we note that dynamin-2 co-immunoprecipitates Shank2 more efficiently than does dynamin-1. Both dynamin isoforms co-immunoprecipitated Myc-tagged Shank1 (Fig. 2 b), and, again, overexpressed dynamin-2 performed better than dynamin-1. We also carried out co-immunoprecipitation assays using rat brain cytosol. Only Shank2 could be examined by this assay because of the insolubility of Shank1. Clear co-immunoprecipitation of Shank2 with dynamin was observed (Fig. 2 c). Shank2 again co-immunoprecipitated with both dynamin-1 and -2, in this case using the carboxyl-terminal anti-dynamin antibodies, but we also note the clear ability of the two endogenous dynamin isoforms to co-immunoprecipitate with either antibody. To test whether cortactin may play an intermediary role in the dynamin-2 and Shank/ProSAP interaction, dynamin-2 was overexpressed in wild-type COS-7 cells, and the immunoprecipitate was tested for endogenous cortactin. As shown in Fig. 2 d, no cortactin was detected in these immunoprecipitates, although a significant amount of cortactin was present in these cells (lysate lanes in Fig.2 d). These results argued further that the interaction between dynamin-2 and Shank2 or Shank1 did not involve cortactin, although other intervening proteins could not be ruled out. To ascertain whether the interaction between dynamin-2 and the Shank family is, indeed, direct, a GST fusion protein containing the region of Shank2 corresponding to the initially identified prey fragment was tested for its ability to bind purified dynamin-2. Clear binding by the GST-Shank2 fusion protein, but not by GST alone, was observed (Fig.3 a). Blot overlays using purified dynamin-2 also revealed specific binding to electrophoretically fractionated Shank1 (Fig. 3 b). Together our data indicate that dynamin-2 specifically associates with a family of known synaptic proteins. However, whether dynamin-2 is present in neurons and what its subcellular distribution may be are unknown. Immunofluorescence microscopy of cultured hippocampal neurons revealed punctate staining along dendrites with both the dynamin-2 and dynamin-1b carboxyl-terminal isoform-specific antibodies (Fig. 4). The dynamin-2 staining exhibited clear co-localization with the synaptic marker synaptophysin (Fig. 4). To examine whether dynamin-2 is present in PSDs, we tested a series of markers for biochemical co-fractionation with these structures. Both dynamin-1 and -2 isoforms were found in the synaptosomal fraction. However, dynamin-2 was clearly found in the purified PSD fraction, whereas dynamin-1 was present at low to undetectable" @default.
• W1537397787 created "2016-06-24" @default.
• W1537397787 creator A5002552436 @default.
• W1537397787 creator A5054709391 @default.
• W1537397787 creator A5055176103 @default.
• W1537397787 creator A5067720072 @default.
• W1537397787 creator A5089071903 @default.
• W1537397787 date "2001-12-01" @default.
• W1537397787 modified "2023-10-03" @default.
• W1537397787 title "Dynamin Isoform-specific Interaction with the Shank/ProSAP Scaffolding Proteins of the Postsynaptic Density and Actin Cytoskeleton" @default.
• W1537397787 cites W1509530066 @default.
• W1537397787 cites W1565336950 @default.
• W1537397787 cites W1570789308 @default.
• W1537397787 cites W1574831557 @default.
• W1537397787 cites W1602952479 @default.
• W1537397787 cites W1608132397 @default.
• W1537397787 cites W1619853111 @default.
• W1537397787 cites W1656075393 @default.
• W1537397787 cites W1677326748 @default.
• W1537397787 cites W1819107323 @default.
• W1537397787 cites W1832262637 @default.
• W1537397787 cites W1961999388 @default.
• W1537397787 cites W1967965496 @default.
• W1537397787 cites W1968046686 @default.
• W1537397787 cites W1972812883 @default.
• W1537397787 cites W1976391336 @default.
• W1537397787 cites W1977779145 @default.
• W1537397787 cites W1978627586 @default.
• W1537397787 cites W1978923083 @default.
• W1537397787 cites W1982334342 @default.
• W1537397787 cites W1983322120 @default.
• W1537397787 cites W1986542931 @default.
• W1537397787 cites W1990516841 @default.
• W1537397787 cites W1991334194 @default.
• W1537397787 cites W1993706961 @default.
• W1537397787 cites W2003959866 @default.
• W1537397787 cites W2006851823 @default.
• W1537397787 cites W2012021856 @default.
• W1537397787 cites W2012606680 @default.
• W1537397787 cites W2013112818 @default.
• W1537397787 cites W2018229588 @default.
• W1537397787 cites W2019873695 @default.
• W1537397787 cites W2022658714 @default.
• W1537397787 cites W2024531257 @default.
• W1537397787 cites W2025929812 @default.
• W1537397787 cites W2029500247 @default.
• W1537397787 cites W2033276495 @default.
• W1537397787 cites W2037956598 @default.
• W1537397787 cites W2039959072 @default.
• W1537397787 cites W2040463675 @default.
• W1537397787 cites W2040987875 @default.
• W1537397787 cites W2042314805 @default.
• W1537397787 cites W2044273755 @default.
• W1537397787 cites W2051410189 @default.
• W1537397787 cites W2055473408 @default.
• W1537397787 cites W2060934876 @default.
• W1537397787 cites W2068240232 @default.
• W1537397787 cites W2071993841 @default.
• W1537397787 cites W2077142887 @default.
• W1537397787 cites W2082530736 @default.
• W1537397787 cites W2084494997 @default.
• W1537397787 cites W2092442184 @default.
• W1537397787 cites W2094180395 @default.
• W1537397787 cites W2096472685 @default.
• W1537397787 cites W2096476993 @default.
• W1537397787 cites W2107801352 @default.
• W1537397787 cites W2108081370 @default.
• W1537397787 cites W2108264768 @default.
• W1537397787 cites W2108836535 @default.
• W1537397787 cites W2109950649 @default.
• W1537397787 cites W2111916392 @default.
• W1537397787 cites W2114911095 @default.
• W1537397787 cites W2139341460 @default.
• W1537397787 cites W2153689647 @default.
• W1537397787 cites W2162446789 @default.
• W1537397787 cites W2169423035 @default.
• W1537397787 cites W2343796797 @default.
• W1537397787 cites W2409928594 @default.
• W1537397787 doi "https://doi.org/10.1074/jbc.m104927200" @default.
• W1537397787 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2715172" @default.
• W1537397787 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11583995" @default.
• W1537397787 hasPublicationYear "2001" @default.
• W1537397787 type Work @default.
• W1537397787 sameAs 1537397787 @default.
• W1537397787 citedByCount "80" @default.
• W1537397787 countsByYear W15373977872012 @default.
• W1537397787 countsByYear W15373977872013 @default.
• W1537397787 countsByYear W15373977872014 @default.
• W1537397787 countsByYear W15373977872015 @default.
• W1537397787 countsByYear W15373977872016 @default.
• W1537397787 countsByYear W15373977872017 @default.
• W1537397787 countsByYear W15373977872018 @default.
• W1537397787 countsByYear W15373977872019 @default.
• W1537397787 countsByYear W15373977872020 @default.
• W1537397787 countsByYear W15373977872021 @default.
• W1537397787 countsByYear W15373977872023 @default.
• W1537397787 crossrefType "journal-article" @default.
• W1537397787 hasAuthorship W1537397787A5002552436 @default.

• next