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Martin L. Dalefield
The D2 dopamine receptor (DAR) is one of the most important drug targets in neurology and psychiatry. The D2 DAR signals through multiple G protein-mediated pathways, which may cause a drug to possess adverse side effects due a lack of selectivity between these functions. GPCR signaling by the D2 DAR is mediated through recruitment of the β-arrestin scaffolding protein, which leads to internalization of the GPCR and termination of signaling. An objective of the Molecular Neuropharmacology Section is to develop high-throughput assays for functionally-selective D2 DAR agents. The laboratory has developed a bioluminescence resonance energy transfer (BRET) assay for measurement of D2 DAR-β-arrestin interactions. In this assay, the D2 DAR is fused to the energy donor Rluc8 (a modified Renilla luciferase) while the β-arrestin protein is fused to the energy acceptor mVenus (a modified YFP). When Rluc8 metabolizes the cell permeable luciferase substrate coelenterazine, light emission occurs with a peak at 480 nm, which in turn excites mVenus, which emits light with a peak at 530 nm. Radiometric reading of emission at both wavelengths allows the calculation of a BRET ratio by division of emission at 530 nm by the emission at 480 nm. This assay was used to characterize a series of drugs derived from the D2 partial agonist aripiprazole, which is used in the treatment of schizophrenia and depression. Two of the drugs in this series have previously been shown to act as D2 partial agonists in an adenylyl cyclase inhibition assay (Vangveravong, et al. 2011). By testing for β-arrestin recruitment, it is possible to determine whether the drugs are functionally selective. All 12 of the drugs were found to be antagonists of β-arrestin recruitment to the D2 DAR with potencies ranging from 12.8 µM to 706 pM. These findings were corroborated with screenings in the Gal-Screen System assay, an established screening tool.
Last Modified December 23, 2013