Functional Assays

Our functional GPCR assays measure second messenger levels, as a result of GPCR activation or inhibition, to characterize receptor pharmacology. We offer fluorescent cAMP, calcium flux and reporter gene assays. Our assays are conducted with primary cell lines and with immortalized/transfected cell lines. We use live cells and, in some cases, membranes. These can either be sent to us, or we can culture and transfect cells as an ancillary service. We also offer ELISAs as an additional service to assess cell surface expression of receptors.

We use this technique to:

• Determine EC₅₀ and E_max of agonists;
• Determine IC₅₀ of antagonists;
• Screen compounds to identify full agonists, partial agonists, inverse agonists, antagonists and allosteric modulators.

cAMP assays are used to measure activation of GPCRs coupled to G_s and G_i/o. Activation of GPCRs coupled to G_s results in adenylyl cyclase stimulation and increased production of the second messenger cyclic adenosine monophosphate (cAMP). Conversely, activation of GPCRs coupled to G_i/o results in a reduction of cAMP production.  cAMP levels are measured using a TR-FRET based assay.

Calcium flux assays measure activation or inhibition of GPCR signalling by monitoring intracellular calcium levels, and is useful for investigating GPCRs coupled to G_q/11. Cells are loaded with a fluorescent dye such as Fluo-8, then stimulated with an agonist, leading to increased intracellular calcium levels. Upon binding to calcium, the fluorescence of the dye is enhanced.

To measure agonist EC_50, cells expressing the target receptor are stimulated with increasing concentrations of the test compound, and second messenger production is determined relative to the maximum stimulated by control compounds, e.g. forskolin or ionomycin, depending on the assay type.

To measure antagonist IC_50, cells expressing the target receptor are stimulated with a fixed concentration of agonist in addition to increasing concentrations of antagonist resulting in a decrease of agonist-stimulated second messenger production.

Example Data: cAMP Agonist and Antagonist Assay

Stimulation of cAMP production by NECA in CHO K1 cells transiently transfected to express human adenosine 2a receptors, EC₅₀ = 7.78 nM (left). Inhibition of NECA-stimulated cAMP production by ZM241385 in HEK293T cells transiently transfected to express human adenosine 2a receptors, IC₅₀ = 63.46 nM (right).

Example Data: Calcium Flux Time-course and Dose-Response Curve

Calcium flux in HEK293T and CHO-K1 cells stimulated with 10 µM ionomycin. Drug was injected 10 seconds after initiating the assay (left). Dose-response curve of intracellular calcium levels for HEK293T cells transiently transfected to express human M1 receptors stimulated with carbachol, EC₅₀ = 3.1 µM (right). Calcium was detected using a Fluo-8 dye (Abcam, ab112128).