Volume 15, Issue 6, Pages 853-865 (September 2004) A Protein Interaction Network Links GIT1, an Enhancer of Huntingtin Aggregation, to Huntington's Disease  Heike Goehler, Maciej Lalowski, Ulrich Stelzl, Stephanie Waelter, Martin Stroedicke, Uwe Worm, Anja Droege, Katrin S. Lindenberg, Maria Knoblich, Christian Haenig, Martin Herbst, Jaana Suopanki, Eberhard Scherzinger, Claudia Abraham, Bianca Bauer, Renate Hasenbank, Anja Fritzsche, Andreas H. Ludewig, Konrad Buessow, Sarah H. Coleman, Claire-Anne Gutekunst, Bernhard G. Landwehrmeyer, Hans Lehrach, Erich E. Wanker  Molecular Cell  Volume 15, Issue 6, Pages 853-865 (September 2004) DOI: 10.1016/j.molcel.2004.09.016

Figure 1 Identification of Y2H Interactions Connected to HD (A) The screening strategy. (B) Identification of interactions by systematic interaction mating. Upper panel, selection of diploid yeast clones on SDII minimal medium. Lower panel, two-hybrid selection of interactions on SDIV minimal medium. The prey proteins HP28 (A5), SH3GL3 (A7), CA150 (B9), HIP15 (B10), PFN2 (B11), HIP13 (C1), CGI125 (C12), and HYPA (D1) were identified as HDexQ51 interactors. Molecular Cell 2004 15, 853-865DOI: (10.1016/j.molcel.2004.09.016)

Figure 2 A Protein Interaction Network for Huntington's Disease (A) Matrix of 186 Y2H interactions between 35 bait and 51 prey proteins. Interactions reported previously (30) or verified in pull down assays (35) are indicated. (B) A comprehensive PPI network for htt. Y2H interactors identified in this study, red diamonds; previously published interactors, blue squares; interactors identified from databases HRPD, MINT, and BIND, bridging any two proteins in the extended network, green triangles and Supplemental Table S6. Htt interactors previously reported and recapitulated in our screens (CA150, HYPA, HIP1, and SH3GL3), red squares. Molecular Cell 2004 15, 853-865DOI: (10.1016/j.molcel.2004.09.016)

Figure 3 Validation of Y2H Interactions by In Vitro Binding Experiments GST-fusion proteins immobilized on glutathione agarose beads were incubated with COS-1 cell extracts containing HA-tagged proteins. After extensive washing, pulled proteins were eluted and analyzed by SDS-PAGE and immunoblotting using anti-htt 4C8 or anti-HA antibodies. Molecular Cell 2004 15, 853-865DOI: (10.1016/j.molcel.2004.09.016)

Figure 4 GIT1 Enhances and Is Critical for htt Aggregation (A) Filter retardation assay for the identification of GIT1 as a promoter of htt aggregation. 48 hr post- transfection, protein extracts were prepared from HEK293 cells coexpressing HD169Q68 and GIT1-CT (aa 249-770). Aggregated proteins retained on the filter were detected with anti-htt (CAG53b) or anti-C-GIT1 antibody. (B) Effect of full-length GIT1 on HD169Q68 aggregation analyzed by the filter retardation assay. (C) Analysis of HD169Q68 aggregation in cells overexpressing GIT1-CT by indirect immunofluorescence microscopy. a, HD169Q68 (red); b, GIT1-CT (green); c, colocalization of GIT1 with the endosomal marker EEA1 (yellow); d–f, colocalization of HD169Q68 (red) and GIT1-CT (green) in COS-1 cells. Scale bars, 10 μm. (D) Silencing of endogenous GIT1 expression. HEK293 cells transfected with the siRNA-GIT1 were analyzed after 48 hr by immunoblotting using anti-C-GIT1 and anti-GAPDH antibodies. (E) Silencing of endogenous GIT1 prevents the accumulation of insoluble htt aggregates. siRNA-GIT1 treated and untreated cells expressing HD169Q68 were analyzed 72 hr post-transfection by filtration. Molecular Cell 2004 15, 853-865DOI: (10.1016/j.molcel.2004.09.016)

Figure 5 Characterization of Interacting Regions in htt and GIT1 (A) Identification of the GIT1 binding region in htt. Deletion constructs of htt (baits) were tested for binding to GIT1 (aa 249-770, prey) in a yeast two-hybrid assay. The N terminus of htt (aa 1-170) binds to GIT1. Htt constructs: aa1-506Q23 (HD1.7), aa1-320Q23 (HDd1.0), aa166-506 (HDd1.3), aa1-92Q20 (HDexQ20), aa1-82Q51 (HDexQ51). (B) Characterization of htt binding region in GIT1. Deletions of GIT1 (preys) were tested for their interaction with HD1.7 (aa1-506Q23; bait). The C terminus of GIT1 mediates the association with htt. β-galactosidase activity: ++ (strong), + (weak), - (no). HIS3/URA3 reporter gene activity: ++ (good growth), + (weak growth), - (no growth). ANK, ankyrin repeats; SHD1, Spa2 homology domain; CC, coiled-coil; PBS, paxillin binding subdomain; polyQ, polyglutamine domain; PRD, proline-rich domain; HEAT, protein binding domain named after the proteins htt, elongation factor 3, regulatory A subunit of protein phosphatase 2A and TOR1. Molecular Cell 2004 15, 853-865DOI: (10.1016/j.molcel.2004.09.016)

Figure 6 Verification of the htt-GIT1 Interaction (A) Coimmunoprecipitation of HD510Q68 and HA-GIT1-CT from COS-1 cell extracts using anti-C-GIT1 antibody. Immunoprecipitated material was analyzed by immunoblotting, using the anti-HA 12CA5 antibody detecting recombinant GIT1 (upper blot) and the htt-4C8 antibody (lower blot). (B) Coimmunoprecipitation of htt and GIT1 from human brain extracts. (C) Subcellular localization of GIT1 and htt in differentiated PC12 (a–c) and SH-SY5Y cells (d–f) by confocal immunofluorescence microscopy. Colocalization of htt and GIT1 are shown in yellow (c and f). Arrows point to cytoplasmic structures recognized by both antibodies. In addition, specific GIT1 labeling was detected at the tip of neurite-like extensions in adhesion foci (arrowheads). Scale bars, 10 μm. Molecular Cell 2004 15, 853-865DOI: (10.1016/j.molcel.2004.09.016)

Figure 7 Detection of GIT1 in Brains of Transgenic Mice and HD Patients (A) Sections of striatum and cortex of R6/1 mice brain labeled with anti-C-GIT1 and anti-htt EM48 antibodies. Arrows point to nuclear inclusions. (B) Neuronal inclusions (arrows) in cortex of HD patients recognized by anti-htt 2B4 and anti-C-GIT1 antibodies. Scale bars, 20 μm. (C) Colocalization of GIT1 and htt in the cortex of HD patients, detected by immunofluorescence microscopy. (D) Detection of N-terminally truncated GIT1 fragments in HD patient brain cortex. Molecular Cell 2004 15, 853-865DOI: (10.1016/j.molcel.2004.09.016)