BRET Assays to Determine Altered Function of a D2DR Variant in G protein-Independent and -Dependent Pathways

Abstract

G protein-dependent and independent signaling pathways perform crucial roles in dopaminerelated physiological and pathological processes. A pivotal role of dopamine in the CNS is the control of locomotion; disruption to dopaminergic systems, such as reduced striatal D2 receptor availability, contributes to the development of movement disorders. Affected members of a family with dystonia, a movement disorder, are heterozygous for a SNP in exon 4 of D2DR (c.634A>T p.I212F), a missense variant located within the third intracellular loop; specifically, the variation is in the middle of a short stretch of residues that have been found to be critical for binding of arrestin and several other dopamine receptor-interacting proteins. The comparative actions between D2-WT and D2-I212F were analyzed in their effects on both the G proteinmediated pathway and the arrestin3 pathway. It was shown that the D2-I212F variant was only 34% as effective as D2-WT at recruiting arrestin3, a deficiency that is likely to have important behavioral consequences. Additionally, quinpirole stimulation of D2-I212F produced dosedependent inhibition of cAMP accumulation; it was observed that lower concentrations of quinpirole were able to fully activate the rare variant. To further investigate whether this missense variant causes movement disorders, a condition that mimics the heterozygosity (D2-WT/D2-I212F), for the SNP affected patients have, was utilized and tested. Co-transfection of D2-WT and D2-I212F trended toward a shift in the EC50, where D2-WT/D2-I212F were different from D2-WT expressed alone, but the difference was not statistically different. This variant is likely to have decreased arrestin-mediated signaling and enhanced G protein-mediated signaling, so behaviors and cellular functions mediated by those two pathways will be altered according to patients with this variant. 3