Our functional GPCR assay service measures second messenger levels as a result of GPCR activation or inhibition to characterize receptor pharmacology. Assays can be conducted with immortalized/transfected cell lines as well as primary cell lines. Assays are carried out with live cells and in some cases on membranes; frozen cells/membranes can be sent to us. Alternatively, our cell culture and transfection ancillary services are available in addition to ELISAs 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 and antagonists.

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

To measure agonist EC_50, cells expressing the target receptor are stimulated with increasing concentrations of the test compound, and cAMP production is determined relative to the maximum stimulated by forskolin.

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 cAMP production.

Example Data: cAMP Antagonist Assay

Inhibition of NECA-stimulated cAMP production by ZM241385 in HEK293T cells transiently transfected to express human adenosine 2a receptors. IC₅₀ = 63.46 nM.