A-kinase anchoring protein 150 controls protein kinase C-mediated phosphorylation and sensitization of TRPV1

Abstract

Post-translational modifications on various receptor proteins have significant effects on receptor activation. For the Transient Receptor Potential family V type 1 (TRPV1) receptor, phosphorylation of certain serine/threonine amino acid residues sensitizes the receptor to activation by capsaicin and heat. Although Protein Kinase C (PKC) phosphorylates TRPV1 on certain serine/threonine residues, it is not completely understood how PKC functionally associates with TRPV1. Recent studies have reported that the A-kinase Anchoring Protein 150 (AKAP150) mediates PKA phosphorylation of TRPV1 in several nociceptive models. Here, we demonstrate that AKAP150 also mediates PKC-directed phosphorylation and sensitization of TRPV1. In cultured rat trigeminal ganglia, immunocytochemical analyses demonstrate co-localization of AKAP150 and PKC isoforms alpha, delta, epsilon, and gamma in TRPV1-positive neurons. Additional biochemical evidence supports immunocytochemical results, indicating that AKAP150 preferentially associates with certain PKC isoforms in rat trigeminal ganglia neurons. Employing siRNA-mediated knock-down of AKAP150 expression, we demonstrate that PKC-mediated phosphorylation of TRPV1 and sensitization to a capsaicin response is dependent upon functional expression of the AKAP150 scaffolding protein. Furthermore, PKC-induced sensitization to a thermal stimulus is abrogated in AKAP150 knock-out animals relative to wild-type. Collectively, the results from these studies indicate that the AKAP150 scaffolding protein functionally modulates PKC-mediated phosphorylation and sensitization of the TRPV1 receptor in rat sensory neurons, suggesting the scaffolding protein to be an integral regulator of peripheral inflammatory hyperalgesia.

Figure 1. PKC isoform co-expression with AKAP150 in TRPV1 (+) trigeminal

neurons. Cultured TG neurons were analyzed for PKC isoform, AKAP150, and TRPV1 expression relationships using immunofluorescence confocal microscopy. TG neurons were probed for TRPV1 (blue, A, E, I, M), AKAP150 (red, B, F, J, N) and PKCα (green, C), PKCδ (green, G), PKCε (green, K) or PKCγ (green, O), with merged images (yellow, D, H, L, P) indicating co-expression (yellow bar = 50 μm). Results are representative of staining relationships observed in 10–15 neurons from 3 coverslips that were stained with each antibody combination.

Figure 2. AKAP150 Co-immunoprecipitates with specific PKC isoforms

A. Cultured TG neurons were harvested, and 350 mg aliquots were immunoprecipitated with antibodies directed against PKCα, PKCε, PKCδ, PKCγ, AKAP150, and TRPV1. Immunoprecipitates and TG cell lysate (TG CL) were resolved by SDS-PAGE, transferred to PVDF, and probed for AKAP150 expression. B. IgG immunoprecipitation control demonstrates specificity of AKAP150 antibody. C. TG CL samples were also probed for PKC isoforms α, ε, δ, and γ to observe relative expression levels in TG cultures.

Figure 3. PKC-mediated phosphorylation of TRPV1 is dependent upon AKAP150 expression

A. Cultured TG neurons were transfected in a mock setting, with scrambled control siRNA, or with AKAP150-specific siRNA. In vitro PKC kinase activity was assayed from cell lysates of cultured TG neurons treated with vehicle (0.1% ethanol) or PDBu (1 mM, 5 min). B. Following siRNA transfection, TG neurons were loaded with ³²P orthophosphate and treated with either veh or PDBu (1 μM, 5 min), and immunoprecipitated TRPV1 was analyzed for ³²P incorporation. Representative autoradiographic and Western blot results are shown. Results are representative of 4 individual trials. * p<0.05, ** p<0.01, NS: no significance, as determined by one-way ANOVA.

Figure 4. PKC-mediated sensitization of capsaicin (CAP)-induced calcium influx is dependent upon the PKC-binding domain of AKAP150

A. Cummulative measurements of calcium accumulation in CHO cells transiently transfected with either AKAP150wt or AKAP150ΔPKC with rTRPV1. Cells were pre-treated with vehicle or PDBu (1 μM, 5 min) and stimulated with CAP (50 nm) to measure calcium accumulation. Results are representative of 45–60 cells per transfection/treatment. B. Co-immunoprecipitation and Western blot analysis of transfected CHO cells (as shown). Results are representative of 3 individual trials. * p<0.05, *** p<0.005, as determined by one-way ANOVA.

Figure 5. PKC-mediated sensitization of capsaicin(CAP)-induced inward current is dependent upon AKAP150 expression

A. Sample CAP-current traces from mock transfected and FITC AKAP150 siRNA-transfected rat TG neurons pre-treated with vehicle (black) or PDBu (blue, 1 μM, 5 min) prior to CAP treatment. B. Cummulative measurements of CAP current from rat TG neurons transfected in a mock setting, with scrambled control siRNA (Scr siRNA), or with FITC-labeled AKAP150-specific siRNA. Results are representative of 6–8 neurons per transfection/treatment. C. Sample CAP-current traces from TG neurons isolated from wild-type (WT) or AKAP150 knock-out (AKAP150 KO) mice, pre-treated with vehicle (black) or PDBu (blue, 1 μM, 5 min) prior to CAP treatment. D. Cummulative measurements of CAP current from TG neurons isolated from wild-type (WT) or AKAP150 knock-out (AKAP150 KO) mice. GF109203x (GFX, 3 μM), a general PKC inhibitor, was co-treated with PDBu, as indicated. Results are representative of 6– 10 neurons per treatment. *** p<0.005, NS: no significance, as determined by student’s t-test.

Figure 6. Genetic ablation of functional AKAP150 protein reduces Protein Kinase C-induced sensitization to thermal hyperalgesia

WT and AKAP150KO mice were injected with 20 μl of 10nM bradykinin (BK) or 1 nmol PDBu in the right rear hindpaw, upon which thermal paw withdrawal latencies were measured by blinded observation using a Hargreaves’ apparatus. Data are depicted as paw withdrawal latency (sec). All plotted data expressed as mean ± SEM, n= 6–8 male animals per group. * p<0.05, ** p<0.01, † p<0.05, as determined by two-way ANOVA. Asterisks denote significance within treatment groups, while crosses denote significance between treatments.