GRP Receptor Internalization Redistribution Assay

• Designed to assay compounds for their ability to modulate internalization of GRPR
• 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

The gastrin releasing peptide (GRP) is the mammalian counterpart of the amphibian peptide bombesin (BB). GRP regulates critical gastrointestinal functions via the GRP receptor (GRPR). Activation of GRPR also stimulates cell proliferation and acts as a growth factor in the pathogenesis of many types of cancer. In addition, GRPR regulates several aspects of neuroendocrine function. GRPR is a Gq coupled G-protein-coupled receptor (GPCR), and when activated by GRP, GRPR mediates downstream activation of the phospholipase C signaling pathway. Following activation by GRP, GRPR and ligand are rapidly internalized into early endosomes. GRP remains in the intracellular space whereas GRPR recycles to the plasma membrane.

Figure 1. Internalization of GRPR-EGFP stimulated with GRP. Cells were treated with 100 nM GRP for 30 min (right panel) or untreated (DMSO control, left panel). Arrows indicate the GRPR-EGFP internalization that is detected by the image analysis algorithm.

Figure 2. Concentration response curves in the GRPR assay: A) GRP and Bombesin concentration response in the GRPR agonist assay (n=8). The EC₅₀ values of GRP and bombesin are ~1 nM and ~0.5 nM respectively. Concentration response was measured in 9 point half log dilution series. Cells were treated with compound for 30 min. Cells were then fixed and receptor internalization was measured using the Cellomics ArrayScan V^TI Reader and the SpotDetectorV3 BioApplication. % activity was calculated relative to the positive (100 nM GRP) and negative control (0.25% DMSO). B) RC-3095 concentration response in the GRPR assay run in antagonist format (n=8). Cells were treated with 10 nM GRP in the presence of a half log dilution series of RC-3095 for 30 min. Cells were then fixed and analyzed on the Cellomics ArrayScan V^TI Reader. The EC₅₀ of RC-3095 is ~100 nM.