Functional Gpcr Assays

Whether inactive GPCRs are pre-coupled to G proteins and how GPCRs can activate heterotrimeric G proteins are issues subject to a lot of speculation. In general, activated GPCRs stimulate heterotrimeric G proteins in membranes in a manner that involves GDP release (a rate-limiting step) and then GTP binding. GTP binding to the a subunit of the G protein leads to a conformational change in its switch regions and promotes dissociation of the G protein complex into a- and pg-subunits. Both entities then have the ability to stimulate or inhibit downstream effectors.2324

The a-subunit defines the G protein subtype; the 16 different a-subunits are classified into four distinct families: Gs, Gi/o, Gq, and G12/13.24-26 Gas activates adenylate cyclase (AC) and therefore leads to an increase in cAMP synthesis; Gai/o inhibits AC. Gaq activates PLCp and increases intracellular calcium levels and PKC activity. Heterotrimeric G12 and G13 have been shown to mediate signals from GPCRs to Rho GTPase activation and to proteins from the ERM family.27

In addition to recognizing effectors for specific subtypes of G proteins, great efforts made to identify novel effectors for G proteins led to discoveries of new effector proteins such as K+ channels, RaplGAP, src tyrosine kinase, Bruton tyrosine kinase, and others. However, their roles and receptor-mediated activation mechanisms of some of these effector proteins are still unclear.28 Functional HTS assays mainly target well-characterized pathways such as GTP binding, cAMP, and Ca2+ assays. When GPCRs are overexpressed, they can display ligand-independent activities (constitutive or basal activities) that can be measured using second messenger or reporter gene assays. Different assay technologies now available allow ligand occupancy of GPCRs to be converted into robust functional assay signals as described in the next section.

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