Cell-based Assays for GPCR Activity

G-protein-coupled receptors (GPCRs), also known as seven-transmembrane receptors, are important signal transducers in many biological processes. These processes include cell-cell communications, response to hormones, immune responses, nerve cell transmission and more.

Given the significance of these processes in health and disease, it should come as no surprise that GPCRs are critical drug targets, too, the activity of which can be measured by commercially available, cell-based assays. Here, we review some of your options. 

Measuring cAMP

GPCRs act through so-called trimeric G proteins, three-component protein complexes called Gα, Gi, Gs and Gq. Each triggers a different intracellular response, leading to changes in the abundance of such second messengers as cyclic adenosine monophosphate (cAMP), phosphatidylinositol and calcium.

Promega’s GloSensor™ assay measures the cAMP response by quantifying the light output of a firefly-luciferase biosensor that is responsive to changes in cAMP levels inside a living cell.

GloSensor is used in both academic settings and large pharmaceutical companies for screening for allosteric modulators of GPCRs. To work with this system, users must transfect HEK293 cells with GloSensor’s firefly luciferase by stable or transient transfection. But because the assay does not require destruction of the cells by lysis, it can be run inside living cells. Bioluminescence is measured kinetically or by endpoint using an automated bioluminescence plate reader.

“In terms of pros, you are going to [be] hard pressed to find a better combination of sensitivity and dynamic range out there,” says Brock Binkowski, senior research scientist II at Promega.

Another approach to measuring cAMP levels as a function of GPCR activity is the ACTOne technology from CodexBioSolutions. Providing real-time changes in intracellular cAMP in a high-throughput format without a cell-lysis step, ACTOne works with cell lines that contain an exogenous Cyclic Nucleotide-Gated (CNG) channel.

“The channel is activated by elevated intracellular levels of cAMP, resulting in ion flux (often detectable by calcium-responsive dyes) and cell-membrane depolarization, which can be detected with a fluorescent ACTOne membrane potential (MP) dye,” says Codex president Jimmy Lu.

Both endpoint and kinetic measurements of Gs- and Gi-coupled GPCR activity are possible with ACTOne, which differs from other technologies in that it does not require receptor modification or engineering. The assay can be read on a fluorescence plate reader, but it is also amenable to high-content screening and single-cell imaging applications.

Intracellular calcium levels

If your GPCR of interest signals by changing intracellular calcium levels, you need an assay that uses sensitive calcium-indicator dyes, which exhibit large fluorescence-intensity increases upon binding to calcium.

Two such assays are the FLIPR® Calcium 6 Kit and Fura-2 QBT™ Calcium Kit from Molecular Devices, both of which rely on the same principle. Drug- or ligand-treated cells exhibit changes in intracellular-calcium levels, which are detected by a calcium-sensitive fluorescent dye that enters cells. A second dye, called a masking dye, remains in the medium to quench autofluorescence.

“The masking dye, in the FLIPR Calcium 6 Kit and the Fura-2 QBT Calcium Kits, does not enter the cell but significantly reduces background originating from residual, extracellular fluorescence of calcium indicator, media and other components,” says Debra Gallant, product manager for reagents at Molecular Devices.

Also enabling measurement of calcium as a second messenger is the ChemiScreen™ product line from EMD Millipore, which funnels various GPCR-based signaling events into a calcium-inducing pathway to enable fluorescent detection of receptor activity.

“A feature that is found in our ChemiScreen stable GPCR cell lines is the endogenous expression of G15 to allow the majority of the receptors to signal through the IP3-calcium pathway for simple calcium flux assays using fluorescent dyes,” says Blaine Armbruster, senior product manager of discovery and development solutions at EMD Millipore.

According to Armbruster, EMD Millipore focuses on “universal signaling through the calcium pathway” and now sports “the largest collection of such assays.” He adds, “The focus on the universal signaling was to allow researchers to have a single assay for any receptor that they were studying.”

(EMD Millipore has also launched a second line of GPCR assays, called ChemiBrite™, for luminescent detection of calcium events.)

Measuring an unknown

When you have no prior knowledge about which second-messenger system a particular GPCR signals through, you can try Life Technologies’ Tango® line of stable cell lines.

The Tango cell lines enable beta-arrestin recruitment assays. Beta arrestin is linked to a protease, and the GPCR is linked to a reporter by a tether that contains a protease-cleavage site. When arrestin is recruited, it clips off the reporter gene, activating it.

“Beta-arrestin recruitment is an almost universal phenomenon for GPCRs. So [Tango] allows you to look at signaling through a GPCR without knowing which messenger is activated,” explains Bonnie Hammer, a senior staff scientist at Life Technologies.

The system, she adds, ignores signaling from endogenous receptors, enabling researchers to focus on the GPCR of interest.

There are other options, of course. Some assays measure GPCR-induced changes in inositol phosphates – Cisbio’s IP-One HTRF® assay, for instance. There also are service providers available for labs that lack the time or resources to do GPCR screening in-house (such as EMD Millipore’s FlexLab™ GPCR Service and Life Technologies’ SelectScreen® Cell-based GPCR Profiling Services).

The point is, if you’re interested in GPCR-based drug development, you should have no problem finding a strategy to meet your needs.

Image: Schematic overview of Life Technologies Tango Assay system for GPCRs.