Mechanisms of GPCR signal propagation: from rhodopsin activation to inhibition of synaptic vesicle fusion by Gβγ-SNARE interactions

Abstract

G-protein-coupled receptor (GPCR) signaling is involved in virtually all physiological processes in vertebrates and contributes to the progression of many diseases. Despite thorough investigation, numerous details regarding the molecular mechanisms of GPCR activation and effector modulation via G-proteins remain unclear. One example is the importance of bulk water in shaping the energy landscape of GPCR activation. To address this, we developed an experimental osmotic stress approach to investigate the relationship between receptor internal hydration and rhodopsin activation. The results indicate that an influx of 80-100 water molecules penetrate the rhodopsin interior upon formation of the active state. Furthermore, dehydration of the rhodopsin interior significantly reduced its affinity for transducin C-terminal peptides. On the basis of these results, we propose that rhodopsin wet/dry cycling is coupled to transducin binding and release. Another aspect of GPCR signaling that is not well understood is the molecular mechanism by which G-protein βγ heterodimers (Gβγ) inhibit synaptic vesicle fusion via interactions with SNARE proteins. To examine this, we characterized the interaction between Gβγ and the ternary SNARE complex both structurally and biochemically. First, we mapped the binding sites on each human Gβ and Gγ isoform for tSNARE and identified the residues critical for the Gβ1γ2-tSNARE interaction using peptide arrays. Next, we discovered that Gβ1γ2 preferentially interacts with ternary SNARE in the partially zipped conformation, as opposed to the fully zipped conformation. To investigate the interaction further, we stabilized the Gβ1γ2-pre-fusion SNARE complex using crosslinking and structurally characterized the complex using single particle cryo-EM. Our preliminary cryo-EM density map suggests that the N-terminal coiled-coil of Gβγ interacts at the C-terminus of the SNARE complex. We next used Chai-1 and Rosetta docking to predict the structure of the Gβ1γ2 N-coiled-coil domain bound to the C-terminus of tSNARE. This structural prediction suggests that the N-termini of both Gβ1 and Gγ2 form an interface with the C-terminal helix of SNAP-25, and that Gγ2 inserts into the C-terminal SNARE helical bundle. These data provide a roadmap for future experiments to further elucidate the molecular details of the interface. Taken together, these studies elucidate novel aspects of GPCR signal propagation.