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. 2014 May 7;289(24):16675–16687. doi: 10.1074/jbc.M114.558668

FIGURE 2.

FIGURE 2.

TRPV1 subunit interaction occurs in the C terminus. A, TRPV1 truncation mutations. Top, stop codons were introduced at residues (in red) Cys-715, Gly-734, Trp-752, and Leu-773; coiled-coil and TADs are identified by colored boxes (blue, coiled-coil; green, TAD). Lower left, truncated mutants were N-terminally tagged with VenusN or VenusC. Lower right, mutant expression was verified by western blot analysis from transfected tsA-201 cell lysates (data are representative of three independent experiments). B, TRPV1 subunit association relies on the C terminus. Confocal images of truncated TRPV1 mutants N-terminally fused to VenusC (as indicated in A) or control empty vector (VenusN-zipper) co-expressed with the full-length VenusN-TRPV1. The fluorescent signal indicates subunit association; scale bar = 100 μm. Lower right, quantitation of field fluorescence indicated that truncating the TRPV1 subunit shorter than Trp-752 results in loss of subunit association (*, p < 0.05; ***, p < 0.001, one-way ANOVA, n = 14–16 images for each mutant and empty vector). RFU, relative fluorescence units. C, co-immunoprecipitation of VenusN-tagged truncated mutants with full-length HA-tagged TRPV1 shows loss of subunit interaction with shorter TRPV1 proteins (n = 3 independent experiments). Upper panel, input membrane immunoblotted (IB) with antibody to GFP which recognizes VenusN protein. Lower panel, HA-TRPV1 was immunoprecipitated with HA antibody, and TRPV1 mutants were detected with GFP antibody. Molecular masses in kDa are indicated on the right. D, subunit interaction measured by BRET-BiFC. Venus-tagged truncated mutants were co-expressed with Venus-tagged and luciferase-tagged full-length TRPV1. BRET-BiFC experiments were conducted as in Fig. 1C except that a fixed amount (500 ng each) of VenusN+ VenusC acceptor was used for the assay. This amount of acceptor was found to achieve BRET-BiFC saturation in the titration curve of WT TRPV1 (VN-TRPV1 + VC-TRPV1. ***, p < 0.01, ANOVA followed by Tukey's multiple comparison test). nBRET = net BRET.