MOR1 Receptor Internalization Redistribution Assay

• Designed to assay compounds for their ability to modulate internalization of MOR1
• Coupled to EGFP for easy monitoring of the cellular translocation event
• Robust cell-based assay for use in high content analysis and fluorescence microscope applications

• Biologically relevant data: Compounds tested in a cellular environment
• Validated: Functionally tested cells provided with an optimized assay protocol
• Easy to use: Just plate cells, add compounds, and image

Opiate drugs such as morphine modulate their activity through opioid receptors and are well known for their ability to produce potent analgesia. However, the clinical use of the opioids is limited by serious side effects such as respiratory depression, constipation, development of tolerance, and physical dependence and addiction liabilities. Three classes of opioid receptors have been pharmacologically characterized; namely the mu (μ), delta (δ), and kappa (κ) opioid receptors, where morphine primarily exerts its effect through binding of mu (μ)-opioid receptors (MORs).

Opioid receptors are G protein-coupled receptors (GPCRs) that are activated both by endogenous opioid peptides and by clinically important alkaloid analgesic drugs such as morphine. Alkaloid- or peptide-mediated MOR activation results in receptor phosphorylation by G protein coupled receptor kinase (GRK) followed by recruitment of arrestin to the receptor. Interaction of arrestins with GPCRs results in an uncoupling of G-protein signaling from receptors (receptor desensitization) and recruitment of the endocytic machinery leading to receptor internalisation. Morphine does not induce endocytosis of the activated receptor, whereas agonists such as DAMGO, etorphine, methadone, and fentanyl mediate internalization of MOR. It is the inability of morphine to promote efficient receptor internalization that has led to suggestions that morphine is particularly prone to promoting MOR desensitization and tolerance.

Figure 2. Concentration response curves in the MOR1 assay: A) DAMGO concentration response in the MOR1 assay (n=12). The EC₅₀ is ~1.4 μM. Concentration response was measured in 9 point half log dilution series. Cells were treated with DAMGO for 30 min. Cells were then fixed and MOR1 internalization was measured using the Cellomics ArrayScan V^TI Reader and the SpotDetectorV3 BioApplication. % activity was calculated relative to the positive (10 μM DAMGO) and negative control (0.25% DMSO). B) Morphine concentration response in the MOR1 assay (n=8). Concentration response was measured in 9 point half log dilution series. Cells were treated with morphine for 30 min. Cells were then fixed and MOR1 internalization was measured using the IN Cell Analyzer 3000 (GE Healthcare). % activity was calculated relative to the positive (10 μM DAMGO) and negative control (0.25% DMSO).