Structural insights into ligand recognition, activation, and signaling of the α2A adrenergic receptor

Abstract

The α_2A adrenergic receptor (α_2AAR) is a G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor that mediates important physiological functions in response to the endogenous neurotransmitters norepinephrine and epinephrine, as well as numerous chemically distinct drugs. However, the molecular mechanisms of drug actions remain poorly understood. Here, we report the cryo-electron microscopy structures of the human α_2AAR-GoA complex bound to norepinephrine and three imidazoline derivatives (brimonidine, dexmedetomidine, and oxymetazoline). Together with mutagenesis and functional data, these structures provide important insights into the molecular basis of ligand recognition, activation, and signaling at the α_2AAR. Further structural analyses uncover different molecular determinants between α_2AAR and βARs for recognition of norepinephrine and key regions that determine the G protein coupling selectivity. Overall, our studies provide a framework for understanding the signal transduction of the adrenergic system at the atomic level, which will facilitate rational structure-based discovery of safer and more effective medications for α_2AAR.

Fig. 1.. Structure and function of α_2AAR agonists.

(A) Chemical structures of norepinephrine (Norepi), brimonidine (BRI) or UK14304, dexmedetomidine (DEX), and oxymetazoline (OXY). (B) Concentration-response curves of different agonists toward G protein activation (blue curve) and β-arrestin-2 recruitment (red curve) measured by a cell-based Gqi-inositol phosphate accumulation assay and the PathHunter assay respectively. Data are presented as means ± SEM of 4 to 10 independent experiments with repeats in duplicate.

Fig. 2.. Cryo-EM structures of Norepi, BRI-, DEX-, and OXY-bound α_2AAR-GoA complexes.

Cryo-EM density maps and models of the α_2AAR-GoA complex bound to Norepi (A), BRI (B), DEX (C), and OXY (D). The densities of the agonists (shown as sticks) are depicted as gray meshes. The maps are colored according to different subunits.

Fig. 3.. Orthosteric binding pocket of active α_2AAR bound to different agonists.

(A) Alignment of four structures of α_2AAR signaling complexes. (B) Superposition of Norepi, BRI, DEX, and OXY from cryo-EM structures. The imidazole moiety is highlighted by the dashed circle. (C to F) Detailed interactions of Norepi (C), BRI (D), DEX (E), and OXY (F) with α_2AAR. Residues within 4 Å of agonist are shown in sticks. The polar interactions are indicated by black dashed lines. (G) Superposition of the α_2AAR orthosteric binding pocket residues bound to four different agonists. (H and I) Concentration-response curves of different agonists for G protein activation (H) and β-arrestin-2 recruitment (I) for wild-type (wt) α_2AAR and receptor mutants D128^3.32A, Y431^7.43A, S215^5.42A, and Y409^6.55A, respectively. Except for the activation of D128^3.32A, which is normalized to DEX for G protein activation and relative to basal for arrestin recruitment, receptor activation is shown relative to the maximum effect of Norepi. Data are presented as means ± SEM of 3 to 11 independent experiments with repeats in duplicate.

Fig. 4.. Comparison of Norepi binding for α_2A and β adrenergic receptors.

(A) Detailed interactions of Norepi with β₁AR [Protein Data Bank (PDB) code: 7BU6]. Residues within 4 Å of agonist are shown in sticks. The polar interactions are indicated by black dashed lines. (B) Superposition of orthosteric pockets of β₁AR-Norepi and β₂AR-Epi (epinephrine) (PDB code: 4LDO). (C) Detailed interactions of dopamine with D1R (PDB code: 7CKZ). The polar interactions are indicated by black dashed lines. (D) Superposition of orthosteric pockets of β₁AR-Norepi and α_2AAR-Norepi. Residues within 4 Å of agonist are shown in sticks.

Fig. 5.. Comparison of inactive and active α_2AAR.

(A) Structural comparison of inactive α_2AAR bound to RS79948 and active α_2AAR bound to Norepi, with changes highlighted as red arrows. The distance is calculated between positions of Cα of residue 6.29 in TM6. (B) Conformational changes within the orthosteric pocket are shown from the extracellular side, with changes highlighted as red arrows. (C) Cross sections of α_2AAR bound to antagonist and agonist are shown, with the interior in black and the exosite highlighted. (D) Concentration-response curves of F427^7.39 mutant for different agonists toward G protein activation and β-arrestin-2 recruitment. Data are presented as means ± SEM of 4 to 10 independent experiments with repeats in duplicate.

Fig. 6.. G protein binding interface.

(A and B) Superposition of the G protein coupling interfaces of α_2AAR-GoA, α_2BAR-GoA, and β₂AR-Gs complexes, using receptor for alignment. (C to H) Detailed interactions of α_2AAR with Goα (C and D), α_2BAR with Goα (E and F), and β₂AR with Gsα (G and H). The polar interactions are indicated by red dashed lines.