Structural genomics of the human dopamine receptor system

Abstract

The dopaminergic system, including five dopamine receptors (D1R to D5R), plays essential roles in the central nervous system (CNS); and ligands that activate dopamine receptors have been used to treat many neuropsychiatric disorders, including Parkinson's Disease (PD) and schizophrenia. Here, we report cryo-EM structures of all five subtypes of human dopamine receptors in complex with G protein and bound to the pan-agonist, rotigotine, which is used to treat PD and restless legs syndrome. The structures reveal the basis of rotigotine recognition in different dopamine receptors. Structural analysis together with functional assays illuminate determinants of ligand polypharmacology and selectivity. The structures also uncover the mechanisms of dopamine receptor activation, unique structural features among the five receptor subtypes, and the basis of G protein coupling specificity. Our work provides a comprehensive set of structural templates for the rational design of specific ligands to treat CNS diseases targeting the dopaminergic system.

Fig. 1. Cryo-EM structures of D1R, D2R, D3R, D4R and D5R signaling complexes.

a, b The cryo-EM density maps (a) and models (b) of the D1R–G_s, D2R–G_i, D3R–G_i, D4R–G_i, and D5R–G_s complexes. c The ligand-binding pockets of the D1R–G_s, D2R–G_i, D3R–G_i, D4R–G_i, and D5R–G_s complexes. Electrostatic surface potential is colored by red (−10 kT/e), blue (+10 kT/e), and white (neutral). d The rotigotine structure in the D1R–G_s, D2R–G_i, D3R–G_i, D4R–G_i, and D5R–G_s complexes. The EM densities of rotigotine in the five structures are shown.

Fig. 2. Structural feature comparison of all active-state dopamine receptors.

a Structural superposition of D1R, D2R, D3R, D4R, and D5R. b Structural alignment of ECL2, TM3, and TM5. c Structural alignment of ECL1 and TM1. d Structural alignment of TM5 and TM6. e Structural alignment of ICL2.

Fig. 3. Rotigotine recognition at all dopamine receptors.

a–e Left, detailed interaction between rotigotine and D1R (a), D2R (b), D3R (c), D4R (d) or D5R (e). Right, effects of mutations of the ligand-binding pocket residues of D1R (a), D2R (b), D3R (c), D4R (d) or D5R (e) on changes in ΔpEC₅₀ in response to stimulation of rotigotine, evaluated using a GloSensor cAMP assay. All data are presented as means ± SEM of three independent experiments for the WT and mutants (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant (two-tailed paired t-test).

Fig. 4. Polypharmacological profile of rotigotine.

a The chemical structure of rotigotine. b The interaction of D1R OBP with rotigotine. c The interaction of D1R EBP with rotigotine. d The affinities (K_i) of rotigotine to different GPCRs as indicated by radioligand competition binding assays and the alignment of OBP-EBP residues. Receptors are listed in order of decreasing rotigotine affinity. e Structural superposition of five dopamine receptors and bound rotigotine. f Structural superposition of five rotigotine-bound dopamine receptors compared with serotonin receptors (gray). 5-HT_1A (PDB: 7E2Y), 5-HT_1B (PDB: 6G79), 5-HT_1D (PDB: 7E32), 5-HT_2B (PDB: 6DRY), and 5-HT5A (PDB: 7X5H). g Structural superposition of five rotigotine-bound dopamine receptors compared with adrenergic receptors (gray). α2A (PDB: 6KUY), α2B (PDB: 6K41), and α2C (PDB: 6KUW).

Fig. 5. Comparison of D1R and D5R in rotigotine binding and PAM binding.

a Structural superposition of D1R–G_s and D5R–G_s complexes when receptors were aligned. b Comparison of rotigotine binding poses in D1R and D5R structures. c Structural comparison of rotigotine recognition between D1R and D5R. d, e Rotigotine-binding pockets of D1R (d) and D5R (e) viewed from the extracellular side. Electrostatic surface potential is colored by red (−10 kT/e), blue (+10 kT/e), and white (neutral). f Comparison of TM6 and TM7 residues for rotigotine recognition between D1R and D5R. g The side chain of W^3.52 residue shows two alternative conformations in the D1R–G_s–rotigotine structure. h The unique conformation of W^3.52 residue is stabilized by a cholesterol molecule in the D5R–G_s–rotigotine structure. i Comparison of TM3, TM4 and ICL2 residues for compound LY3154207 recognition between D1R and D5R.

Fig. 6. The binding of rotigotine in D4R is regulated by cholesterol.

a Cholesterol molecules at the surface of D4R. b A cholesterol molecule is located between TM1 and TM7 of D4R and stabilizes rotigotine binding through residues W435^7.40 and F91^2.61. c–f Structural comparison of the TM1–TM7 region and residue 2.61 of D1R (c), D2R (d), D3R (e), and D5R (f) show differences from those of D4R. g Concentration response of WT and W^7.40A mutant of all five dopamine receptors stimulated by rotigotine.

Fig. 7. G protein coupling of dopamine receptors.

a The structures of the D1R–G_s, D2R–G_i, D3R–G_i, D4R–G_i, and D5R–G_s complexes. b Comparison of the G protein conformations among the structures of D1R–G_s, D2R–G_i, D3R–G_i, D4R–G_i, and D5R–G_s complexes. c Structural comparison focused on the α5 helix of the Gα subunit bound to dopamine receptors. d Comparison of the G protein conformations among the structures of D1R–G_s and D5R–G_s complexes. e Comparison of the G protein conformations among the structures of D2R–G_i, D3R–G_i, and D4R–G_i complexes.