MOR1 Receptor Activation Redistribution Assay

• Designed to assay compounds for their ability to inhibit a cAMP response through the MOR1 receptor
• 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 modulate their activity through opioid receptors and are known for their ability to produce potent analgesia. Three classes of opioid receptors have been pharmacologically characterized; the mu (μ), delta (δ), and kappa (κ) opioid receptors. The opiate morphine works primarily by binding to the mu (μ)-opioid receptors (MORs), which are G protein coupled receptors (GPCR) coupling to Gi. MOR activation results in receptor phosphorylation by GPCR kinase (GRK) followed by recruitment of arrestin to the receptor. When arrestins bind to GPCRs, the G-protein signaling is blocked and the endocytic machinery is recruited which leads to receptor internalization.

Morphine does not induce endocytosis of the activated MOR, whereas other agonists such as DAMGO do. In this assay, the MOR1 receptor is stably transfected into a GPCR Reporter Assay cell line for Gs/Gi-coupled receptors. This assay system measures translocation of protein kinase A (PKA) in response to changes in the cytoplasmic cAMP concentration. Binding of a MOR agonist to the receptor causes activation of the Gi complex at the intracellular face of the receptor. This leads to inhibition of adenylate cyclase, which catalyzes the formation of cAMP from ATP. In cells stimulated with forskolin, which induces high levels of cAMP, PKAcat-GFP is found in a diffuse localization in the cytoplasm. MOR1 activation leads to inhibition of adenylate cyclase and thereby less cAMP. This, in turn, leads to aggregation of PKAcat-GFP in cytoplasmic foci.

Figure 1. Cytoplasmic foci formation of PKAcat-EGFP. Cells expressing the MOR1 receptor were treated with 40 μM forskolin for 15 min in the presence (right panel) or absence (left panel) of 1 μM DAMGO. Activation of the receptor inhibits the forskolin-induced dispersion of PKAcat-EGFP aggregates. Arrows indicated the cytoplasmic foci detected by the image analysis algorithm.

Figure 2. DAMGO and morphine concentration response in the MOR1:PKA assay. Cells were treated with test compound in the presence of forskolin for 15 min. Cells were then fixed and cytoplasmic spot formation was measured using the Cellomics ArrayScan V^TI Reader and the SpotDetectorV3 BioApplication. % activity was calculated relative to the positive (1 μM DAMGO) and negative control (0.25% DMSO). The EC₅₀ of DAMGO is ~10 nM (n=16), while the EC50 of morphine is ~100 nM (n=8).