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Cell-autonomous regulation of Mu-opioid receptor recycling by substance P.

Bowman SL, Soohoo AL, Shiwarski DJ, Schulz S, Pradhan AA, Puthenveedu MA - Cell Rep (2015)

Bottom Line: SP, through activation of the neurokinin 1 (NK1R) receptor, increases the post-endocytic recycling of the mu-opioid receptor (MOR) in trigeminal ganglion (TG) neurons in an agonist-selective manner.SP-mediated protein kinase C (PKC) activation is both required and sufficient for increasing recycling of exogenous and endogenous MOR in TG neurons.Our results define a physiological pathway that cross-regulates opioid receptor recycling via direct modification of MOR and suggest a mode of homeostatic interaction between the pain and analgesic systems.

View Article: PubMed Central - PubMed

ABSTRACT
How neurons coordinate and reprogram multiple neurotransmitter signals is an area of broad interest. Here, we show that substance P (SP), a neuropeptide associated with inflammatory pain, reprograms opioid receptor recycling and signaling. SP, through activation of the neurokinin 1 (NK1R) receptor, increases the post-endocytic recycling of the mu-opioid receptor (MOR) in trigeminal ganglion (TG) neurons in an agonist-selective manner. SP-mediated protein kinase C (PKC) activation is both required and sufficient for increasing recycling of exogenous and endogenous MOR in TG neurons. The target of this cross-regulation is MOR itself, given that mutation of either of two PKC phosphorylation sites on MOR abolishes the SP-induced increase in recycling and resensitization. Furthermore, SP enhances the resensitization of fentanyl-induced, but not morphine-induced, antinociception in mice. Our results define a physiological pathway that cross-regulates opioid receptor recycling via direct modification of MOR and suggest a mode of homeostatic interaction between the pain and analgesic systems.

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PKC Requires S363 and T370 to Regulate MOR Recycling(A) Schematic of PKC phosphorylation sites on C-terminal tail of MOR (S363, T370, and S375).(B) Kymographs of SpH-MOR single exocytic events for MOR S363A, T370A, and S375A after SP.(C) Quantitation of percentage recycling across cells (n = 20, 14, 18, and 22) in HA-NK1R-expressing cells with MOR mutants S363A and T370A, and S375, compared to wild-type. Dashed line shows number of events in same cells prior to SP normalized to 100%.(D) Percentage recycling with PMA-treated cells expressing MOR mutants S363A, T370A, and S375A (n = 52, 29, 18, and 17).(E) Ratiometric recycling assay in TG neurons for S363A with and without SP. Scale bar represents 10 μm.(F) Ratio quantitation across multiple cells (mean ± SEM; n = 35 surf ctrl, 38 intern, 33 rec, 41 rec + SP) between the washout without and with SP for S363A.(G) Tukey box plots of Pearson's coefficients from S363A cells in (F) show no increase in correlation with SP.(H) Ratiometric recycling assay in TG neurons for T370A with and without SP.(I) Ratio quantitation (n = 47, 27, 29, and 27) without and with SP for T370A.(J) Tukey box plots of Pearson's coefficients from T370A cells in (I) show no increase in correlation with SP. Scale bars represent 5 μm.All error bars are ±SEM.
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Figure 5: PKC Requires S363 and T370 to Regulate MOR Recycling(A) Schematic of PKC phosphorylation sites on C-terminal tail of MOR (S363, T370, and S375).(B) Kymographs of SpH-MOR single exocytic events for MOR S363A, T370A, and S375A after SP.(C) Quantitation of percentage recycling across cells (n = 20, 14, 18, and 22) in HA-NK1R-expressing cells with MOR mutants S363A and T370A, and S375, compared to wild-type. Dashed line shows number of events in same cells prior to SP normalized to 100%.(D) Percentage recycling with PMA-treated cells expressing MOR mutants S363A, T370A, and S375A (n = 52, 29, 18, and 17).(E) Ratiometric recycling assay in TG neurons for S363A with and without SP. Scale bar represents 10 μm.(F) Ratio quantitation across multiple cells (mean ± SEM; n = 35 surf ctrl, 38 intern, 33 rec, 41 rec + SP) between the washout without and with SP for S363A.(G) Tukey box plots of Pearson's coefficients from S363A cells in (F) show no increase in correlation with SP.(H) Ratiometric recycling assay in TG neurons for T370A with and without SP.(I) Ratio quantitation (n = 47, 27, 29, and 27) without and with SP for T370A.(J) Tukey box plots of Pearson's coefficients from T370A cells in (I) show no increase in correlation with SP. Scale bars represent 5 μm.All error bars are ±SEM.

