Characterization of the GTPγS release function of a G protein-coupled receptor.

G protein-coupled receptor (GPCR) signaling is one of the most ubiquitous and sensitive forms of cell surface reception. GPCRs stabilize the nucleotide-free state of heterotrimeric guanine-nucleotide binding proteins (G proteins); however, this state is produced at the cost of relieving the G protein of its stable inhibitor, GDP. Upon agonist binding to receptor, the G protein binds GTP and signal transduction ensues. Herein we demonstrate that the agonist can also stimulate the release of GTP. This receptor-mediated mechanism permits dissociation and reassociation of the G protein as the receptor acts as a catalyst for two different reactions. We demonstrate that this mechanism requires a unique, selective active state in addition to the active state that promotes GDP release. The release reaction is competitive with antagonists and we demonstrate operational efficacy. Further, we show that agonists have the potential to preferentially stimulate GTP binding or GTP release. This release selectivity may serve as a form of receptor signaling and reshape our understanding of G protein-coupled receptor signaling. Finally, we demonstrate that these reactions can be recapitulated in human spinal cord dorsal horn, providing an avenue for investigating state selectivity in physiologically significant samples.