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GPCRs

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.

GPCR functional assay

As an extension to our receptor binding assay platform, we have developed functional assays in-house to screen the GPCR targets. Ligand binding to GPCR promotes G-Protein coupling, initiating signal transduction pathways, which 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 has capabilities for performing Guanine nucleotide binding assay ([35S] GTPγS assay) for Gi coupled receptors.

GPCR functional assay

 

  • cAMP assay (See more)
    For GPCRs coupled with Gs G-protein, cAMP assay is employed to determine the functionality of the expressed protein. In GVK BIO we employ cAMP Glo assay and Alpha screen technology to perform cAMP assay.
  • Ca2+release assay (See more)
    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 (See more)
    The principle of radioligand binding assay is based on labeled radioligand molecules binding to the 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.
  • GTPγS assay (See more)
    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 signaling. Non-hydrolyzable 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.