G Protein Selectivity in Dopamine Receptors is Determined before GDP Release

Dopaminergic signaling in neurophysiological processes utilizes multiple G proteins. The dopamine receptor subtypes D1R/D5R selectively couple to G_s/olf proteins, while D2R/D3R/D4R is selective for G_i/o proteins. The molecular mechanisms underlying this selectivity are not clear, so structural models of D1R and D2R were built in complex with their cognate and noncognate G proteins, in either GDP-bound or nucleotide-free states. These eight complexes were relaxed in a membrane environment through 2 μs-long molecular dynamics (MD) simulations. A thermodynamic analysis of these complexes provided free energies of G protein binding to the receptors that was consistent with D1R’s preference for G_s protein and D2R’s preference for G_i protein, but only for the GDP-bound states of the G proteins, suggesting that G_s vs G_i selectivity happens before GDP release. Biophysical measurements of receptor preassociation with G proteins in cells were also consistent with these preferences. The role of the Gα protein’s α5-helix in G protein selectivity was probed by switching the last 18 residues of Gα between Gα_s and Gα_i to create chimeric G_i18s and G_s18i proteins. Thermodynamic analysis of MD-relaxed chimeric complexes revealed a complete switch in G protein binding selectivity for both D1R and D2R receptors, but again only for the GDP-bound G proteins. Biophysical measurements of receptor preassociation with G proteins in cells also overall supported this selectivity alteration. These studies have shown that G protein selectivity for dopamine receptors is conferred before GDP release; however, additional molecular events may be needed for a productive coupling to enable a successful GDP/GTP exchange.