by Laura Lane
Richard Mailman isn’t exactly complaining when he explains how life became complicated in the 1980s. Rather, his voice brims with enthusiasm when he explains that targeting G protein coupled receptors (GPCR) goes far beyond simplistic binding assays. That’s what he learned while developing a novel compound to target the dopamine-binding D2 receptor.
“We stumbled on a compound that acted exactly like dopamine in one assay and, in another system, acted like a pure antagonist,” says Mailman, professor of pharmacology and psychiatry at the University of North Carolina, Chapel Hill. “It was clear evidence that GPCRs could signal through different signaling pathways.”
Now, two decades after those initial findings, the market is offering the types of products that allow life scientists to make similar inroads in understanding GPCRs, which represent the most abundant type of receptors on the cell surface that are involved in signal transduction. You may use new kits to probe functional selectivity, the phenomenon that Mailman observed and named years ago. Or, you could be interested in exploring other aspects of GPCR signal transduction. Whatever your goal, the current generation of technologies helps to maintain your cells in physiological environments and to improve your ability to focus on specific receptors.
“It’s widely recognized that this complicates drug discovery and makes the task more difficult,” he says. “It complicates life, but also offers tremendous opportunity, which the [pharmaceutical] industry needs to find the next generation of medications.”
Pathway puzzle
Traditionally, researchers have followed the paradigm that successful ligand binding could be assessed by measuring subsequent changes within the cell, usually the presence of cyclic AMP (cAMP), calcium and inositol triphosphate (IP3), and other second messengers that reflect the activation of G proteins. The great variety of assays—of all colors and creeds—that have proliferated over the years has helped researchers to make major inroads. However, the drying pipeline of new drugs proves the weakness of this relatively primitive strategy.
“People are realizing that GPCR signaling is very complicated and that no single assay is going to give all the information that you need, so you need to look at different readouts,” says Tom Wehrman, director of cell biology at DiscoveRx.
Arrestin development
In the last few years, researchers have been turning to a class of proteins known as beta-arrestins and its integral relationship with GPCRs. These proteins bind to an activated GPCR, causing it to detach from G protein, and targeting the receptor for endocytosis. Thus, beta-arrestins control the rate at which GPCRs are recycled and ready for additional binding. In addition, beta-arrestins couple with many major signaling pathways, such as MAP kinase, p38, and PI-3 kinase.
“Experts on the field will know that beta-arrestin biology as been around for several years, but only recently have companies put fabulous cell-based assays on the market,” says Ron Herzig, Invitrogen’s business area director for membrane targets.
These assays enable researchers to fine-tune their observation of GPCR activation. Instead of measuring the reaction of all the GPCRs within a cell, beta-arrestin assays are designed to focus the observation of the targeted GPCR.
“That’s functional selectivity,” Herzig says.
Time to tango
Invitrogen offers the Tango GPCR Assay System, which involves engineering a specific cleavage site into specific GPCRs along with a downstream transcription factor. The beta-arrestin is fused with a protease, which acts on the GPCRs’ cleavage site, freeing the transcription factor to return to the nucleus and promoting the expression of beta-lactamase. By adding a substrate to the live cell culture, the beta-lactamase creates a signal based on fluorescence resonance energy transfer.
Tango assay kits are available for a variety of GPCRs, including Gq-, Gs-, and Gi/o-coupled receptors, orphan GPCRs, hormone receptors, neurotransmitter receptors, peptide receptors, and chemokine receptors.
“Tango enables companies in drug discovery to look at a side of the coin they haven’t been able to look at before,” he says. “They may have a very important compound that’s in their library but they didn’t pull it out because they didn’t look at beta-arrestin.”
Path to discovery
DiscoveRx offers beta-arrestin assays based on its proprietary Enzyme Fragment Complementation (EFC). Essentially, the system’s two, inactive beta-galactosidase fragments—the enzyme acceptor (EA) fused to beta-arrestin and the small, 4 kDa enzyme donor (ProLink; PK) fused to the GPCR—come together to form active beta-galactosidase upon GPCR activation, which goes on to hydrolyze a substrate to produce a chemiluminescent signal. By reading chemiluminescence, you can avoid the noise of naturally fluorescent compounds and achieve a high signal-to-noise ratio.
The company uses EFC in its PathHunter Beta-Arrestin Assay Kits. You can purchase cell lines for 130 different known GPCRs and 83 different orphan GPCRs. These cell lines carry the beta-arrestin-EA fusion protein and a GPCR fused to the PK tag. Upon ligand binding, the GPCR is phosphorylated and beta-arrestin binds to the GPCR, bringing EA and PK together to reconstitute beta-galactosidase. Alternatively, you can purchase clones with just the beta-arrestin-EA fusion protein. These clones come with a vector containing the Prolink tag into which you can clone any GPCR.
“In [conventional] second messenger assays, if endogenous GPCRs are present in the cell, you’ll get a positive signal if any ligands hit any receptors,” Wehrman says. “Our system doesn’t give a signal unless the ligand has bound to the [specific] GPCR with the ProLink tag.”
Second generation, second messenger
Revelations of beta-arrestins doesn’t mean that second messengers are now second-class citizens. Technologies for improving the assays remain in full swing. Companies are working to increase detection sensitivity and the duration of the signal—all the while improving efficiency and ease.
Reaching these goals with assays for second messengers means assessing something less fleeting than calcium and IP3. For Cisbio, the solution is inositol-1-phosphate (IP1), a downstream metabolite of IP3. With a longer half-life than IP3, IP1 accumulates in the cell upon the activation of the Gq receptor. The addition of lithium chloride stabilizes IP1. For detection, add labeled monoclonal antibody to IP1 and d2-labeled IP1, which act as a donor-acceptor fluorescence pair.
The newest generation of this assay uses terbium cryptate as a label, which is a fluorescence donor that allows energy transfer much more efficiently than its predecessor, europium cryptate. The exogenous labeled IP1 competes with the endogenous IP1 to pair with the cryptate-conjugated antibody. The resulting signal is inversely proportional to the amount of endogenous IP1 created from Gq activation.
“IP1 comes as the ideal complement to cAMP to investigate the phospholipase C activation pathway,” says François Degorce, head of Cisbio’s HTRF marketing & business development. “The new assays provide functional information that directly correlates to events at the pathway level, unlike more indirect measurements such as calcium sensing or gene reporter techniques.”
Launched in April, terbium cryptate provides increased sensitivity that should help researchers who want to study GPCRs in their natural environment, Degorce says. These technological advances should enable researchers to study GPCR activation pathways not only through functional assays based measuring secondary messenger accumulation, but also to address GPCR structural mechanisms at the cell surface, such as receptor dimerization.
The electric touch
Roche and partner Acea Biosciences are preparing to launch a device that minimizes disruption of the cell’s natural environment. Called the xCelligence, the device allows the culturing of cells in 96-well plates that stay in the incubator. About 80 percent of the plate’s surface area is covered by a network of electrodes that measures changes in the electrical impedance. The measurement reflects cell morphology, the number of cells, and the cells’ ability to adhere to the plate.
“You can monitor cells from the point of seeding, through the initial challenge, through downstream events and experiments as long as you want,” says Jeff Emch, marketing manager for Roche Diagnostics.
With impedance’s correlation with morphology, xCelligence can monitor the cytoskeletal changes that result from ligand binding to endogenous levels of GPCRs. Different ligands will cause different morphological changes.
“There’s no need for additional reagents,” Emch says. “That’s one of the beauties of this system.”
