Functional Cell Profiling of Endogenous GPCRs using the xCELLigence System
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.
Since 1925, ATCC has operated as an independent, nonprofit entity that has helped life science organizations and academic institutions produce innovations that have benefited all of us: life saving vaccines; nutritional improvements; cancer research breakthroughs; and safer, more effective medications. With distribution to more than 140 countries and a working relationship with 12 distribution partners, ATCC has the experience, knowledge, rigorous methodologies, standards, longevity and the global reach to serve academic institutions, government agencies, biotech, biopharma, and research organizations around the world.
For more information, visit www.atcc.org.
Roche Diagnostics Deutschland GmbH
Headquartered in Basel, Switzerland, Roche is a leader in researchfocused healthcare with combined strengths in pharmaceuticals and diagnostics. Roche is the world's largest biotech company with truly differentiated medicines in oncology, virology, inflammation, metabolism and CNS. Roche is also the world leader in invitro diagnostics, tissuebased cancer diagnostics and a pioneer in diabetes management. Roche's personalised healthcare strategy aims at providing medicines and diagnostic tools that enable tangible improvements in the health, quality of life and survival of patients. In 2009, Roche had over 80'000 employees worldwide and invested almost 10 billion Swiss francs in R&D. The Group posted sales of 49.1 billion Swiss francs. Genentech, United States, is a wholly owned member of the Roche Group. Roche has a majority stake in Chugai Pharmaceutical, Japan. For more information: www.roche.com.
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