Journal: Nature chemical biology

Article Title: Profiling the proximal proteome of the activated mu opioid receptor

doi: 10.1038/s41589-024-01588-3

Figure Lengend Snippet: a. Receptor signaling was measured using a commercial cAMP biosensor (pGloSensor-20F). cAMP accumulation was measured after ~10 minutes of DAMGO/isoproterenol incubation and normalized to isoproterenol alone. Data from three independent experiments are presented as mean ± SEM. b. Comparison of agonist-stimulated receptor internalization as assayed by loss of cell surface immunoreactivity and measured by flow cytometry comparing untreated (control) and treated samples (10 μM DAMGO, 30 min). Data from four independent experiments are presented as mean ± SEM. c. Comparison of cell surface recovery of receptors (‘recycling’) following 30 min of DAMGO application (10 μM), agonist removal, and a 30 min recovery period in the presence of antagonist (10 μM). Data from four independent experiments are presented as mean ± SEM. d. The heatmap shows all significantly enriched gene ontology terms (adjusted p-value < 0.05) among the proteins that significantly change in the proximal protein environment of the μOR upon activation with DAMGO, morphine, or PZM21 including the number of proteins that match the gene ontology terms. Cluster 1–4 refer to the clustering of the heatmap in Figure 1b. e. Colocalization of μOR with endosomes to monitor receptor trafficking following activation. HEK293 cells stable expressing the μOR with an N-terminal Flag-tag were activated with 10μM DAMGO, morphine, or PZM21 for 10min. The receptor was imaged using anti-Flag. Endosomes were marked with anti-EEA1. n = 3 independent biological replicates, representative example shown, Scale bar is 10μm.

Article Snippet: 10 μM DAMGO ([D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin acetate salt, Sigma-Aldrich), morphine (Morphine sulfate, Sigma-Aldrich), or PZM21 (synthesized by Enamine at 98% purity as tested by NMR and LC-MS) was added for the noted period of time.

Techniques: Incubation, Comparison, Flow Cytometry, Control, Activation Assay, Expressing, FLAG-tag

Journal: Nature chemical biology

Article Title: Profiling the proximal proteome of the activated mu opioid receptor

doi: 10.1038/s41589-024-01588-3

Figure Lengend Snippet: a. Experimental design of the study. μOR-APEX constructs were stably expressed in HEK cells. The receptor was activated with 10μM DAMGO, morphine and PZM21 over a time course of 60 min. Cells were pretreated with biotinphenol for 30 min and biotinylation was initiated by the addition of H2O2 for 30 sec at indicated time points after agonist treatment. Following cell lysis, biotinylated proteins were enriched using streptavidin and subsequently quantified using global and targeted mass spectrometry approaches. b. Global agonist-dependent changes in proximal proteome of the μOR. Heatmap visualizing all proteins with a significant (p < 0.01, log2 fold change > 1) change in biotin labeling for at least one of the ligands across the time course. The heatmap was clustered according to the significance score, calculated as a combination of the −log10 p-value and log2 fold change. Gene ontology enrichment analysis was performed for individual clusters and significant gene ontology terms for the individual clusters as well as the matching genes are indicated in dark purple. Data from three biological replicates are presented as mean. c. Targeted proteomics analysis of agonist-dependent biotinylation by μOR-APEX2 of selected localization markers for plasma membrane (top), early endosome (middle), and late endosome/lysosome (bottom). Data from three biological replicates for DAMGO (red), morphine (yellow), and PZM21 (green) are presented.

Article Snippet: 10 μM DAMGO ([D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin acetate salt, Sigma-Aldrich), morphine (Morphine sulfate, Sigma-Aldrich), or PZM21 (synthesized by Enamine at 98% purity as tested by NMR and LC-MS) was added for the noted period of time.

Techniques: Construct, Stable Transfection, Lysis, Mass Spectrometry, Labeling, Membrane

Journal: Nature chemical biology

Article Title: Profiling the proximal proteome of the activated mu opioid receptor

doi: 10.1038/s41589-024-01588-3

Figure Lengend Snippet: a. Ligand-dependent proximal protein interaction networks of μOR. All proteins are visualized in the heatmap that showed a significant difference in biotin labeling (log2FC > 1 and p-value < 0.001) before and after location-specific detrending for at least one of the ligands across the time course. The heatmap was clustered according to the significance score calculated as a combination of the −log10 p-value and log2 fold change. Data from three biological replicates are presented as mean. b. Gene ontology enrichment analysis for proteins of the ligand-dependent μOR proximal interaction networks. The heatmap shows all significantly enriched gene ontology terms (adjusted p-value < 0.05) among the proteins present in the proximal interaction network of the μOR including the number of proteins that match the gene ontology terms. c. Temporal profile for ARRB2. Line charts represent the log2 fold change over the time course of receptor activation with DAMGO (red), morphine (yellow) and PZM21 (green) after data detrending. Data from three biological replicates are presented as mean ± SEM. d. Correlation between the maximum location coefficient calculated for the Endosome (Endo) and the maximum ARRB2 log2FC over the time course of μOR activation with DAMGO (red), morphine (yellow) and PZM21 (green).

Article Snippet: 10 μM DAMGO ([D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin acetate salt, Sigma-Aldrich), morphine (Morphine sulfate, Sigma-Aldrich), or PZM21 (synthesized by Enamine at 98% purity as tested by NMR and LC-MS) was added for the noted period of time.

Techniques: Labeling, Activation Assay

Journal: Nature chemical biology

Article Title: Profiling the proximal proteome of the activated mu opioid receptor

doi: 10.1038/s41589-024-01588-3

Figure Lengend Snippet: a. Temporal profile for KCTD12 and EYA4. Line charts represent the log2 fold change over the time course of receptor activation with DAMGO (red), morphine (yellow) and PZM21 (green) after data detrending. Data from three biological replicates are presented. b. Schematic of APEX-based proximity labeling of the plasma membrane spatial reference construct (PM-APEX) was performed in a cell line expressing the μOR in the presence and absence of the Gɑi inhibitor Pertussis toxin (PTX) and after activation of μOR using DAMGO. c. Line charts depict protein intensity for KCTD12 and EYA4 in PM-APEX experiment determined by quantitative MS over the DAMGO time course in the presence (grey) and absence of PTX (red). Data from four biological replicates are presented. d. Interaction network of high-confidence interactors of EYA4 determined by affinity purification. Split nodes indicate protein abundance differences for the high-confidence interactors upon introduction of SIX-binding mutant (A633R) and phosphatase-dead mutant (D375N). Data from three biological replicates are presented as mean. e. APEX-based proximity labeling of EYA4 was performed in a cell line expressing the μOR in the presence and absence of the Gɑi inhibitor Pertussis toxin (PTX) and after activation of the μOR using DAMGO. f. Gene set enrichment analysis (GSEA) in log2FC depicted in Extended Data Fig. 8 using the location-specific sets of proteins defined in Human Cell Map.

Article Snippet: 10 μM DAMGO ([D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin acetate salt, Sigma-Aldrich), morphine (Morphine sulfate, Sigma-Aldrich), or PZM21 (synthesized by Enamine at 98% purity as tested by NMR and LC-MS) was added for the noted period of time.

Techniques: Activation Assay, Labeling, Membrane, Construct, Expressing, Affinity Purification, Binding Assay, Mutagenesis