GPCR Molecular Pharmacology

The ß–Galactoisidase Gene Assay enables high-throughput screening of novel ligands of GPCRs through the use of the ß–Galactoisidase reporter gene (see original publication: doi.org/10.1006/abio.1995.1235) . Our stably transfected (receptor) cells are transfected with the ß–Gal gene plasmid, which produces ß–Galactosidase enzyme upon GPCR stimulation via cAMP-dependent CREB/CRE promotor amplification. The resulting enzyme is then able to release colorimetric O-Nitrophenol from an ONPG substrate upon development, yielding a linear correlative response between receptor agonism and assay output.

The AlphaScreen® (PerkinElmer) is a high-throughput assay utilized to directly measure the intracellular levels of cAMP generated by receptor stimulation. Our stably transfected (receptor) cells are fully compatible with this orthogonal method that utilizes a higher-throughput 384-well plate format to measure receptor signaling with even greater sensitivity.

Radioligand binding assays allow us to correlate observed activity in the ß–Gal or AlphaScreen® assays to orthogonal or allosteric binding modes on the GPCR. Radioactively labeled known ligands are saturated at the receptor and then titrated off with our unknown lead ligands. As more and more labeled ligand is displaced, the resulting radioactivity can be measured and specific orthogonal binding can be calculated.