Mentions: Considering that PKC was required and sufficient for heterologous regulation of MOR recycling through SP, we sought to identify the target of PKC. The MOR itself presented an interesting candidate. PKC can phosphorylate three sites on the C-terminal tail of MOR: serine 363, threonine 370, and serine 375 (Figure 5A; Doll et al., 2011; Feng et al., 2011). To test whether MOR phosphorylation was required, we mutated each of these sites to alanine to block phosphorylation (Feng et al., 2011) and quantified SP-mediated regulation of MOR recycling. SP did not increase the percentage of recycling events per unit time when either S363 or T370 was mutated (Figures 5B and 5C). In contrast, the recycling of S375A increased to a level comparable to wild-type in response to SP (Figures 5B and 5C). This indicates that S363 and T370 are required for SP-mediated regulation, but S375 is not (Figures 5B and 5C). Additionally, PMA increased SpH-MOR exocytic events for S375A, but not S363A or T370A, comparable to wild-type MOR (Figure 5D). In TG neurons, SP failed to increase S363A or T370A recycling (Figures 5E–5G for S363A and Figures 5H–5J for T370A), indicating that both S363 and T370 are required for PKC to regulate MOR recycling.


Cell-autonomous regulation of Mu-opioid receptor recycling by substance P.

Bowman SL, Soohoo AL, Shiwarski DJ, Schulz S, Pradhan AA, Puthenveedu MA - Cell Rep (2015)

PKC Requires S363 and T370 to Regulate MOR Recycling(A) Schematic of PKC phosphorylation sites on C-terminal tail of MOR (S363, T370, and S375).(B) Kymographs of SpH-MOR single exocytic events for MOR S363A, T370A, and S375A after SP.(C) Quantitation of percentage recycling across cells (n = 20, 14, 18, and 22) in HA-NK1R-expressing cells with MOR mutants S363A and T370A, and S375, compared to wild-type. Dashed line shows number of events in same cells prior to SP normalized to 100%.(D) Percentage recycling with PMA-treated cells expressing MOR mutants S363A, T370A, and S375A (n = 52, 29, 18, and 17).(E) Ratiometric recycling assay in TG neurons for S363A with and without SP. Scale bar represents 10 μm.(F) Ratio quantitation across multiple cells (mean ± SEM; n = 35 surf ctrl, 38 intern, 33 rec, 41 rec + SP) between the washout without and with SP for S363A.(G) Tukey box plots of Pearson's coefficients from S363A cells in (F) show no increase in correlation with SP.(H) Ratiometric recycling assay in TG neurons for T370A with and without SP.(I) Ratio quantitation (n = 47, 27, 29, and 27) without and with SP for T370A.(J) Tukey box plots of Pearson's coefficients from T370A cells in (I) show no increase in correlation with SP. Scale bars represent 5 μm.All error bars are ±SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494997&req=5

