Agonist-selective recruitment of engineered protein probes and of GRK2 by opioid receptors in living cells

  1. Miriam Stoeber  Is a corresponding author
  2. Damien Jullié
  3. Joy Li
  4. Soumen Chakraborty
  5. Susruta Majumdar
  6. Nevin A Lambert
  7. Aashish Manglik
  8. Mark von Zastrow  Is a corresponding author
  1. University of California, San Francisco, United States
  2. University of Geneva, Switzerland
  3. Washington University School of Medicine, United States
  4. St Louis College of Pharmacy, United States
  5. Medical College of Georgia, Augusta University, United States
6 figures, 1 table and 1 additional file

Figures

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) …

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
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 …

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
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 …

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
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 …

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 …

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
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
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 …

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
DesignationSource or referenceIdentifiersAdditional
information
Cell line (human, female)HEK293ATCCCRL-1573; RRID: CVCL_0045Human embryonic kidney
AntibodyMouse anti-FLAG (M1)Sigma-AldrichF-3040; RRID: AB_439712(1:1000)
AntibodyRabbit anti-GFPInvitrogenA-11122; RRID: AB_221569(1:100)
Recombinant DNA reagentEGFP-Nb33(Stoeber et al., 2018)N/AEGFP-C1 backbone
Recombinant DNA reagentpmApple-Nb33(Stoeber et al., 2018)N/ApmApple-C1 backbone
Recombinant DNA reagentNES-Venus-mGsi(Wan et al., 2018)N/ApcDNA3 backbone
Recombinant DNA reagentNES-Venus-mGs(Wan et al., 2018)N/ApcDNA3 backbone
Recombinant DNA reagentsignal sequence FLAG (ssf)-MOR, murine(Stoeber et al., 2018)N/ApcDNA3 backbone
Recombinant DNA reagentssf-KOR, murine(Chu et al., 1997)N/ApcDNA3 backbone
Recombinant DNA reagentssf-KOR-TPD (S356A, T357A, T363A, S369A)This studyN/ApcDNA3 backbone, see Materials and methods
Recombinant DNA reagentSuper ecliptic pHluorin (SEP) -KOR, murineThis studyN/ApCAGGS-SE backbone, see Materials and methods
Recombinant
DNA reagent
GRK2-EGFP, murineThis studyN/ApCAGGS-SE backbone, see Materials and methods
Recombinant DNA reagentGRK2-pmApple, murineThis studyN/ApCAGGS-SE backbone, see Materials and methods
Recombinant DNA reagentGRK2-C-tail- EGFP (aa 546–670)This studyN/ApCAGGS-SE backbone, see Materials and methods
Peptide, recombinant proteinDynorphin A (1–17, DynA)AnaspecAS-24298
Peptide, recombinant proteinDAMGO, [D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin acetate saltSigma-AldrichE7384
Chemical compound, drugU-69593 (U69)Cayman Chemical13255
Chemical compound, drugU-50488 hydrochloride (U50)Tocris0495
Chemical compound, drugGNTI dihydrochloride (5’GNTI)Axon Med Chem1226
Chemical compound, drugEtorphine-HClNIDAN/A
Chemical compound, drugMorphine sulfate (MS)Sigma-Aldrich1448005
Chemical compound, drugNaloxone hydrochloride dihydrateSigma-AldrichN7758
Chemical compound, drugPZM21EnamineN/Acustom synthesis
Chemical compound, drugmitragynine pseudoindoxyl (MP)This studyN/A(Váradi et al., 2016)
Chemical compound, drugCompound101 (Cmpd101)HelloBioHB2840
Chemical compound, drugPertussis toxinSigma-AldrichP7208
Commercial assay or kitAlexa Fluor 647 Protein Labeling KitThermo Fisher ScientificA20173
Software, algorithmPrismGraphPad8.1.1
Software, algorithmImageJImagej.net/contributors2.0.0-rc-54/1.51 g
Software, algorithmMATLABMathWorksR2014b
Software, algorithmPyMOLSchrödingerv1.7.4.5

Additional files

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