Dissecting the molecular basis for the modulation of neurotransmitter GPCR signaling by GINIP

Abstract

It is well-established that activation of heterotrimeric G-proteins (G{beta}{gamma}) by G-protein-coupled receptors (GPCRs) stimulated by neurotransmitters is a key mechanism underlying neuromodulation. Much less is known about how G-protein regulation after receptor-mediated activation contributes to neuromodulation. Recent evidence indicates that the neuronal protein GINIP shapes GPCR inhibitory neuromodulation via a unique mechanism of G-protein regulation that controls neurological processes like pain and seizure susceptibility. However, the molecular basis of this mechanism remains ill-defined because the structural determinants of GINIP responsible for binding Gi subunits and regulating G-protein signaling are not known. Here, we combined hydrogen-deuterium exchange mass-spectrometry, protein folding predictions, bioluminescence resonance energy transfer assays, and biochemical experiments to identify the first loop of the PHD domain of GINIP as an obligatory requirement for Gi binding. Surprisingly, our results support a model in which GINIP undergoes a long-range conformational change to accommodate Gi binding to this loop. Using cell-based assays, we demonstrate that specific amino acids in the first loop of the PHD domain are essential for the regulation of Gi-GTP and free G{beta}{gamma} signaling upon neurotransmitter GPCR stimulation. In summary, these findings shed light onto the molecular basis for a post-receptor mechanism of G-protein regulation that fine-tunes inhibitory neuromodulation.