G-Protein Coupled Receptors (GPCRs), also known as 7-transmembrane Receptors are important targets for drugs to treat various disorders. Disruption in their regulation causes inflammatory and respiratory diseases, cancer, cardiovascular diseases and CNS disorders like Alzheimer’s, Parkinson’s disease, etc.
To address the need of the hour, we have established various cell lines expressing GPCRs and validated the assays.
DRC of reference compound using dopamine D2 receptor
DRC of reference compound using dopamine D2L receptor
As an extension to our receptor binding assay platform, we have developed in-house functional assays to screen the GPCR targets. Ligand binding to GPCR promotes G-Protein coupling, initiating signal transduction pathways that trigger a series of cellular responses.
At GVK BIO, we have capabilities for measuring GPCR functional responses by developing cAMP assays for Gs coupled receptors and Ca2+ measurements for Gq coupled receptors. We also have capabilities for performing Guanine nucleotide binding assay ([35S] GTPγS assay) for Gi coupled receptors.
For GPCRs coupled with Gs G-protein, cAMP assay is employed to determine the functionality of the expressed protein. In GVK BIO we employ cAMPGlo assay and Alpha screen technology to perform cAMP assay.
Ca2+ Release Assay
Ca2+ release assay is used for GPCRs couple with Gq G-protein. A fluorometric assay using FLIPR is employed to determine the Ca2+ release by GPCRs. The Ca2+ released when GPCR is activated by specific ligand binds to a dye and emits fluorescence at specific wavelength.
Radioligand Binding Assays
The principle of radioligand binding assay is based on labelled radioligand molecules binding to specific receptors, transporters, enzymes or any protein of interest. Measuring the rate and extent of competitive binding between the radiolabelled ligand and unlabelled compounds provides us information on the affinity of the compounds to the specific protein.
GPCR occupation by agonists leads to guanine nucleotide exchange on the G protein α-βγ complex; GDP bound to Gα subunits dissociates and is immediately replaced by GTP. GTP-bound Gα subunit then detaches from the Gαβγ complex and the dissociated βγ and Gα-GTP subunits are capable of downstream signalling. Non-hydrolysable analogues of GTP, such as [35S] GTPγS and [Eu] GTPγS allow measurement of GPCR agonist induced GTP incorporation onto Gα subunits. GTPγS incorporation is an index of GPCR activity based on agonist activation and antagonist inhibition.
DRC of carbachol using CHO cell Lines over
expressing muscarinic M3 receptor