Figure 5: PKC Requires S363 and T370 to Regulate MOR Recycling(A) Schematic of PKC phosphorylation sites on C-terminal tail of MOR (S363, T370, and S375).(B) Kymographs of SpH-MOR single exocytic events for MOR S363A, T370A, and S375A after SP.(C) Quantitation of percentage recycling across cells (n = 20, 14, 18, and 22) in HA-NK1R-expressing cells with MOR mutants S363A and T370A, and S375, compared to wild-type. Dashed line shows number of events in same cells prior to SP normalized to 100%.(D) Percentage recycling with PMA-treated cells expressing MOR mutants S363A, T370A, and S375A (n = 52, 29, 18, and 17).(E) Ratiometric recycling assay in TG neurons for S363A with and without SP. Scale bar represents 10 μm.(F) Ratio quantitation across multiple cells (mean ± SEM; n = 35 surf ctrl, 38 intern, 33 rec, 41 rec + SP) between the washout without and with SP for S363A.(G) Tukey box plots of Pearson's coefficients from S363A cells in (F) show no increase in correlation with SP.(H) Ratiometric recycling assay in TG neurons for T370A with and without SP.(I) Ratio quantitation (n = 47, 27, 29, and 27) without and with SP for T370A.(J) Tukey box plots of Pearson's coefficients from T370A cells in (I) show no increase in correlation with SP. Scale bars represent 5 μm.All error bars are ±SEM.
Mentions: Considering that PKC was required and sufficient for heterologous regulation of MOR recycling through SP, we sought to identify the target of PKC. The MOR itself presented an interesting candidate. PKC can phosphorylate three sites on the C-terminal tail of MOR: serine 363, threonine 370, and serine 375 (Figure 5A; Doll et al., 2011; Feng et al., 2011). To test whether MOR phosphorylation was required, we mutated each of these sites to alanine to block phosphorylation (Feng et al., 2011) and quantified SP-mediated regulation of MOR recycling. SP did not increase the percentage of recycling events per unit time when either S363 or T370 was mutated (Figures 5B and 5C). In contrast, the recycling of S375A increased to a level comparable to wild-type in response to SP (Figures 5B and 5C). This indicates that S363 and T370 are required for SP-mediated regulation, but S375 is not (Figures 5B and 5C). Additionally, PMA increased SpH-MOR exocytic events for S375A, but not S363A or T370A, comparable to wild-type MOR (Figure 5D). In TG neurons, SP failed to increase S363A or T370A recycling (Figures 5E–5G for S363A and Figures 5H–5J for T370A), indicating that both S363 and T370 are required for PKC to regulate MOR recycling.

Bottom Line: SP, through activation of the neurokinin 1 (NK1R) receptor, increases the post-endocytic recycling of the mu-opioid receptor (MOR) in trigeminal ganglion (TG) neurons in an agonist-selective manner.SP-mediated protein kinase C (PKC) activation is both required and sufficient for increasing recycling of exogenous and endogenous MOR in TG neurons.Our results define a physiological pathway that cross-regulates opioid receptor recycling via direct modification of MOR and suggest a mode of homeostatic interaction between the pain and analgesic systems.

View Article: PubMed Central - PubMed

ABSTRACT
How neurons coordinate and reprogram multiple neurotransmitter signals is an area of broad interest. Here, we show that substance P (SP), a neuropeptide associated with inflammatory pain, reprograms opioid receptor recycling and signaling. SP, through activation of the neurokinin 1 (NK1R) receptor, increases the post-endocytic recycling of the mu-opioid receptor (MOR) in trigeminal ganglion (TG) neurons in an agonist-selective manner. SP-mediated protein kinase C (PKC) activation is both required and sufficient for increasing recycling of exogenous and endogenous MOR in TG neurons. The target of this cross-regulation is MOR itself, given that mutation of either of two PKC phosphorylation sites on MOR abolishes the SP-induced increase in recycling and resensitization. Furthermore, SP enhances the resensitization of fentanyl-induced, but not morphine-induced, antinociception in mice. Our results define a physiological pathway that cross-regulates opioid receptor recycling via direct modification of MOR and suggest a mode of homeostatic interaction between the pain and analgesic systems.

Show MeSH
Related in: MedlinePlus