Novel screening assay for the selective detection of G-protein-coupled receptor heteromer signaling.

Drugs targeting G-protein-coupled receptors (GPCRs) make up more than 25% of all prescribed medicines. The ability of GPCRs to form heteromers with unique signaling properties suggests an entirely new and unexplored pool of drug targets. However, current in vitro assays are ill equipped to detect heteromer-selective compounds. We have successfully adapted an approach, using fusion proteins of GPCRs and chimeric G proteins, to create an in vitro screening assay (in human embryonic kidney cells) in which only activated heteromers are detectable. Here we show that this assay can demonstrate heteromer-selective G-protein bias as well as measure transinhibition. Using this assay, we reveal that the δ-opioid receptor agonist ADL5859, which is currently in clinical trials, has a 10-fold higher potency against δ-opioid receptor homomers than δ/µ-opioid receptor heteromers (pEC(50) = 6.7 ± 0.1 versus 5.8 ± 0.2). The assay enables the screening of large compound libraries to identify heteromer-selective compounds that could then be used in vivo to determine the functional role of heteromers and develop potential therapeutic agents.

A bioluminescent-based, HTS-compatible assay to monitor G-protein-coupled receptor modulation of cellular cyclic AMP.

We have developed a novel assay for monitoring changes in intracellular cyclic AMP (cAMP) concentration with high sensitivity (30 +/- 5 fmol [mean +/- standard error of the mean] of cAMP per well) and reproducibility (Z' of > 0.8). The assay is of format amenable to high throughput screening (HTS) in 96-, 384-, and 1,536-well plates, and as a bioluminescent assay is potentially less prone to interferences originating from fluorescent compounds. Because of its high sensitivity, fewer numbers of cells (1,000 cells per well) in low-volume 384-well plates are required to screen for changes in cAMP concentrations. The assay does not rely on the use of antibodies, and thus it does not suffer from changes in the affinity or quality of the antibodies. The assay is based on the fact that cAMP is a potent activator of cAMP-dependent protein kinase (PKA), and activation of PKA can be monitored by measuring ATP utilization in a kinase reaction. The amount of ATP consumed can be measured using a luciferase/luciferin luminescent reaction. Since the amount of relative luminescence units (RLU) generated is a measure of the remaining ATP, a reciprocal relationship between RLU and both the activity of PKA and the intracellular concentration of cAMP is observed. Thus, the functional activity of agents that modulate the activity of Galpha(s) or Galpha(i) forms of G-protein-coupled receptors (GPCRs), which cause change in intracellular cAMP, can be monitored by the change in the activity of PKA and the amount of RLU readout. The assay can be performed in two steps and requires only 30 min after cell lysis for completion. The assay has been successfully used to generate 50% effective concentration (EC(50)) values for forskolin, a known direct activator of cellular adenylate cyclases, and EC(50) values for agonists and 50% inhibitory concentration values for antagonists modulating GPCRs that alter adenylate cyclase activity (Galpha(s) and Galpha(i)). Finally, adherent, suspension, and frozen cells have been successfully used in this assay, thus offering flexibility and convenience for many HTS applications.

A 1536-well cAMP assay for Gs- and Gi-coupled receptors using enzyme fragmentation complementation.

Guanosine triphosphate binding protein (G protein)-coupled receptors (GPCRs) are a large class of pharmaceutical drug targets. With the increasing popularity of functional assays for high throughput screening, there arises an increasing need for robust second messenger assays that reflect GPCR activation and are readily amenable for miniaturization. GPCRs that upon agonist stimulation modulate adenylyl cyclase activity, and, consequently, cellular cyclic adenosine monophosphate (cAMP) levels, via the G protein Gs or Gi, form a subset of therapeutic targets. While there are several cAMP assays currently available, most are not scalable for miniaturization into the 1536-well format employed for automated high throughput screening of large chemical libraries. Here, we describe a cAMP assay based on the enzyme fragmentation complementation (EFC) of beta-galactosidase. In this assay, recombinant cells expressing Gs- or Gi-coupled receptors exhibit robust and reproducible pharmacology for agonists and antagonists, as measured by cAMP levels. Furthermore, the EFC cAMP assay offers sufficient sensitivity to be used with cells expressing endogenous GPCRs. We demonstrate the miniaturization of this assay into a 1536-well format with comparable sensitivity and plate statistics to those of the 384-well assay for both Gs- and Gi-coupled receptors, and its suitability for miniaturized high throughput screening.

The beta2-adrenergic receptor specifically sequesters Gs but signals through both Gs and Gi/o in rat sympathetic neurons.

Beta(2)-adrenergic receptors (beta(2)-AR) and CB1 cannabinoid receptors share the property of being constitutively active. The CB1 cannabinoid receptor can also sequester G(i/o) proteins; however, it is not known whether the beta(2)-AR can also sequester G proteins. Beta(2)-ARs were heterologously expressed in rat superior cervical ganglion neurons by microinjection of cDNA and studied using the patch-clamp technique. The beta-AR agonist isoproterenol increased the Ca(2+) current 25.9+/-1.6% in neurons microinjected with 100 ng/microl beta(2)-AR cDNA but was without effect on control neurons. Pretreatment with cholera toxin (CTX) abolished the effect of isoproterenol, indicating coupling via G(s) proteins. In neurons microinjected with 200 ng/microl beta(2)-AR cDNA, isoproterenol had the opposite effect of inhibiting the Ca(2+) current 36.5+/-2.0%. Inhibition of the Ca(2+) current was sensitive to pertussis toxin, indicating beta(2)-AR coupling to G(i/o) proteins. Pretreatment with CTX resulted in a greater 54+/-3.8% inhibition of the Ca(2+) current, indicating that G(s) coupling masks the full effect of G(i/o) coupling. Expression of beta(2)-ARs abolished signaling by G(s)-coupled receptors for vasoactive intestinal polypeptide (VIP). VIP inhibited the Ca(2+) current 49.5+/-0.5% in control neurons but had no effect in neurons expressing beta(2)-ARs. In contrast, expression of beta(2)-ARs had no effect on signaling by the G(i/o)-coupled alpha(2)-adrenergic receptor. This study demonstrates that the beta(2)-AR couples to both G(s) and G(i/o) proteins but specifically sequesters G(s) proteins, preventing their interaction with another G(s)-coupled receptor. beta(2)-adrenergic receptors thus have the potential to prevent other G(s)-coupled receptors from transducing their biological signals.