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CDK5 inhibits the clathrin-dependent internalization of TRPV1 by phosphorylating the clathrin adaptor protein AP2μ2

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Science Signaling  11 Jun 2019:
Vol. 12, Issue 585, eaaw2040
DOI: 10.1126/scisignal.aaw2040

Figures

  • Fig. 1 Constitutive internalization of TRPV1.

    (A) Schematic diagram of the biotinylation internalization assay. (B) Representative Western blot for TRPV1, transferrin receptor (TFR), and clathrin heavy chain (CHC) in biotinylation internalization assays in HEK293 cells transfected with EGFP-TRPV1 plasmid. Internalized proteins were analyzed by treating the cells with mesna solution to remove the surface biotin. β-Actin is a loading control. The bar graph shows quantitation of the ratio of internalized TRPV1 normalized to the negative control (−) in which the biotinylated cells were incubated at 37°C for 0 min. As a positive control (+), there was no mesna solution treatment to remove surface biotinylation after internalization. Data represent means ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test. ns, not significant. (C) Schematic diagram of the antibody feeding assay to measure internalization of endogenous TRPV1. (D) Representative images and quantification of the antibody feeding assay in primary DRG neurons. Scale bar, 10 μm. Data represent means ± SEM of 29 neurons (4°C group) and 41 neurons (37°C group) from three independent experiments. ***P < 0.001 by the two-tailed paired t test. (E and F) Representative traces showing the typical average change in Ca2+ influx in HEK293 cells (E) or primary DRG neurons (F) in each treatment group. Application of capsaicin (CAP) and KCl is indicated by arrows. Data represent means ± SEM of 50 cells for each group from three independent experiments; *P < 0.05, ***P < 0.001 by two-way ANOVA.

  • Fig. 2 TRPV1 internalization depends on clathrin and dynamin.

    (A) Representative Western blot for TRPV1 and TFR in biotinylation internalization assays in HEK293 cells expressing EGFP-TRPV1 and subjected to hypertonic treatment. The ratio of internalized TRPV1 or TFR normalized to the control group was quantified. Data represent means ± SEM of three independent experiments. *P < 0.05, **P < 0.01 by the one-tailed paired t test. (B) Representative images of HEK293 cells showing the distribution of EGFP-TRPV1 and clathrin. Cells stained with immunoglobulin G (IgG) are a negative control for clathrin staining. Scale bar, 10 μm. n = 20 cells from three independent experiments. (C) Schematic diagram of the biotinylation assay to quantify TRPV1 on the cell surface. (D) Representative Western blot for TRPV1, TFR, and clathrin in biotinylation assays in HEK293 cells coexpressing EGFP-TRPV1 and siRNA targeting CHC (siCHC). Surface TRPV1, surface TFR, and total CHC were quantified and normalized to the nonsilencing siRNA (NS) group. Data represent means ± SEM of three independent experiments. *P < 0.05 by the one-tailed paired t test. (E) Representative Western blot for TRPV1, TFR, and clathrin in biotinylation assays in HEK293 cells expressing EGFP-TRPV1 and treated with the dynamin inhibitor dynasore or vehicle (DMSO). Surface TRPV1 and TFR were quantified and normalized to the DMSO control group. Data represent means ± SEM of three independent experiments. *P < 0.05, ***P < 0.001 by the one-tailed paired t test. (F) Representative Western blot for TRPV1, TFR, clathrin, and EGFP in biotinylation assays in HEK293 cells coexpressing EGFP-TRPV1 plus EGFP-dynamin (WT), EGFP-DN-dynamin (K44A), or EGFP vector alone (EGFP). Surface TRPV1 and surface TFR were quantified and normalized to the EGFP vector control group. Data represent means ± SEM of three independent experiments. *P < 0.05, **P < 0.01, #P < 0.05, and ##P < 0.01 by the one-tailed paired t test.

  • Fig. 3 TRPV1 internalization depends on AP2μ2.

    (A) Representative images showing the distribution of TRPV1 and AP2μ2 in HEK293 cells. Cells stained with IgG is a negative control for the AP2μ2 antibody. Scale bar, 10 μm. n = 20 cells from three independent experiments. (B) Western blot of in vitro pull-down assays using purified recombinant His-TRPV1-NT, His-TRPV1-CT, GST-AP2μ2, and GST alone. The immunoblots were probed with an antibody against the His tag. Blot is representative of three independent experiments. (C) Western blot of GST pull-down assays in HEK293 cells coexpressing GST-TRPV1 and either His-Myc-AP2μ2 or the His-Myc vector alone. The immunoblot was probed with antibodies against GST, AP2μ2, and the His tag. Blot is representative of three independent experiments. (D and E) Representative Western blot for biotinylation assay in HEK293 cells cotransfected with EGFP-TRPV1 and either His-Myc-AP2μ2 plasmids (D) or AP2μ2 siRNA (E). Surface TRPV1, surface TFR, and total AP2μ2 were quantified and normalized to the His-Myc vector alone or nonsilencing siRNA (NS) control group. Data represent means ± SEM of three independent experiments. *P < 0.05, ***P < 0.001 by the one-tailed paired t test.

  • Fig. 4 CDK5 negatively regulates TRPV1 internalization by phosphorylating AP2μ2.

