Signaling-Biased and Constitutively Active Dopamine D2 Receptor Variant

Abstract

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 Gα_i1β₁γ₂ 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-I²¹²F compared to D2-WT was considerably lower without overexpressed GRK2 than with added GRK2. D2-I²¹²F exhibited higher basal activation of Gα_oA than Gα_i1 but little or no increase in the potency of quinpirole relative to D2-WT. Other signs of D2-I²¹²F constitutive activity for G protein-mediated signaling, in addition to basal activation of Gα_i/o, were enhanced basal inhibition of forskolin-stimulated cyclic AMP accumulation that was reversed by the inverse agonists sulpiride and spiperone and a ∼4-fold increase in the apparent affinity of D2-I²¹²F 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-I²¹²F 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 α-helices that constrains many G protein-coupled receptors in an inactive conformation spontaneously breaks in D2-I²¹²F. Overall, these results confirm that D2-I²¹²F 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.

Keywords: D2 receptor; Dopamine; G protein; allelic variant; arrestin; biased signaling; constitutive activity.