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First published online 3 June 2003
doi: 10.1242/jcs.00603

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The intracellular domain of teneurin-2 has a nuclear function and represses zic-1-mediated transcription

Friedrich Miescher Institute, Novartis Forschungsstiftung, PO Box 2543, CH-4002 Basel, Switzerland

View larger version (19K): [in a new window]   Fig. 1. Schematic models of all teneurin-2 proteins used in this study. BD stands for Gal4 DNA-binding domain and AD for NFB activation domain.

View larger version (92K): [in a new window]   Fig. 2. The teneurin-2 intracellular domain colocalizes with PML. Microscopic analysis of HT1080 cells transfected with teneurin-2 variants IT (a), I (b-k), PML (g-i) and PML-RAR (j-l). Staining of non-permeabilized cells with an anti-HA antibody detecting the C-terminal tag of construct IT. (a) Staining of permeabilised cells with anti-VSV detecting the C-terminal tag of construct I (b) and same field analysed in phase contrast (c). Double-immunofluorescence of I transfected cells for I (anti-VSV; d) and endogenous PML (anti-PML; e) with an overlap shown in f. Double immunofluorescence staining of cells co-transfected with I and PML for I (anti-VSV; g) and PML (anti-PML; h) with the overlap shown in i. Double-immunofluorescence staining of cells co-transfected with I and PML-RAR for I (anti-VSV; j) and PML (anti-PML; k). For comparison, l shows PML-RAR single transfected cells (anti-PML). Bar, 50 µm (a-c) or 10 µm (d-l).

View larger version (46K): [in a new window]   Fig. 3. Zic promotes degradation of the teneurin-2 intracellular domain. Western blot analysis of cell extracts of COS-7 cells transfected with teneurin-2 I (A, left side), the recombinant transcription factor BDAD (pBD-NFB) (B, left side) and zic (C and D, left side) alone and in combination, as indicated above the lanes (A-D right sides). Extracts of cells without lactacystin treatment were compared to extracts made from cells grown with the addition of lactacystin for the time periods indicated before harvesting. In A, the teneurin-2 intracellular domain was detected by anti-VSV, in B, BDAD was detected by anti-Gal4, and in C and D, zic was detected by anti-myc. Zic caused downregulation of the cotransfected teneurin-2 I but not of BDAD. Immunofluorescence staining of cells transfected with teneurin-2 I (anti-VSV antibody) and zic (anti-myc antibody) alone (E) and after co-transfection (F) revealed that the anti-VSV signal in PML bodies is lost in nuclei showing high expression levels of zic. Bar, 10 µm.

View larger version (19K): [in a new window]   Fig. 4. Teneurin-2 attenuates the transcriptional activity of zic. (A) Expression of teneurin-2 I could be induced by addition of increasing concentrations of ponasterone in EcR-293 cells stably transfected with the teneurin-2 construct I, and the addition of ALLN resulted in the stabilization of the teneurin-2 I protein. (B) Transcriptional activity of zic was studied by transfecting pXP2- APOE189 alone (- zic) or together with a zic-expressing construct (+ zic) into the teneurin-2 I stable transfectant EcR-293-I cells in the absence or presence of proteasome inhibitor ALLN (-/+ ALLN). Luciferase activity was dependent on the presence of zic, and induction of teneurin-2 I by ponasterone resulted in decreased luciferase expression, which was even more pronounced in the presence of ALLN, which is known to stabilize teneurin-2 I. (C) Transcriptional activity of zic after transfecting pXP2-APOE189 alone (- zic) or together with a zic expressing construct (+ zic) into EcR-control cells. Addition of ponasterone had no effect on the resulting luciferase activity measured.

View larger version (66K): [in a new window]   Fig. 5. Expression of fusion proteins between teneurin-2 and the recombinant transcription factor BDAD. Western blot analysis (A) and immunofluorescence staining (B) of BDAD and three different BDAD-teneurin-2 fusion constructs in HT1080 cells using anti-Gal4 antibodies. (A) Transient transfection with constructs pBD-NFB expressing BDAD, BDAD-I, BDAD-ITE and BDAD-ITEY in the presence (+) or absence (-) of ALLN, as indicated above the lanes. (B) Immunofluorescence analysis of permeabilized cells transfected with BDAD-I and of non-permeabilized cells with either BDAD-ITE or BDAD-ITEY using anti-Gal4 to detect BDAD-I and anti-teneurin-2 for BDAD-ITE and BDAD-ITEY. Staining of BDAD-ITE- or BDAD-ITEY-transfected cells with anti-Gal4 after permeabilization resulted in addition to the cell surface staining in ER and Golgi staining, but no nuclear staining could be detected (data not shown). BDAD-I did not accumulate in PML bodies but was diffusely distributed throughout the nucleus and was strongly protected from degradation in the presence of the proteasome inhibitor ALLN. Bar, 20 µm.

View larger version (31K): [in a new window]   Fig. 6. The intracellular domain of teneurin-2 is released from the cell membrane. (A) Detection of nuclear activity of transmembrane BDAD-teneurin-2 fusion proteins by induction of a luciferase reporter gene. HT1080 cells transfected with various BDAD-teneurin-2 fusion constructs and a construct expressing only BD (negative control) were analysed for luciferase activity of the co-transfected luciferase reporter plasmid. (B) Luciferase activity obtained by transfection of BDAD-ITEY (left bars) or BDAD-IT (right bars) into HT1080 control cells (bars a, HT-control) or cells stably expressing TEY (bars b, TEY cells). The ratio of the values obtained for each construct in TEY cells versus the values obtained in HT-control cells is given above the bars. (C) A model is proposed for the activation of the release of the intracellular domain by homophilic interaction between the C-terminal parts of the teneurin-2 extracellular domains on the basis of the data presented in this figure and in Table 1, showing a 6.5-fold induction of luciferase activity of BDAD-ITEY/BDAD-IT in TEY cells, whereas no difference is obtained when the same experiments are performed in TE cells (data of Table 1).

View larger version (27K): [in a new window]   Fig. 7. Proteasome inhibitors stabilize the cleaved intracellular domain of teneurin-2. (A) Comparison of luciferase activities induced by transfection of BDAD-teneurin-2 (BDAD-ITEY) in the absence (-) or presence of protease inhibitors (A, ALLN; L, lactacystin). (B) Western blot analysis of ITE- and I-transfected COS-7 cells showing cleavage products of ITE, which were stabilized by ALLN. Each lane was loaded with the same amount of cell extracts from parallel cultures treated or not treated with ALLN to ensure equal protein loading. Proteins were detected by anti-VSV antibodies. One of the stabilized cleavage products corresponded to the size of the entire intracellular domain (arrow) and another one to a smaller fragment (arrowhead).