Article Figures & Data
Figures
- Fig. 1.
The opioid agonist DAMGO increases postendocytic recycling of MOR. (A) HEK293 cell expressing SpH-MOR imaged with total internal reflection fluorescence microscopy after DAMGO addition. The appearance of an individual exocytic recycling event is denoted by red boxes. Images are 100 ms apart. Scale bar, 5 µm. (B) Profile of an individual exocytic event (puff) over 1 s. Frames are 100 ms apart. Scale bar, 1 μm. The event begins as a defined spot of fluorescence intensity that appears suddenly. The fluorescence diffuses on the cell membrane as shown by the heat map surface plot. (C) Experimental paradigm to study MOR postendocytic recycling. (D) The number of recycling events normalized to cell area (square micrometer) over time ± DAMGO washout after the baseline recording. In the −washout condition, P > 0.999 for baseline versus +1 minute and P = 0.306 for baseline versus +6 minutes (n = 15 cells). In the +washout condition, ****P < 0.0001 for both baseline versus 1-minute washout and baseline versus 6-minute washout (n = 27 cells). Mean and S.E.M. are plotted for each time point. (E) The percentage of recycling events in each condition (±washout) was normalized to the baseline events for each condition. ****P < 0.0001 for −washout 1 minute versus +washout 1 minute. ****P < 0.0001 for −washout 6 minutes versus +washout 6 minutes (−washout: n = 15 cells; +washout: n = 27 cells.) Box and whisker plots are shown with all points from each condition.
- Fig. 2.
The agonist-mediated increase in MOR recycling requires G protein signaling. (A) Representative images of HEK293 cell expressing SpH-MOR imaged with total internal reflection fluorescence microscopy before and after DAMGO addition. Cells pretreated with PTX 14–16 hours before imaging. Scale bar, 5 µm. (B) Following DAMGO addition, SpH-MOR clusters on the cell surface before internalizing in both control (Ctrl) and PTX-treated cells. Scale bar, 2.5 µm. (C) Quantification of percentage of normalized fluorescence in control and PTX conditions for the first 5 minutes following DAMGO addition. Values are normalized to the first frames following DAMGO addition (Ctrl: n = 10 cells; PTX: n = 8 cells). (D) Number of recycling events per cell area (square micrometer) over time in response to DAMGO washout ± PTX. P = 0.0064 for control baseline versus PTX baseline (Ctrl: n = 10 cells; PTX: n = 8 cells). Mean and S.E.M. are plotted for each time point. (E) Percentage of baseline recycling events/min at washout 6 minutes for each condition; ***P = 0.0003 for control washout 6 minutes versus PTX washout 6 minutes (Ctrl: n = 10 cells; PTX: n = 8 cells). Box and whisker plots are shown with all points from each condition. (F) Percentage of baseline recycling events/min in response to KT5720 (KT; n = 14 cells), forskolin (Fsk; n = 14 cells), or Fsk + KT (n = 14 cells); treatment normalized to initial baseline recycling events (one-sample t test: P = 0.908 for baseline vs. +KT; P = 0.9758 for baseline vs. +Fsk; P = 0.7886 for baseline vs. +Fsk+KT). Box and whisker plots are shown with all points from each condition. ns, not significant.
- Fig. 3.
