Tools For Studying G-Protein-Coupled Receptors: It’s All About Detection

As the largest protein family in the human genome, perhaps it’s not surprising that GPCRs are involved in a myriad of vital functions in our physiology. As hot targets in drug discovery, they’ve already been identified as key players in heart disease, cancer, diabetes, inflammation, pain, and central nervous system disorders. And more is to come: there are more than one hundred so-called orphan receptors—GPCRs whose ligands and functions remain undiscovered—for researchers to investigate.

But as transmembrane proteins, GPCRs are not always easy to work with. When a ligand binds the extracellular portion, the protein transduces a signal to the intracellular portion, which initiates a signal within the cytoplasm. This is typically achieved by activating a second messenger cascade, such as causing a rise in intracellular calcium, or releasing another cytoplasmic messenger. Therefore most tools used to study GPCRs involve detecting a change in intracellular signaling cascades following receptor activation. And the toolbox for GPCR researchers is quickly becoming more refined, with the progress driven in part by the search for new GPCR drug targets.

Sometimes the devil is in the details. For example, Long believes that “cAMP assays are best employed to study Gs-coupled GPCR targets and also many Gi-coupled receptors, although the latter target class can prove challenging when looking for antagonists of a Gi, as signal windows and Z values can be small.” In addition, he points out that no matter how glitzy your assay system, mechanical problems can bring you down. “Often one of the biggest hurdles in successfully optimizing a cAMP assay, using cells for high throughput screening applications, is the automated liquid handling component,” he says. “Reliable and accurate dispensing of viscous cellular suspensions, or addition of compounds and reagents to adherent cells, can prove challenging to ensure cell viability is maintained whilst achieving adequate mixing of reagents.”

PerkinElmer’s recent acquisition of EuroScreen allows it to offer the latter’s GPCR screening technology in its GPCR portfolio. EuroScreen developed AequoScreen, suitable for high throughput screening of agonist, antagonist, and allosteric modulators of GPCRs, based on the calcium indicator aequorin. Isolated from luminescent jellyfish, the photoprotein aequorin emits blue light (469 nm) upon binding calcium ions. This luminescent calcium signal has no autofluorescence, giving a high signal to noise ratio, and measures calcium concentration over a wide dynamic range with little buffering effect.

BioVision offers a colorimetric cAMP assay kit, whose advantage is that the equipment needed for it is present in almost every lab, unlike assays using luminescence or fluorescence. This extremely sensitive assay can detect cAMP over the range of 1 – 100 fmol. The assay takes about three hours and uses 96-well plates. Gloria Zheng, product manager of BioVision, says that the sensitivity and low background noise inherent in their assay stems from their unique protein-G-coated plate system: “The protein G coated plate efficiently absorbs the cAMP polyclonal antibody to the plate. cAMP-HRP conjugate directly competes with cAMP from the sample for bindin

Bypassing the measurement of second messengers altogether, DiscoveRx offers its cell-based PathHunter platform, which can measure the activation of Gi, Gs, and Gq coupled receptors directly. PathHunter is based on DiscoveRx's proprietary technology called enzyme fragment complementation (EFC), which they used to develop biochemical high-throughput screening assays for GPCR, kinases, and proteases. DiscoveRx converted this into a cell-based format called PathHunter, for which they received the 2006 Frost and Sullivan Award for Technology Innovation. DiscoveRx claims that it’s the first chemiluminescence assay technology that can measure protein trafficking directly inside the cell; it’s meant to serve as an important link between in vitro biochemical assays and more complex imaging methods.

An invaluable tool in GPCR research, DiscoveRx’s beta-arrestin assays detect the chemiluminescent signal resulting from the proximity of two beta-galactosidase fragments during an interaction between a ligand-activated GPCR and recruited beta-arrestin. DiscoveRx just released a 30 second version of this assay for beta-arrestin-based screening called the PathHunter Flash Detection Kit. The speed of this detection, according to DiscoveRx, permits the screening of one million compounds in 48 hours. Another way to speed up your screening is to run PathHunter beta-arrestin and intracellular calcium assays simultaneously, in the same wells, provided your instrumentation has real-time fluorescence and flash chemiluminescence capabilities.

Invitrogen also offers GPCR cell lines equipped with their GeneBLAzer beta-lactamase reporter technology, which uses fluorescence resonance energy transfer (FRET)-based radiometric detection to increase the signal to noise ratio. Invitrogen offers their GeneBLAzer Validated GPCR Assays, or CellSensor Cell Lines and Reporter Vectors, to get you up and running quickly. These tools are particularly well suited to cell-based target validation in GPCR drug discovery. The GeneBLAzer tools are available in 96, 384, 1536 & 3456-well plate formats. PerkinElmer also offers a range of stably transfected GPCR cell lines. All cell lines are tested for receptor expression levels, and some are also tested for whole-cell functional assays including cAMP or inositol triphosphate levels. As with the GPCR cell lines from other vendors, these cells can be used in binding and functional whole-cell studies, regulation of receptors and signaling, secondary screening assays, assay development, and large-scale production of recombinant GPCR membrane preparations.

Hopefully, the rate of advances in GPCR tools is helping you to find a new avenue of GPCR investigation.