(R4511) A novel variant of the human dopamine D2 receptor, associated with a hyperkinetic movement disorder, mediates enhanced activation of G proteins

Dayana Rodriguez-Contreras (Oregon Health & Science University, Oregon Health & Science University)| Alec Condon (Oregon Health & Science University)| Dave Buck (VA Portland Health Care System)| Naeem Asad (New York University Abu Dhabi)| Timothy Dore (New York University Abu Dhabi)| Dineke Verbeek (University of Groningen)| Marina Tijssen (University of Groningen)| Ujwal Shinde (Oregon Health & Science University)| John Williams (Oregon Health & Science University)| Kim Neve (VA Portland Health Care System, VA Portland Health Care System)

A novel DRD2 variant with a mutation in exon 5 of DRD2 (c.634A>T; p.I212F) has been identified in a Dutch family. Ile212 is at the N-terminus of the D2 receptor (D2R) 3rd cytoplasmic loop. D2-I212F 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-I212F causes the movement disorder. Molecular dynamics (MD) simulations of human D2R models suggested that the presence of Phe212 in D2R separates the side chains of Arg132, at the cytoplasmic end of the 3rd transmembrane domain (TM3), and Glu368, at the cytoplasmic end of TM6, spontaneously breaking the ionic lock in the inactive D2-I212F. 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. In vitro and ex vivo studies supported the hypothesis that D2-I212F is a constitutively active receptor: We expressed D2L/S-I212F in HEK293 cells and assayed activation of Gαi1 and GαoA by bioluminescence resonance energy transfer. We observed increased quinpirole potency to activate Gαi1 by D2L/S-I212F (2.8 or 3.5 nM for D2L or D2S, respectively) compared to D2L/S-WT (19 or 27 nM) whereas quinpirole potency for activating GαoA was not substantially changed by the I212F mutation. On the other hand, D2L/S-I212F exhibited increased basal activation of both Gαi/o proteins compared to D2L/S-WT (set as 0%); basal GαoA activation by D2L/S-I212F (43 or 57% of maximal stimulation, respectively) was significantly higher than D2L/S-I212F-mediated basal Gαi1 activation (25% and 35%, respectively). We then used AAV to express Cre recombinase-regulated D2S-WT or D2S-I212F 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-I212F (-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-I212F, indicating that most of the tonic current was due to endogenous dopamine to which D2-I212F may be more sensitive, but that D2-I212F also displayed some constitutive activity in the presumed absence of dopamine. Thus, our in silico, in vitro, and ex vivo studies all indicate that the mutation enhances constitutive and agonist-induced activation of G proteins by D2R