Andrew Huang, Patricia Yeung, Radhika Venkat, Helena Mancebo and Shengwen Zhang *
Multispan, Inc., 26219 Eden Landing Road, Hayward, CA 94545-3718
- Correspondence: email@example.com
G-protein coupled receptors (GPCRs) are among the hottest targets in drug discovery due to their broad involvement in many physiological functions and pathological conditions. We have developed stable mammalian cell lines expressing over 200 GPCRs that are suitable for high throughput cell-based assays including calcium mobilization, cAMP and IP1 assays. However, many GPCRs can couple to different G proteins and therefore activate separate downstream signaling pathways, and more and more compounds have been discovered to selectively activate a subset of these pathways for particular receptors. Screening compounds with one type of readout may generate a significant number of false negatives. In addition, for orphan GPCRs or those without known signal transduction mechanisms, these assays are not effective in identifying receptor activators. On the other hand, many GPCRs undergo ligand-dependent desensitization, a decrease in the responsiveness to ligand, accompanied by receptor aggregation on the cell surface and internalization via clathrin-coated pits to intracellular compartments. Based on this more universal trafficking property of GPCRs, we sought to develop a generalized cell-based drug screening method. Using proprietary recombinant cell technologies, we overexpressed a number of GPCRs with a FLAG tag at their N-terminus and examined their expression on the cell surface. Using live cell flow cytometry, we found that surface expression of the GPCRs decreased upon their agonist treatment in time- and dose-dependent manners, and their pharmacological profiles closely correlated to those obtained by second messenger measurements. Furthermore, the agonist-induced effects can be reversed by receptor-specific antagonists. Our results support that measuring GPCR internalization can be a universal high throughput cell-based approach in identifying drugs that act through the receptors without bias toward any signaling pathway or the knowledge of cellular mechanisms of orphan receptors.
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