news
RESEARCH PROFILES Real-time assay for GPCRs Currently, ∼50% of the drugs in the market are targeted against some type of G-protein-coupled receptor (GPCR). Most assays for GPCRs require labeling cells with dyes, which can alter the natural environment of the cells, and engineering cells recombinantly so that they express promiscuous G proteins. As a result, traditional assays may not give a realistic assessment of GPCR function and pharmacology. To overcome these limitations, Yama Abassi and colleagues at ACEA Biosciences and Euroscreen (Belgium) have developed a new assay for GPCRs that does not require labeling or engineering cells. As described in this issue of Analytical Chemistry (pp 35– 43), the new assay involves specially designed microwell plates with microelectrodes embedded in the bottom of the wells. Cells are placed in the wells, and the impedance is measured. The impedance measurement correlates with cell number, cell morphology, and quality of cell adhesion. When a particular agonist or ligand is added, the GPCR is activated and the morphology of the cell changes. This change in morphology alters the impedance in such a way that even minute changes in cell morphology can be detected. Readouts can be obtained in as little as 50 ms/well; this allows for the precise measurement of the rate of change, magnitude, and duration of the response. To demonstrate the assay, Abassi and colleagues seeded the electronic microwells with either CHO-K1 cells expressing the human H1 histamine receptor or 1321-N1 cells expressing the human vasopressin receptor. They then stimulated the cells with histamine and vasopressin, respectively. The addition of histamine and vasopressin induced an increase in impedance within 5 min. Although the researchers used previously identified receptors and agonists to validate the system, they have an ongoing project to screen for orphan receptors, which have no known ligands. The pharmaceutical industry is interest12
Add cell
Baseline impedance
Add agonist
Increased impedance by cell
Further increase in impedance is proportional to the extent of morphological change induced by the agonist
Cell–electrode impedance is used to noninvasively measure GPCR-mediated changes in cell morphology.
ed in “deorphanizing” these receptors because they are potential drug targets, says Abassi. The new assay has several advantages over traditional assays for GPCRs. The primary advantage is that it does not require labeling. Another advantage is that “you don’t necessarily need to engineer the cells. You can use endogenous proteins—either GPCRs or G-proteins that are expressed naturally in the cell itself,” says Abassi. In addition, the assay can be used to assess the function of a particular GPCR, regardless of the signaling pathway the GPCR is linked to. Other assays use different technologies, depending on the signaling pathway. For example, “if you are interested in a G-protein coupled receptor, then you would use an appropriate readout for that GPCR. It could be cAMP, calcium, or IP3,” says Abassi. Another advantage of the new assay is that it is noninvasive, which allows you to stimulate the GPCR as many times as you want, says Abassi. “That is really important with regards to GPCRs because they undergo what is called ‘desensitization’,” he adds. Desensitization occurs when GPCRs are modified (e.g., phosphorylated) so that they no longer recognize the agonist or when GPCRs are internalized into the cell itself. “It takes a while for the cell to recycle these re-
A N A LY T I C A L C H E M I S T R Y / J A N U A R Y 1 , 2 0 0 6
ceptors back to the surface,” says Abassi. Several researchers are interested in how long the desensitization response lasts. Because the new assay is noninvasive, measurements can be taken over time. “After an initial stimulation, you can come in after 10, 20, or 30 h and restimulate to see if the signaling has been restored,” says Abassi. Such information is important if you are designing a new drug, he adds. The new assay can also be used to examine what is referred to as “cross talk” between two different signaling pathways. Several GPCRs are linked to other types of receptors. “If you activate one particular G-protein-coupled receptor, that receptor can then activate another receptor or another signaling pathway not directly linked to the receptor,” says Abassi. It is important to know whether activating one GPCR will also activate another signaling pathway for a different GPCR, he says. Real-time electronic cell sensing can be used for more than just studying the function of GPCRs, says Abassi. For example, it can be used to look at cell proliferation or to determine the effects of cytotoxic compounds on cells, he adds. “It’s really nice because it gives you real-time information about what is going on [inside the cell].” a —Britt Erickson
