Mechanistic insights into allosteric regulation of the A(2A) adenosine G protein-coupled receptor by physiological cations

Cations play key roles in regulating G-protein-coupled receptors (GPCRs), although their mechanisms are poorly understood. Here, (19)F NMR is used to delineate the effects of cations on functional states of the adenosine A(2A) GPCR. While Na(+) reinforces an inactive ensemble and a partial-agonist stabilized state, Ca(2+) and Mg(2+) shift the equilibrium toward active states. Positive allosteric effects of divalent cations are more pronounced with agonist and a G-protein-derived peptide. In cell membranes, divalent cations enhance both the affinity and fraction of the high affinity agonist-bound state. Molecular dynamics simulations suggest high concentrations of divalent cations bridge specific extracellular acidic residues, bringing TM5 and TM6 together at the extracellular surface and allosterically driving open the G-protein-binding cleft as shown by rigidity-transmission allostery theory. An understanding of cation allostery should enable the design of allosteric agents and enhance our understanding of GPCR regulation in the cellular milieu.