Functional Cell Profiling of Endogenous GPCRs using the xCELLigence System

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Gcoupled protein receptors (GPCRs), also known as 7transmembrane proteins, constitute the single largest class of therapeutic targets for clinical and investigational drugs. There are ~800 predicted members of this class in the human genome, involved in diverse signaling pathways in a wide array of cells and tissue types. Modulation of GPCR function has proven to have therapeutic benefit in a wide variety of diseases in immunology, neurology, cardiology, and oncology.

Roche Applied Science (SIX: RO, ROG; OTCQX: RHHBY) partnered with ATCC to research realtime endogenous GPCR function using the xCELLigence System from Roche and cells from ATCC. Cells are seeded onto plates containing microelectrodes, allowing precise measurement of subtle changes in cytoskeletal structure and cellular contraction induced by GPCR activation without using exogenous labels. GPCRs transmit extracellular signals by binding coupled guanine nucleotidebinding proteins, or G proteins, in the cytoplasm. The major second messenger pathways coupled to G proteins include cyclic AMP/protein kinase A, calcium/phospholipase C, betaarrestin/MAPK and the Rho family GTPases, all of which can result in morphological changes easily detected by cellular impedance recording.

In contrast to traditional assays that use engineered cell lines, morphological impedancebased measurements can capture the aggregate effect of multiple signaling pathways. The advantages of assaying endogenous GPCR function include assessing the target receptor at its normal expression level; analyzing the natural interaction of receptors with regulatory partners including homo- or heterodimers; and permitting the native coupling to intracellular G proteins. Use of labelfree assay systems also significantly reduces reagent costs, because a single assay can measure all the second messenger pathways a given GPCR activates. Impedancebased realtime kinetic recordings can thus detect all the GPCR responses during the course of the experiment.

In a recent study (1), the xCELLigence System proved to be a sensitive and robust assay for continually measuring endogenous GPCR function. Control GPCR agonists produced large morphological responses with high sensitivity (by IC50 value determination) and excellent robustness (by Z factor determination). A panel of 43 ligands encompassing 24 therapeutically relevant receptor families was examined. Functional GPCR profiles were created for the frequently used and therapeutically relevant cell lines, HeLa, U-2 OS, SH-SY5Y and CHO-K1 (ATCC CCL-2, HTB-96, CRL-2266 and CCL-61), as well as two primary cell types, human vascular endothelial cells (ATCC PCS-100-010) and mixed renal primary epithelial cells (ATC C PCS-400-012).

It was shown that the function of a wide variety of GPCRs can be assayed using the xCELLigence System in tumor cell lines and primary cells. Most of the receptor target GPCR families tested produced robust responses greater than three standard deviations above the mean relative to cells in control wells, in one or more of the cell lines used in the present study, without any additional receptorspecific assay optimization. Since some of the ligands tested most likely activate multiple members of the same GPCR family, additional experiments using selective agonists and antagonists combined with gene expression profiling and siRNA knockdown of individual receptors should allow for identification of the specific receptor subtypes responsible for the morphological changes detected using the xCELLigence System.

1. Focus Application Note 7, Roche Applied Science, 2010.

2. Kenakin TP. (2009) Nat Rev Drug Discov. 8:617-26.

3. Yu N, Atienza JM, Bernard J, Blanc S, Zhu J, Wang X, Xu X, Abassi YA (2006). Anal Chem. 78:35-43.

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