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The PDZ-interacting domain of TRPC4 controls its localization and surface expression in HEK293 cells

¹ Department of Physiology, University of Saarland, D-66421 Homburg, Germany
² Department of Clinical Pharmacology, University of Bern, Murtenstrasse 35, 3010 Bern, Switzerland
³ Department of Geriatrics, Geneva University Hospital, CH-1211 Geneva 14, Switzerland

View larger version (38K): [in a new window]   Fig. 5. Quantification of relative plasma membrane content of myc-TRPC4 and myc-TRL in HEK293 cells by surface biotinylation. (A) myc-TRPC4 or myc-TRL were transiently expressed in HEK293 cells together with YFP-EBP50. 48 hours after transfection, plasma membrane were biotinylated followed by cell lysis and incubation of lysates with avidin-agarose to recover biotinylated proteins. Total (left panel) and surface fraction (right panel) were separated by electrophoresis, electroblotted on PVDF and probed with an anti-myc antibody. Reactive bands were quantified with a densitometer. Optical densities of four dilutions of each sample were plotted against the amount of total (left panel) or biotinylated (right panel) proteins loaded. Lines indicate curves of best linear fit. The slopes of the correlations were determined. RT (left panel) and RS (right panel) were calculated by dividing SWT, the slope obtained from the myc-TRPC4-expressing cells (filled circles, solid line) by STRL, the slope derived from the myc-TRL-expressing cells (opened circles, dotted line). RT and RS represent the relative total and surface contents, respectively of myc-TRPC4 and myc-TRL in transfected HEK293 cells. (B) The RS/RT ratio was determined from densitometry of three blot pairs similar to those presented in A and is shown as mean±s.e.m. (C) Degree of contamination with YFP (used as a marker of cytosolic proteins) or with non-biotinylated proteins. Monolayers were surface biotinylated (left panel) or exposed to the buffer alone (right panel) at 4°C for 30 minutes prior lysis. The cell extracts were incubated with avidin-agarose and the bound material, together with aliquots of the cell lysates, were separated by SDS-PAGE, transferred to PVDF and probed with either an anti-YFP (left panel) or an anti-myc (right panel) antibody. The absence of detectable avidin-bound YFP (lane 2), myc-TRPC4 (lane 5) or myc-TRL (lane 6) suggests no contamination by biotinylated cytosolic proteins or intracellular myc-tagged channels. T, cell lysate (30 µg of protein); B, avidin-bound (40 µl of precipitate). Results shown are representative of three independent experiments.

View larger version (64K): [in a new window]   Fig. 1. TRPC4 associates with both PDZ domains of EBP50 through its C-terminal TRL motif. Triton extracts of HEK293 cells expressing either myc-TRPC4 or myc-TRL were incubated for 3 hours at 4°C with glutathione-sepharose beads loaded with either GST, GST-PDZ1 or GST-PDZ2. After extensive washing, bound proteins were analyzed by SDS-PAGE, transferred to PVDF and probed with an anti-myc antibody (right panel). The middle panel demonstrates that myc-TRPC4 and myc-TRL were expressed at comparable levels in the transfected cells. Note that control cells (lane 4) transfected with the empty vector failed to react with the anti-myc antibody, implying that the immunoreactivity is specific for the myc epitope. The left panel shows the amount of GST or GST-fusion proteins used, as revealed by Coomassie Blue staining. Lanes 4-6 contain 10% of the extract used for the binding assay and lanes 7-12 contain 40% of the eluate. Results shown are representative of three independent experiments.

View larger version (103K): [in a new window]   Fig. 2. Subcellular distribution of YFP-EBP50 in human JEG-3 cells. (A,B) Human JEG-3 cells, grown on coverslips, were fixed with PFA, permeabilized with triton and probed with antibodies directed against either ezrin (A) or NHERF/EBP50 (B). The cells were then stained with a secondary antibody conjugated to either Alexa 594 (A) or Alexa 488 (B). (C,D) JEG-3 cells transiently expressing YFP-EBP50 (C) or the YFP alone (D) fixed with PFA. Images were acquired with a 100x oil immersion objective and filter sets for Texas Red (A) and FITC (B-D). Bar, 20 µm.

View larger version (21K): [in a new window]   Fig. 3. YFP-tagged EBP50 co-immunoprecipitates with myc-TRPC4 but not with the mutant lacking the last three C-terminal residues. myc-TRPC4 or myc-TRL were transiently expressed in HEK293 cells together with YFP alone or YFP-EBP50. Detergent extracts of the transfected cells were incubated with an anti-YFP antibody and protein G-coated agarose beads. 40% of the precipitated proteins were separated by SDS-PAGE, transferred to PVDF and immunoblotted with an anti-myc antibody (lower panel). Middle panel: cell lysates (17% of the input used for the co-immunoprecipitation) probed with anti-myc antibody to detect myc-TRPC4 and myc-TRL Upper panel: cell lysates probed with anti-YFP antibody to detect YFP and YFP-EBP50. Size markers indicate the molecular mass in kDa. IB, immunoblot; IP, immunoprecipitation. Results shown are representative of three independent experiments.

