Fig 1.
TGFα-shedding assay can evaluate TRP channel activation.
(A) Schematic representation of the TGFα-shedding assay for detecting transient receptor potential (TRP) channel activation. TRPV1 is activated upon capsaicin stimulation, inducing AP-TGFα ectodomain shedding. Released AP-TGFα can be quantified by measuring AP activity in the supernatant based on the production of para-nitrophenol (p-NP) from para-nitrophenyl phosphate (p-NPP). (B) Schematic of the assay protocol: HEK293 cells transiently expressing AP-TGFα with or without TRP channel expression are reseeded onto 96-well plates and stimulated with a ligand. After the supernatant (sup) is transferred to a blank plate, AP-TGFα release is quantified by a colorimetric reaction to measure AP activity, using p-NPP as a substrate. AP-TGFα release (%) is calculated as the ratio of AP activity in the supernatant to the total AP activity. ΔAbs405 was calculated by subtracting the absorbance at 405 nm measured at 0 h [Abs405 (0 h)] from the absorbance at 405 nm measured at 1 h [Abs405 (1 h)], using 1.25 as a correction factor for the amount of supernatant transferred (80 of 100 μL). See Methods for details. (C) Concentration–response curve for the TGFα-shedding responses induced by TRPV1 activation upon capsaicin stimulation. The vehicle-treated condition is set as the baseline. Mock-transfected cells expressing only the AP-TGFα reporter were used as a control. (D–F) Concentration–response curves for the TGFα-shedding responses induced by TRPA1 (C), TRPM8 (D), and TRPV3 (E) activation upon AITC, menthol, and 2-APB stimulation, respectively. (G, H) Evaluation of antagonist activity (G) and inverse agonist activity (H) for capsazepine (CPZ). CPZ antagonism was examined in the presence of 100 nM capsaicin. Note that inverse agonism is shown without subtracting the vehicle-treated basal responses. In all panels, the symbols and error bars represent the mean and SEM, respectively, for three independent experiments performed in triplicate. For many data points, the vertical error bars are smaller than the symbols and, thus, are not visible.
Fig 2.
TRP channel activation induces ectodomain shedding of EGFR ligands via both ADAM10 and ADAM17.
(A–C) Concentration–response curves showing the TGFα-shedding response in ADAM17-deficient HEK293 cells (ΔADAM17 cells). H1R (A) and TRPV1 (C) were evaluated as representatives of Gq-coupled GPCRs and TRP channels, respectively. TPA-induced shedding responses (B) were evaluated in cells expressing only AP-TGFα without receptors. (D) Concentration–response curve of TGFα-shedding responses induced by TRPV1 activation in ADAM10-siRNA-transfected parent cells or ΔADAM17 cells. (E–G) Comparisons of shedding responses induced by TRPV1 (E) and H1R (F) activation using AP-EGF and AP-BTC as reporters. The TPA-induced shedding response (G) was evaluated in cells expressing only AP-TGFα, AP-EGF, or AP-BTC, without receptors. (H, I) Concentration–response curves for the EGF- and BTC-shedding responses induced by TRPA1 (H) and TRPV3 (I) activation. In all figures, the symbols and error bars represent the mean and SEM, respectively, for three independent experiments performed in triplicate. For many data points, the vertical error bars are smaller than the symbols and, thus, are not visible.
Fig 3.
Possible mechanisms underlying ectodomain shedding of EGFR ligands by TRP channel and GPCR activation.
Putative molecular mechanisms for ectodomain shedding of EGFR ligands by TRP channels and Gq-coupled GPCRs activation. ADAM, a disintegrin and metalloproteinase domain-containing protein; ANO6, anoctamin 6; BTC, betacellulin; EGF, epidermal growth factor; GPCR, G protein–coupled receptor; PKC, protein kinase C; PS, phosphatidylserine; TGFα, transforming growth factor alpha; TRP, transient receptor potential.