Roche describes the functional cell profiling of endogenous G-coupled protein receptors (GPCRs) using the Xcelligence System.
Modulation of GPCR function has proven to have therapeutic benefit in a variety of diseases in immunology, neurology, cardiology and oncology.
GPCRs, also known as seven-transmembrane proteins, constitute the single largest class of therapeutic targets for clinical and investigational drugs.
There are around 800 predicted members of this class in the human genome, involved in diverse signalling pathways in a range of cells and tissue types.
Roche Applied Science partnered with ATCC to research real-time 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 nucleotide-binding 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, beta-arrestin/MAPK and Rho 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 impedance-based measurements can capture the aggregate effect of multiple signalling pathways.
The advantages of assaying endogenous GPCR function include assessing the target receptor at its normal expression level; analysing the natural interaction of receptors with regulatory partners including homo- or heterodimers; and permitting the native coupling to intracellular G proteins.
Use of label-free assay systems also significantly reduces reagent costs, because a single assay can measure all the second messenger pathways a given GPCR activates.
Impedance-based real-time kinetic recordings can thus detect all the GPCR responses during the course of the experiment.
In a recent study, 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 variety of GPCRs can be assayed using the Xcelligence System in tumour cell lines and primary cells.
Most of the receptor target GPCR ranges 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 receptor-specific assay optimisation.
Since some of the ligands tested most likely activate multiple members of the same GPCR range, 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.
