Conformational plasticity of the intracellular cavity of GPCR−G-protein complexes leads to G-protein promiscuity and selectivity

While the dynamics of the intracellular surface in agonist-stimulated GPCRs is well studied, the impact of GPCR dynamics on G-protein selectivity remains unclear. Here, we combine molecular dynamics simulations with live-cell FRET and secondary messenger measurements, for 21 GPCR−G-protein combinations, to advance a dynamic model of the GPCR−G-protein interface. Our data show C terminus peptides of Gα_s, Gαi, and Gα_q proteins assume a small ensemble of unique orientations when coupled to their cognate GPCRs, similar to the variations observed in 3D structures of GPCR−G-protein complexes. The noncognate G proteins interface with latent intracellular GPCR cavities but dissociate due to weak and unstable interactions. Three predicted mutations in β₂-adrenergic receptor stabilize binding of noncognate Gα_q protein in its latent cavity, allowing promiscuous signaling through both Gα_s and Gα_q in a dose-dependent manner. This demonstrates that latent GPCR cavities can be evolved, by design or nature, to tune G-protein selectivity, giving insights to pluridimensional GPCR signaling.