Agonist-selective recruitment of engineered protein probes and of GRK2 by opioid receptors in living cells
- University of California, San Francisco, United States
- University of Geneva, Switzerland
- Washington University School of Medicine, United States
- St Louis College of Pharmacy, United States
- Medical College of Georgia, Augusta University, United States
Figures
Figure 1
Comparative detection of direct probe recruitment by opioid receptors in living cells.
(A) Crystal structures of the DAMGO-bound MOR (red) - Gi (green/blue) complex (PDB: 6DDF) and the BU27-bound MOR (red) – nanobody (green) complex (PDB: 5C1M). Ligands are shown in blue. (B) …
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Figure 1—source data 1
Concentration-dependent recruitment of mGsi and Nb33 probes to KOR in response to DynA, U69, and U50 (Figure 1I-K).
- https://cdn.elifesciences.org/articles/54208/elife-54208-fig1-data1-v1.csv
Figure 2
Selective recruitment of protein probes by KOR upon activation by etorphine.
(A) mGsi intensity during TIR-FM time-lapse series of a HEK293 cell, co-expressing Venus-mGsi and KOR, adding increasing concentrations of etorphine (1 nM - 10 μM), followed by reference compound …
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Figure 2—source data 1
ET concentration-dependent recruitment of mGsi and Nb33 probes to KOR, or to KOR-TPD, or to KOR in the presence of Cmpd101.
- https://cdn.elifesciences.org/articles/54208/elife-54208-fig2-data1-v1.csv
Figure 3
Agonist-selective protein probe recruitment by MOR.
(A) 7 min kymograph traced inside a cell expressing Venus-mGsi, mCherry-Nb33, and FLAG-MOR (labeled with anti-FLAG M1-AF647) and treated with increasing concentrations of DAMGO (agonist), followed …
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Figure 3—source data 1
Concentration-dependent recruitment of mGsi and Nb33 probes to MOR in response to DAMGO, ET, morphine, or PZM21.
- https://cdn.elifesciences.org/articles/54208/elife-54208-fig3-data1-v1.csv
Figure 4
Receptor-proximal probe recruitment across agonists and receptors.
(A) Table summarizing mGsi and Nb33 recruitment efficacies to KOR and MOR upon different agonists. Intrinsic activities (‘I.A.’, maximal response) for both probes and each agonist are given (average …
Figure 5
Agonist-selective modes of GRK2 recruitment to the plasma membrane.
(A) Schematic depicting two modes of GRK2 recruitment from the cytosol to the plasma membrane upon KOR activation: one involves interaction with G beta-gamma subunits exposed upon G protein …
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Figure 5—source data 1
Concentration-dependent recruitment of GRK2 to the plasma membrane in response to DynA or ET (Figure 5D).
- https://cdn.elifesciences.org/articles/54208/elife-54208-fig5-data1-v1.csv
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Figure 5—source data 2
Recruitment behavior of GRK2 to the plasma membrane in response to ET and DynA (Figure 5E–H).
- https://cdn.elifesciences.org/articles/54208/elife-54208-fig5-data2-v1.csv
Figure 6
Agonist-selective modes of GRK2 recruitment by KOR.
(A) Schematic of receptor clustering in the plasma membrane using cross-linking by polyclonal antibody. (B) TIR-FM images of a cell expressing GRK2-mCherry and SEP-KOR. KOR was cross-linked with …
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Figure 6—source data 1
Recruitment of GRK2 to KOR clusters upon DynA or ET treatment (Figure 6D).
- https://cdn.elifesciences.org/articles/54208/elife-54208-fig6-data1-v1.csv
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (human, female) | HEK293 | ATCC | CRL-1573; RRID: CVCL_0045 | Human embryonic kidney |
| Antibody | Mouse anti-FLAG (M1) | Sigma-Aldrich | F-3040; RRID: AB_439712 | (1:1000) |
| Antibody | Rabbit anti-GFP | Invitrogen | A-11122; RRID: AB_221569 | (1:100) |
| Recombinant DNA reagent | EGFP-Nb33 | (Stoeber et al., 2018) | N/A | EGFP-C1 backbone |
| Recombinant DNA reagent | pmApple-Nb33 | (Stoeber et al., 2018) | N/A | pmApple-C1 backbone |
| Recombinant DNA reagent | NES-Venus-mGsi | (Wan et al., 2018) | N/A | pcDNA3 backbone |
| Recombinant DNA reagent | NES-Venus-mGs | (Wan et al., 2018) | N/A | pcDNA3 backbone |
| Recombinant DNA reagent | signal sequence FLAG (ssf)-MOR, murine | (Stoeber et al., 2018) | N/A | pcDNA3 backbone |
| Recombinant DNA reagent | ssf-KOR, murine | (Chu et al., 1997) | N/A | pcDNA3 backbone |
| Recombinant DNA reagent | ssf-KOR-TPD (S356A, T357A, T363A, S369A) | This study | N/A | pcDNA3 backbone, see Materials and methods |
| Recombinant DNA reagent | Super ecliptic pHluorin (SEP) -KOR, murine | This study | N/A | pCAGGS-SE backbone, see Materials and methods |
| Recombinant DNA reagent | GRK2-EGFP, murine | This study | N/A | pCAGGS-SE backbone, see Materials and methods |
| Recombinant DNA reagent | GRK2-pmApple, murine | This study | N/A | pCAGGS-SE backbone, see Materials and methods |
| Recombinant DNA reagent | GRK2-C-tail- EGFP (aa 546–670) | This study | N/A | pCAGGS-SE backbone, see Materials and methods |
| Peptide, recombinant protein | Dynorphin A (1–17, DynA) | Anaspec | AS-24298 | |
| Peptide, recombinant protein | DAMGO, [D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin acetate salt | Sigma-Aldrich | E7384 | |
| Chemical compound, drug | U-69593 (U69) | Cayman Chemical | 13255 | |
| Chemical compound, drug | U-50488 hydrochloride (U50) | Tocris | 0495 | |
| Chemical compound, drug | GNTI dihydrochloride (5’GNTI) | Axon Med Chem | 1226 | |
| Chemical compound, drug | Etorphine-HCl | NIDA | N/A | |
| Chemical compound, drug | Morphine sulfate (MS) | Sigma-Aldrich | 1448005 | |
| Chemical compound, drug | Naloxone hydrochloride dihydrate | Sigma-Aldrich | N7758 | |
| Chemical compound, drug | PZM21 | Enamine | N/A | custom synthesis |
| Chemical compound, drug | mitragynine pseudoindoxyl (MP) | This study | N/A | (Váradi et al., 2016) |
| Chemical compound, drug | Compound101 (Cmpd101) | HelloBio | HB2840 | |
| Chemical compound, drug | Pertussis toxin | Sigma-Aldrich | P7208 | |
| Commercial assay or kit | Alexa Fluor 647 Protein Labeling Kit | Thermo Fisher Scientific | A20173 | |
| Software, algorithm | Prism | GraphPad | 8.1.1 | |
| Software, algorithm | ImageJ | Imagej.net/contributors | 2.0.0-rc-54/1.51 g | |
| Software, algorithm | MATLAB | MathWorks | R2014b | |
| Software, algorithm | PyMOL | Schrödinger | v1.7.4.5 |
