Structural insights into the high basal activity and inverse agonism of the orphan receptor GPR6 implicated in Parkinson's disease

Abstract

GPR6 is an orphan G protein-coupled receptor with high constitutive activity found in D2-type dopamine receptor-expressing medium spiny neurons of the striatopallidal pathway, which is aberrantly hyperactivated in Parkinson's disease. Here, we solved crystal structures of GPR6 without the addition of a ligand (a pseudo-apo state) and in complex with two inverse agonists, including CVN424, which improved motor symptoms in patients with Parkinson's disease in clinical trials. In addition, we obtained a cryo-electron microscopy structure of the signaling complex between GPR6 and its cognate G_s heterotrimer. The pseudo-apo structure revealed a strong density in the orthosteric pocket of GPR6 corresponding to a lipid-like endogenous ligand. A combination of site-directed mutagenesis, native mass spectrometry, and computer modeling suggested potential mechanisms for high constitutive activity and inverse agonism in GPR6 and identified a series of lipids and ions bound to the receptor. The structures and results obtained in this study could guide the rational design of drugs that modulate GPR6 signaling.

Fig. 1.. Top and side views of GPR6 structures in different conformational states.

(A) The inactive state (blue) stabilized by IAG 3h (orange spheres). (B) The active-like state (grey) stabilized by a putative lipid agonist (green spheres). (C) Signaling complex of GPR6 (salmon) with G_s heterotrimer (Gα_s – teal, Gβ – gold, Gγ – lime) stabilized by Nb35 (grey) and an unknown ligand. Water molecules are shown as red spheres, membrane boundaries are shown as dashed lines, annular lipids and PEG400 are shown as purple and yellow sticks, respectively. The unmodelled density of a cognate agonist in the GPR6-G_s structure is contoured at 3.0 σ and shown in green mesh. GPR6 helices are identified with roman numerals and G_s subunits are labeled respectively.

Fig. 2.. Structural details of IAG binding to GPR6.

(A, B) Sections through the orthosteric ligand-binding pocket in GPR6 with residues interacting with IAGs 3h (A) and CVN424 (B) within 4.5 Å of the ligand shown as sticks. Refined 2mF_o-DF_c electron density around the contoured at 1.0 σ is shown as blue mesh. The right sides of each panel show 2D schematic of the corresponding IAG chemical structures and their interactions with residues within 4.5 Å. π-stacking and hydrogen bonding interactions are shown as grey and red dashed lines, respectively. (C, D) Specific binding of [³H]-RL-983 was measured at different GPR6 mutants. Data points represent mean ± SD of N=3 independent experiments (N=6 for WT) carried out in quadruplicates.

Fig. 3.. Structural details of lipid-like agonist binding to GPR6.

(A) Section through the ligand-binding site of the pseudo-apo GPR6 structure depicting a continuous channel accessible from both the extracellular side and the lipid bilayer. The mFo-DFc ligand omit electron density (green mesh) is contoured at 3 σ. (B) Ligand-binding pocket in the pseudo-apo GPR6 structure. The putative ligand is modeled as oleic acid (OLA) and shown as green sticks. The lipid chain entering the pocket from the lipid bilayer is shown in grey sticks. The 2mFo-DFc electron density (blue mesh) is contoured at 1 σ around both lipids. Residues within 4.5 Å of the putative ligand are shown as thin grey sticks. Water molecules are shown as red spheres. (C) MD simulation snapshots of the GPR6 active conformation stabilized by OLA (lime sticks). Key amino acids that interact with the OLA’s headgroup are shown as sticks. (D) Sequence similarity of the OLA-interacting-residues in GPR6 with its two paralogs GPR3 and GPR12. The color code represents the property of the amino acid residues, yellow – hydrophobic, green – aromatic, blue – positive charge, tan – cysteine, purple – polar uncharged. (E) Basal activity of GPR6 mutants. Bars correspond to means from N=3 independent experiments. All data points are shown. Data are normalized by the basal activity of the WT receptor and analyzed using the one-sided t-test based on a linear mixed effects model with P values adjusted for multiple testing to determine significance compared to the WT GPR6. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (data and P values are shown in tables S2 and S3).

Fig. 4.. Structural insights into GPR6 activation.

(A) Superposition of GPR6 structures: inactive state GPR6-3h (blue), active-like pseudo-apo GPR6 (grey), active GPR6 (salmon) in complex with G_s (Gα_s – teal, Gβ – gold, Gγ – lime). (B) Intracellular view of overlaid 7TM bundles from GPR6-3h, GPR6-pseudoapo, and GPR6-G_s structures highlights hallmarks of activation. (C-F) Magnified view of microswitches involved in GPR6 activation: Phe152^3.36 and toggle switch Trp292^6.48 (C), toggle-switch Trp292^6.48 and Val^3.40-Val^5.50-Phe^6.44 motif (D), sodium-binding pocket (E), DR^3.50Y and NPxxY^7.53 motifs (F).

Fig. 5.. GPR6 basal activation profile from MD simulations.

(A) Free energy landscape of apo GPR6 calculated from the microstate MSM is shown by projecting MD snapshots onto the first two TICs. The stationary macro-state probabilities are annotated as numbers at the centroid positions of the membership MD snapshots. MD snapshots nearest to each centroid (colored) are superposed to the active-like (grey, upper panel) or inactive (grey, lower panel) crystal structures. The net fluxes between the most populated inactive macro-state I5 and the most populated active macro-state A2 are visualized as the thicknesses of the connecting curves. The flux graph was simplified by discarding connections with the lowest fluxes. (B) Free energy landscape projected to a plot of root-mean-squared deviation (r.m.s.d.) of the NPxxY motif from the inactive crystal structure and the distance between Arg166^3.50 and Thr274^6.30. Positions of the crystal structures are indicated by asterisks, I_Xrc refers to the inactive GPR6-3h crystal structure, A_Xrc refers to the active-like pseudo-apo crystal structure, A_Cryo refers to the G_s protein-bound cryo-EM structure. (C, D) Violin plots showing distribution of distances between Arg166^3.50 and Thr274^6.30 (C) and r.m.s.d. of NPxxY motif from the inactive crystal structure (D). Stationary probabilities of each macro-state are shown in parenthesis. Macro-states 0–2 correspond to A0-A2, macro-states 3–5 correspond to I3-I5.

Fig. 6. Structural insights into GPR6 coupling to G_s protein.

(A, B) Two side views of the GPR6 cytoplasmic cavity that accommodates G_s, and interactions of GPR6 residues with residues of the α5 helix from Gα_s subunit (teal) that mediate this binding. GPR6 and G_s residues involved in interactions are shown as sticks. (C) Interface of G_s with ICLs 1 and 2 from GPR6. Residues of Gβ (gold) and the N helix of Gα_s interacting with ICL1 and ICL2, respectively, are shown as sticks. (D) Basal activity of GPR6 mutants in HEK293 cells. Bars correspond to means from N=3 independent experiments. All data points are shown. Data are normalized by the basal activity of the WT receptor and analyzed using the one-sided t-test based on a linear mixed effects model with P values adjusted for multiple testing to determine significance compared to the WT GPR6. *P<0.05, ***P < 0.001, ****P < 0.0001 (data and P values are shown in table S3).