Enhancing the Signaling of GPCRs via Orthosteric Ions

Abstract

G protein-coupled receptors play essential roles in cellular processes such as neuronal signaling, vision, olfaction, tasting, and metabolism. As GPCRs are the most important drug targets, understanding their interactions with ligands is of utmost importance for discovering related new medicines. In many GPCRs, an allosteric sodium ion next to the highly conserved residue D^2.50 has been proposed to stabilize the inactive receptor state by mediating interactions between transmembrane helices. Here, we probed the existence of internal and functionally important sodium ions in the dopamine D2 receptor, using molecular dynamics simulations. Besides a new sodium ion at the allosteric ligand binding site, we discovered an additional sodium ion, located close to the orthosteric ligand binding site. Through cell-based activation assays, the signaling of D2 receptor with site-specific mutations was tested against a series of chemically modified agonists. We concluded an important structural role of this newly discovered orthosteric sodium ion in modulating the receptor signaling: It enables the coordination of a polar residue in the ligand binding site with an appropriately designed agonist molecule. An identical interaction was also observed in a recently released high-resolution crystal structure of mu-opioid receptor, which was reresolved in this work. Probably because of similar interactions, various metal ions have been found to increase the signaling of many other GPCRs. This unique principle and strategy could be used to optimize the drug activity of GPCR. Our findings open a new mechanistic opportunity of GPCR signaling and help design the next generation of drugs targeting GPCRs.

Figure 1

Allosteric Na⁺ ion next to D80^2.50 in D2R. (A) Entrance pathway of a Na⁺ ion from the extracellular receptor interface toward the allosteric site D80^2.50 of apo D2R. Zone I (gray): extracellular receptor interface at which the Na⁺ ion is in the bulk environment. Zone II (red): transition zone in the vicinity of D114^3.32. Zone III (blue): allosteric site in the vicinity of D80^2.50. Yellow spheres: Na⁺ ion positions during MD simulations. (B) Distance between allosteric Na⁺ ion and D80^2.50. (C) The final position of the allosteric Na⁺ ion at the end of MD simulations. The oxygen atoms in D80^2.50 and S121^3.39 as well as those of three water molecules establish a dedicated coordination network with the allosteric Na⁺ ion.

Figure 2

A second Na⁺ ion has been found in the orthosteric site of D2R using all-atom long-time MD simulations. (A) The entrance pathway of the orthosteric Na⁺ toward agonist molecule MLS1547. Yellow spheres: Na⁺ ion trajectories in the MD simulations. Black ball-and-stick: MLS1547. (B) The distance between orthosteric Na⁺ ion and H393^6.55 (C) The final position of the orthosteric Na⁺ ion at the end of MD simulations. MLS1547 and H393^6.55 as well as two water molecules establish a dedicated coordination network with the orthosteric Na⁺ ion.

Figure 3

Interaction fingerprint between distinct residues in D2R and MLS1547. (A) The 2D interaction diagram of D2R in complex with MLS1547. Green ball-and-stick: MLS1547. (B) The interaction fingerprint between residues in D2R and MLS1547. It represents the interaction frequency during the MD simulations. (C) Arrestin recruitment for D2R variants showing mutations on key residues predicted by MD simulations. All mutated receptors show noticeably decreased signaling.

Figure 4

Synthesis and biological activity of MLS1547 derivatives. (A) Scheme for synthesis of compound MLS-d1. (B) Activation of D2R mediated signaling induced by dopamine, MLS1547- and MLS1547-derivatives.

Figure 5

Two different roles of Na⁺ in D2 receptor activation. Left panel: an allosteric Na⁺ (yellow sphere) stabilizes the inactive state of receptor. Blue ball-and-sphere: dopamine molecule. Right panel: an orthosteric Na⁺ enhances the activation of receptor via a coordinating interaction with both H393^6.55 and agonist molecule MLS1547 (black ball-and-stick).