Gβγ activation is required and sufficient to increase MOR recycling. (A) Number of MOR recycling events per cell area (square micrometer) over time in response to a DAMGO washout in control (Ctrl) and gallein conditions. Cells were treated with gallein 30 minutes prior to imaging. P = 0.0046 for control baseline versus gallein baseline (Ctrl: n = 9 cells; gallein: n = 25 cells). Mean and S.E.M. are plotted for each time point. (B) Percentage of baseline recycling events/min at washout 6 minutes for each condition: P = 0.0015 for control washout 6 minutes versus gallein washout 6 minutes (Ctrl: n = 9 cells; gallein: n = 25 cells). Box and whisker plots are shown with all points from each condition. (C) Number of MOR recycling events per cell area (square micrometer) over time in response to a DAMGO washout in control, mSIRK, and 12155 conditions. Cells were treated acutely with mSIRK or 12155 during the washout. Mean and S.E.M. are plotted for each time point (Ctrl: n = 10 cells; mSIRK: n = 12 cells; 12155: n = 12 cells). (D) Percentage of baseline recycling events/min at washout 1 minute for each condition: ***P = 0.0003 for control washout 1 minute versus mSIRK washout 1 minute; *P = 0.0488 for control washout 1 minute versus 12155 washout 1 minute (Ctrl: n = 10 cells; mSIRK: n = 12 cells; 12155: n = 12 cells). Box and whisker plots are shown with all points from each condition. (E) Changes in surface MOR fluorescence over time measured after DAMGO addition and washout. MOR fluorescence decreased upon receptor internalization after DAMGO addition and returned upon recycling after DAMGO washout. Gβγ activation by either mSIRK or 12155 increased the rate of recovery of fluorescence. Scale bar, 10 μm. (F) Quantification of fluorescence recovery over 30 minutes following DAMGO treatment normalized to the baseline fluorescence. (G) Quantification of fluorescence recovery normalized to the fluorescence loss before washout for control, mSIRK, or 12155 conditions. (H) Box and whisker plots showing the fluorescence after 15 minutes of agonist washout in control, mSIRK, or 12155 conditions. Gβγ activation by either mSIRK or 12155 increased the receptors recycled; ****P < 0.0001 for control versus mSIRK, **P = 0.0058 for control versus 12155 (Ctrl: n = 48 fields; mSIRK: n = 44 fields; 12155: n = 38 fields, all across three independent experiments).
- Fig. 4.
Homologous regulation of MOR recycling by MOR phosphorylation at serine 363. (A) Percentage of baseline recycling events/min in response to U73122 (n = 17 cells) or U73343 (n = 21 cells) treatment normalized to initial baseline recycling events. One-sample t test: ***P = 0.0002 for baseline versus U73122; P = 0.8910 for baseline versus U73343. Box and whisker plots are shown with all points from each condition. (B) Percentage of baseline recycling events/min in response to chelerythrine (n = 20 cells) or Gö6983 (n = 21 cells) treatment normalized to initial baseline recycling events. One-sample t test: ***P = 0.0007 for baseline versus chelerythrine; ****P < 0.0001 for baseline versus Gö6983. Box and whisker plots are shown with all points from each condition. (C) Number of recycling events per cell area (square micrometer) over time in response to DAMGO washout in control (Ctrl), S363A, S363A + gallein, or S363D conditions. P = 0.0392 for control baseline vs. S363A baseline; P = 0.0429 for control baseline vs. S363A + gallein baseline; P = 0.7203 for control baseline vs. S363D baseline; P = 0.9391 for S363D baseline vs. S363D washout 6 minutes (Ctrl: n = 18 cells; S363A: n = 15 cells; S363A + gallein: n = 19 cells; S363D: n = 10 cells). Mean and S.E.M. are plotted for each time point. (D) Percentage of baseline recycling events/min at washout 6 minute for each condition: ***P = 0.0003 for control washout 6 minute versus S363A washout 6 minutes; **P = 0.0077 for control washout 6 minutes versus S363A + gallein washout 6 minutes; ****P < 0.0001 for control washout 6 minutes versus S363D washout 6 minutes (Ctrl: n = 18 cells; S363A: n = 15 cells; S363A + gallein: n = 19 cells; S363D: n = 10 cells). Box and whisker plots are shown with all points from each condition. (E) Proposed model of self-regulation of postendocytic recycling of MOR. ns, not significant.
Additional Files
Data Supplement
- Supplemental Data -
Supplementary Figure 1 - Clustering and Internalization of MOR across conditions.
Supplementary Figure 2 - Raw MOR recycling events across all conditions.
- Supplemental Movie -
Supplementary Movie 1 - An example movie of exocytic events in a cell following agonist treatment.
- Supplemental Data -
In this issue
G Proteins Regulate Mu Opioid Receptor Recycling