    (A) Representative Western blot for biotinylation internalization assay in HEK293 cells transfected with EGFP-TRPV1 and treated with roscovitine (Ros) or vehicle (DMSO). Internalized TRPV1 was quantified and normalized to the vehicle control group. (B) Representative Western blot for GST pull-down assay in HEK293 cells transfected with GST-TRPV1 plasmids and treated with roscovitine or vehicle. AP2μ2 was quantified and normalized to the vehicle control group. (C) Representative Western blot for biotinylation internalization assay in HEK293 cells cotransfected with EGFP-TRPV1 and shRNAs against CDK5 (shCDK5), P35 (shP35), or a scrambled control. Internalized TRPV1 was quantified and normalized to the scrambled control group. (D) Representative Western blot and autoradiogram for in vitro kinase assay using Cdk5 immunoprecipitated from mouse brain lysates incubated with the indicated GST-tagged fragments of AP2μ2 in the presence of radiolabeled adenosine 5′-triphosphate (ATP). (E) Representative Western blot for the His tag in GST pull-down assays using purified recombinant GST-AP2μ2 and His-CDK5. (F) Representative Western blot and autoradiogram for in vitro kinase assay using Cdk5 immunoprecipitated from mouse brain lysates and incubated with GST-AP2μ2 1 to 100 amino acids or mutant GST-AP2μ2 1 to 100 amino acids (S45A) in the presence of radiolabeled ATP. (G) Representative Western blot for Cdk5 in GST pull-down assays using GST-AP2μ2 1 to 100 amino acids or mutant GST-μ2 1 to 100 amino acids (S45A) against mouse brain lysates. (H) Representative Western blot and quantification of AP2μ2 phosphorylated at Ser45 (p-AP2μ2) in HEK293 cells treated with roscovitine or vehicle. (I) Representative Western blot and quantification of AP2μ2 phosphorylated at Ser45 in HEK293 cells cotransfected with EGFP-TRPV1 and shRNAs against CDK5. For all panels, n = 3 independent experiments. Data represent means ± SEM. *P < 0.05 by the one-tailed paired t test.

  • Fig. 5 The TAT-S45 fusion peptide enhances TRPV1 internalization.

    (A) Schematic diagram of the TAT-S45 peptide and TAT-S45A control peptide. (B) Representative Western blot and autoradiogram for in vitro kinase assay. P35 was immunoprecipitated from mouse brain lysates and incubated with GST-CDK5 and GST-μ2 1 to 100 amino acids in the presence of radiolabeled ATP plus the TAT-S45 or TAT-S45A peptide. P-AP2μ2 1 to 100 amino acids was quantified and normalized to the TAT-S45A control group. Data with means ± SEM of three independent experiments; *P < 0.05 by the one-tailed paired t test. (C and D) Representative Western blots for biotinylation assays (C) or biotinylation internalization assays (D) in HEK293 cells transfected with EGFP-TRPV1 plasmids after TAT-S45 or TAT-S45A peptide treatment. Surface TRPV1 (C) and internalized TRPV1 (D) were quantified and normalized to the TAT-S45A control group. Data represent means ± SEM of three independent experiments; *P < 0.05, **P < 0.01 by the one-tailed paired t test. (E) Representative Western blot showing GST-TRPV1 and AP2μ2 in GST pull-down assays from lysates of HEK293 cells expressing GST-TRPV1 and treated with TAT-S45 or TAT-S45A peptide. AP2μ2 and clathrin were quantified and normalized to the TAT-S45A control group. Data with means ± SEM of three independent experiments; *P < 0.05 by the one-tailed paired t test. (F) Representative traces showing the typical average change in Ca2+ influx of HEK293 cells in each group. Application of capsaicin (CAP) and KCl is indicated by arrows. Data with means ± SEM of 50 cells for each group from three independent experiments; ***P < 0.001 by two-way ANOVA.

  • Fig. 6 Phosphorylation of AP2μ2 at Ser45contributes to the development of inflammatory thermal hyperalgesia in rats.

    (A and B) Representative Western blot and quantification of Ap2μ2 phosphorylated at Ser45 (p-AP2μ2) in rat DRG tissues at the indicated times after injection with 25% CFA (A) or 6 hours after intrathecal injection with roscovitine or DMSO, followed by injection with 25% CFA (B). For quantitative analysis, p-AP2μ2 abundance was normalized to the 0-hour (A) or DMSO control (B) group. Data represent means ± SEM of three independent experiments. *P < 0.05 by the one-tailed paired t test. (C) Representative Western blot for TRPV1 in rat DRG tissues after intrathecal injection of TAT-S45A and TAT-S45 peptides. The bar graph shows the abundance of TRPV1 normalized to that in the TAT-S45A group. Data represent means ± SEM of three independent experiments. *P < 0.05 by the two-tailed paired t test. (D) Paw lifting time in the first 5 min after capsaicin injection into rats intrathecally injected with the TAT-S45A or TAT-S45 peptide. Data were analyzed by the two-tailed unpaired t test; **P < 0.01; n = 8 rats for each group. (E to H) Representative time course of the PWL of the ipsilateral hind paw as measured by radiant heat stimuli before and after intrathecal injection of 10 μg (E and F), 3 μg (G), or 30 μg (H) of TAT-S45 or TAT-S45A peptides, followed by no treatment (E) or injection of 25% CFA (F to H). Data represent means ± SEM of six to eight rats; *P < 0.05 by the two-way ANOVA followed by Bonferroni’s post hoc tests.

  • Fig. 7 Schematic model representing the TRPV1 internalization process and its regulation by CDK5.

    (A) Under basal conditions, AP2μ2 binds to the N terminus (NT) of TRPV1 and stimulates TRPV1 internalization through a clathrin- and dynamin-dependent pathway. (B) Cdk5 activity is increased in the context of peripheral inflammation, leading to the Cdk5-mediated phosphorylation of AP2μ2 at Ser45, which negatively regulates TRPV1 internalization. (C) The TAT-S45 peptide competes with endogenous AP2μ2 for phosphorylation by Cdk5, thus antagonizing the Cdk5-dependent inhibition of TRPV1 internalization and reducing inflammatory hyperalgesia.

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