Universal HTS Assays for GPCRs

In general, the set of G proteins normally coupled to a target GPCR determines whether one employs the cAMP versus the IP3/Ca++ assays to measure activation. However, more generalized assays which can be used as universal GPCR screening technologies have also been developed which can be used for most, if not all receptors. One such universal screening approach for GPCR activity measures mitogen activated protein (MAP) kinase stimulation, since the MAP kinases are believed to be a point of convergence of the vast majority of GPCRs in regulating cell function. A commercially available assay to measure the activity of one of the MAP kinases, ERK, is the AlphaScreen SureFire ERK marketed by Perkin Elmer. This is a cell-based, homogenous, nonradioactive assay that measures phosphorylated ERK1 and ERK2 [34].

Another universal GPCR assay measures p-arrestin translocation in response to agonist stimulation [35]. This technology is based on the hypothesis that activation of many if not most GPCRs results in the translocation of a family of protein kinases referred to as G protein receptor kinases (GRKs) to the receptor which phosphorylate the GPCRs. This promotes the translocation of p-arrestin to the receptor that interrupts GPCR/G protein signaling. While other protein kinases such as protein kinase C can phosphorylate GPCRs, it is believed that activation of only the GRK/p - arrestin system selectively leads to phosphorylation and modulation of the agonist-bound receptor whereas other protein kinases are more pervasive in their actions, and their phosphorylation of a receptor is not dependent on agonist binding to that receptor. For example, stimulation of the p - adrenergic receptor with agonist can lead to activation of a specific GRK, leading to phosphorylation of the p- adrenergic receptor and association of specific p - arrestins. However, this process (GRK and ß-arrestin activation) does not lead to specific phosphorylation of other GPCRs which are free of agonist. In fact, activation of protein kinase C can cause phosphorylation of multiple GPCRs at a time.

The technology was originally designed to measure ß-arrestin translocation using confocal microscopy but has been modified to be detected by BRET [36, 37] . More recently, Wehrman et al. [38] have developed an enzyme fragment complementation assay to measure ß-arrestin translocation in a format adapted for HTS by DiscoveRx. Here, two fragments of the enzyme ß-galactosidase are employed, a small N-terminal fragment (Prolink) and a large truncated form of ß-galactosidase (EA). Each fragment alone is inactive but when added together, they recombine to form an active ß - galactosidase enzyme that can produce thousand of luminescent molecules in a short time. In the GPCR assay format, Prolink is tagged to the GPCR C-terminus and EA is tagged to ß-arrestin. When agonist stimulates the GPCR, the ß - arrestin - EA associates with the receptor- Prolink and complementation occurs, generating a highly luminescent response. Like the MAP kinase assays, ß - arrestin - based assays such as the EFC-GPCR are employed for HTS of both known and orphan GPCRs, and as they are not dependent on G protein-mediated signaling events, they can also be considered a universal approach for identifying agonists, inverse agonists, and antagonists.

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