View larger version (54K): [in a new window]   Fig. 4. Subcellular distribution of CD4, myc-TRPC4 and myc-TRL in HEK293 cells co-expressing YFP-EBP50. (A-F) Non-permeabilized cells fixed with PFA. (G-L) Cells permeabilized with Triton X-100 after fixation. (M-R) Cells pre-extracted with digitonin before fixation. Upper panels: cells co-expressing YFP-EBP50 (A,G,M) and CD4 (B,H,N). Middle panels: cells co-expressing YFP-EBP50 (C,I,O) and myc-TRPC4 (D,J,P). Lower panels: cells co-expressing YFP-EBP50 (E,K,Q) and myc-TRL (F,L,R). The arrows in L and R point to regions of myc-TRL accumulation. To detect the transmembrane proteins, the transfected cells were probed with an anti-CD4 (upper panels) or an anti-myc (middle and lower panels) antibody and then stained with an Alexa 594-conjugated secondary antibody. Images were collected with a 100x oil immersion objective and filter sets for FITC (to detect YFP-EBP50) and Texas Red (to detect CD4 and the myc-tagged channels), using identical settings. Bar, 20 µm.

View larger version (119K): [in a new window]   Fig. 6. Deletion of the ERM-binding domain modifies the subcellular distribution of YFP-EBP50 in human JEG-3 cells. JEG-3 cells transiently expressing YFP-EBP50 (A) or YFP-ERM (C) were fixed with PFA, permeabilized with triton, stained with an anti-ezrin antibody and then with an Alexa 594-conjugated secondary antibody. Images were acquired with a 100x oil immersion objective and filter sets for FITC (A,C) and Texas Red (B,D). Bar, 20 µm.

View larger version (31K): [in a new window]   Fig. 7. (A) The YFP-EBP50 mutant lacking the ERM-binding site still associates with TRPC4 in HEK293 cells. Extracts of HEK293 cells expressing myc-TRPC4 together with YFP-EBP50 or YFP-ERM were incubated with an anti-YFP antibody and precipitated using Protein-G-coated agarose beads. Bound proteins were resolved on SDS-PAGE, transferred to PVDF and probed with an anti-myc antibody (lower panel). The upper and middle panels show the levels of expression of myc-TRPC4 and the YFP-tagged proteins, respectively, in the transfected cells. (Upper panel) 15% input; (Lower panel) 40% precipitate. IB, immunoblot; IP, immunoprecipitation. (B) Deletion of the ERM-binding site decreases the membrane-associated fraction of YFP-EBP50. Western blots of total extracts (T.E.) and crude membrane fractions (Mb.) prepared from HEK293 cells expressing myc-TRPC4 or myc-TRL together with either YFP-EBP50 or YFP-ERM. The amount of loaded proteins is indicated (µg). (Upper panels) Blots probed with an anti-myc antibody to detect myc-TRPC4 and myc-TRL; (Lower panels) blots probed with an anti-YFP antibody to detect YFP-EBP50 and YFP-ERM. Molecular weights as revealed by prestained protein markers are indicated to the right (kDa). Results shown are representative of three independent experiments.

View larger version (18K): [in a new window]   Fig. 8. Cell surface expression of myc-TRPC4 is altered by co-expression of the YFP-ERM mutant. Association of myc-TRPC4 with the plasma membrane in HEK293 cells expressing YFP-EBP50 or YFP-ERM was analyzed by cell surface biotinylation, as described in the legend to Fig. 5. (A) Representative western blots of total (left panel) and biotinylated proteins (right panel) extracted from HEK293 cells expressing myc-TRPC4 together with YFP-EBP50 or YFP-ERM. SEBP50 corresponds to the slope of the linear fit obtained from the YFP-EBP50-expressing cells (filled circles, solid line) and SERM represents the slope of the linear fit derived from the YFP-ERM-expressing cells (open circles, dotted line). (B) Relative total (RT) and surface (RS) contents of myc-TRPC4, in YFP-EBP50- and YFP-ERM-expressing cells were calculated as described in the legend to Fig. 5. RS/RT was deduced from three blot pairs similar to those presented in A and is shown as mean±s.e.m.

View larger version (100K): [in a new window]   Fig. 9. Localization of myc-TRPC4 and myc-TRL in HEK293 cells expressing YFP-ERM. HEK293 cells expressing YFP-ERM together with myc-TRPC4 (upper panels) or myc-TRL (lower panels) were fixed with PFA, permeabilized with triton, stained with an anti-myc antibody and then with an Alexa 594-conjugated antimouse secondary antibody. Images were acquired with a 100x oil immersion objective and filter sets for Texas Red [to detect myc-TRPC4 (A) and myc-TRL (C)] and for FITC [to detect YFP-ERM (B,D)]. Bar, 20 µm.

View larger version (80K): [in a new window]   Fig. 10. Immunostaining of cytoskeletal elements in HEK293 cells co-expressing YFP-ERM and myc-TRPC4. HEK293 cells transiently expressing myc-TRPC4 together with YFP-ERM were fixed with PFA, permeabilized with triton, stained with either an anti-ß-tubulin antibody and then with an Alexa 594-conjugated anti-mouse secondary antibody (A-C) or with phalloidin conjugated to Texas Red (D-F). Images were acquired with a 100x oil immersion objective and filter sets for FITC and Texas Red. YFP-ERM (A,D) and either ß-tubulin (B) or Factin (E) are overlaid in the panels on the right. Bar, 20 µm.

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