Signaling-Biased and Constitutively Active Dopamine D2 Receptor Variant

A dopamine D2 receptor mutation was recently identified in a family with a novel hyperkinetic movement disorder. Compared to the wild type D2 receptor, the novel allelic variant D2-I212F activates a G alpha(i1)beta(1)gamma(2) heterotrimer with higher potency and modestly enhanced basal activity in human embryonic kidney (HEK) 293 cells and has decreased capacity to recruit arrestin3. We now report that omitting overexpressed G protein-coupled receptor kinase-2 (GRK2) decreased the potency and efficacy of quinpirole for arrestin recruitment. The relative efficacy of quinpirole for arrestin recruitment to D2-(IF)-F-212 compared to D2-WT was considerably lower without overexpressed GRK2 than with added GRK2. D2-(IF)-F-212 exhibited higher basal activation of G alpha(oA) than G alpha(i1) but little or no increase in the potency of quinpirole relative to D2-WT. Other signs of D2-(IF)-F-212 constitutive activity for G protein-mediated signaling, in addition to basal activation of G alpha(i/o), were enhanced basal inhibition of forskolin-stimulated cyclic AMP accumulation that was reversed by the inverse agonists sulpiride and spiperone and a similar to 4-fold increase in the apparent affinity of D2-(IF)-F-212 for quinpirole, determined from competition binding assays. In mouse midbrain slices, inhibition of tonic current by the inverse agonist sulpiride in dopamine neurons expressing D2-(IF)-F-212 was consistent with our hypothesis of enhanced constitutive activity and sensitivity to dopamine relative to D2-WT. Molecular dynamics simulations with D2 receptor models suggested that an ionic lock between the cytoplasmic ends of the third and sixth a-helices that constrains many G protein-coupled receptors in an inactive conformation spontaneously breaks in D2-(IF)-F-212. Overall, these results confirm that D2-(IF)-F-212 is a constitutively active and signaling-biased D2 receptor mutant and also suggest that the effect of the likely pathogenic variant in a given brain region will depend on the nature of G protein and GRK expression.