Compartment-specific opioid receptor signaling is selectively modulated by different dynorphin peptides
- Cellular and Molecular Biology Training Program, University of Michigan Medical School, United States
- Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, United States
- Department of Pharmacology, University of Michigan Medical School, United States
Figures
Figure 1 with 1 supplement
Initial activation and internalization of KOR by Dynorphins are comparable.
(A) Schematic of the regions of Pro-dynorphin from which Dynorphin A8 (Dyn A8), Dynorphin A (Dyn A), Dynorphin B (Dyn B), and α-neoendorphin (α-neo) peptides are generated, showing that Dyn A and …
Figure 1—figure supplement 1
Ligand-mediated decreases in intracellular cAMP levels and endocytosis of KOR saturates at 1 µM for Dyn A and Dyn B.
(A) Dyn A- and Dyn B-mediated decreases in intracellular cAMP levels were measured by carrying out doseresponse curves (0–10 µM) in PC12 cells stably expressing SpH-KOR treated for 30 min at 37°C. (B…
Figure 2 with 1 supplement
The post-endocytic fate of KOR is determined by the specific Dynorphin that activates it.
(A) Frames from a time lapse movie of a representative region of a PC12 cell stably expressing SpH-KOR (SpH-KOR cells) shown in (B), treated for 5 min with Dyn B (1 μM) and imaged in TIR-FM, showing …
Figure 2—figure supplement 1
The differences in KOR recycling between Dyn A and Dyn B cannot be explained entirely by peptide degradation.
(A) Quantitation of the number of exocytic events/μm2/min in SpH-KOR PC12 cells shows a significantly higher number of events for Dyn B compared to Dyn A (1 µM) in the continued presence of protease …
Figure 3 with 1 supplement
Dyn A selectively drives KOR signaling from late endosomal compartments.
(A) Representative image of a PC12 cell expressing FLAG-KOR and Rab7-GFP, treated with 1 µM Dyn A for 20 min. Yellow arrows denote KOR endosomes that colocalize with Rab7. (B) SpH-KOR cells treated …
Figure 3—figure supplement 1
Differential receptor sorting between Dyn A and Dyn B persists even after agonists are washed out from the surface.
(A) Representative images of PC12 cells expressing FLAG KOR treated with Dyn A in the presence of 40 µM Dyngo4A for 30 min to block endocytosis. KOR (magenta in merge) fluorescence is restricted to …
Figure 4 with 1 supplement
Dyn A-specific late endosomal localization and signaling is conserved in striatal neurons.
(A) The number of discrete exocytic events quantitated in rat medium spiny neuron (MSN) expressing SpH-KOR shows increased recycling for 1 μM Dyn B compared to Dyn A (***p<0.001, n = 8 cells). (B) …
Figure 4—figure supplement 1
mTOR signaling does not show significant differences between Dyn A and Dyn B.
(A) Representative blots showing phosphorylated (pS6K) and total (tS6K) S6K levels in PC12 cells stably expressing SpH-KOR treated with 1 µM Dyn A or Dyn B for 5 and 20 min. (B) Quantitation of the …
Author response image 1
BAM22 drives noticeably less KOR recycling than DynB, even though they are both sensitive to ECE2 inhibition.
(A) Recycling of HA-KOR to the cell surface after 30 min treatment with 100 nM Dyn B or BAM22 and washout for 0-120 min. Cell surface receptors were quantified by ELISA as described in Methods. …
Author response image 2
Dyn B is dominant over Dyn A for KOR endocytic trafficking.
(A) Quantitation of the number of exocytic events/μm2/min, as in Figure 2, in SpH-KOR PC12 cells co-treated with 1μM of Dyn A and 1μM of Dyn B, compared to either on its own. The co-treatment mimics …
Tables
Table 1
Displacement binding parameters for Dynorphin peptides at PC12 SPH-KOR and CHO-KOR cells.
| PC12 SpH-KOR cells | CHO-KOR cells | |||||||
|---|---|---|---|---|---|---|---|---|
| Ligand | Low Ki (nM) | High Ki (nM) | % Bmax at 10 μM | nH | Low Ki (nM) | High Ki (nM) | % Bmax at 10 μM | nH |
| Dyn A8 | 56.9 ± 0.3 | 0.020 ± 1.90 | 18.30 ± 1.48 | 36.9% | 563 ± 0.1 | 0.41 ± 0.15 | 21.51 ± 1.54 | 41.6% |
| Dyn A17 (Dyn A) | 52.4 ± 0.3 | 0.016 ± 0.47 | 15.22 ± 1.56 | 39.1% | 119 ± 0.2 | 0.26 ± 0.54 | 18.72 ± 1.09 | 29.4% |
| Dyn B13 (Dyn B) | 37.8 ± 0.2 | 0.010 ± 0.31 | 11.13 ± 1.07 | 33.4% | 355 ± 0.1 | 0.38 ± 0.11 | 17.16 ± 0.82 | 42.2% |
| a-neo-endorphin | 39.1 ± 0.1 | 0.011 ± 0.20 | 13.32 ± 1.84 | 38.7% | 591 ± 0.1 | 0.18 ± 0.34 | 20.55 ± 1.57 | 21.4% |
Additional files
-
Source data 1
Numerical data.
- https://cdn.elifesciences.org/articles/60270/elife-60270-data1-v2.xlsx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/60270/elife-60270-transrepform-v2.docx
