A novel variant of the human dopamine D2 receptor, associated with a hyperkinetic movement disorder, mediates enhanced activation of G proteins

A novel variant with a mutation in exon 5 of (c.634A>T; p.I212F)has been identified in a Dutch family. Ile is at the N‐terminus of the D2 receptor (D2R) 3 cytoplasmic loop. D2‐I F is associated with a novel hyperkinetic movement disorder characterized by progressive chorea and cervical dystonia. We hypothesized that determining the effect of the mutation on D2R function might explain how D2‐I F causes the movement disorder. Molecular dynamics (MD) simulations of human D2R models suggested that the presence of Phe in D2R separates the side chains of Arg , at the cytoplasmic end of the 3 transmembrane domain (TM3), and Glu , at the cytoplasmic end of TM6, spontaneously breaking the ionic lock in the inactive D2‐I F. This ionic lock contributes to maintaining unliganded receptors in an inactive conformation, and it is broken in agonist‐activated receptors. Conversely, preventing formation of the lock frequently creates a constitutively active receptor. and studies supported the hypothesis that D2‐I F is a constitutively active receptor: We expressed ‐I F in HEK293 cells and assayed activation of Gα and Gα by bioluminescence resonance energy transfer. We observed increased quinpirole potency to activate Gα by D2 ‐I F (2.8 or 3.5 nM for D2 or D2 , respectively) compared to D2 ‐WT (19 or 27 nM) whereas quinpirole potency for activating Gα was not substantially changed by the I F mutation. On the other hand, D2 ‐I F exhibited increased basal activation of both Gα proteins compared to D2 ‐WT (set as 0%); basal Gα activation by D2 ‐I F (43 or 57% of maximal stimulation, respectively) was significantly higher than D2 ‐I F‐mediated basal Gα activation (25% and 35%, respectively). We then used AAV to express Cre recombinase‐regulated D2 ‐WT or D2 ‐I F in dopamine neurons of D2 autoreceptor knockout mice and characterized D2R activation of G protein‐regulated inward‐rectifying potassium channels (GIRKs). When CyHQ‐sulpiride (5 μM) was circulated over a midbrain slice, photolytic release of sulpiride produced a small inhibition of a tonic GIRK current in cells expressing D2‐WT (‐9 pA), and a much larger inhibition in cells expressing D2‐I F (‐62 pA). The enhanced inhibition of tonic current by the mutant receptor could reflect constitutive activation of G proteins or a heightened sensitivity of the mutant to dopamine. To distinguish between these possibilities, midbrain slices were treated with reserpine to deplete endogenous dopamine. This treatment abolished the response to sulpiride in cells expressing D2‐WT and greatly decreased the response in cells expressing D2‐I F, indicating that most of the tonic current was due to endogenous dopamine to which D2‐I F may be more sensitive, but that D2‐I F also displayed some constitutive activity in the presumed absence of dopamine. Thus, our , and studies all indicate that the mutation enhances constitutive and agonist‐induced activation of G proteins by D2R

Duke Scholars

Author John Wiley Williams Jr. Medicine, General Internal Medicine