G-Protein-Coupled Receptor Activation Mediated By Internal Hydration

Rhodopsin is a canonical member of the Class A (rhodopsin-like) G-protein-coupled receptors and is responsible for scotopic vision under dim light in retinal rod cells. Utilizing rhodopsin as a model GPCR, we explored the role of water in the structural activation mechanism of GPCRs using osmotic stress techniques. We hypothesized that rhodopsin activation is hydration mediated as shown by nanosecond molecular dynamics simulations [1], which reveal a bulk influx of water into the protein core following photoactivation. To experimentally validate these results, we subjected the protein within native lipid membranes to environments of varying osmotic pressures, using different molecular-weight polyethylene glycols as osmolytes. By photoactivating the protein while recording time-dependent UV-visible spectra, we measured the fraction of protein in the active metarhodopsin-II (MII) conformation, the receptor state capable of activating the G-protein. High-molecular weight osmolytes comprehensively favored the more closed, inactive metarhodopsin-I (MI) conformation by withdrawal of water from the protein core. By contrast, small osmolytes penetrated into the protein core, and stabilized the active MII conformation until a quantifiable saturation point was reached. A universal osmotic response was discovered for the limit of increasing osmolyte size and maximal polymer exclusion from the protein. By measuring the thermodynamic dependence of the MI-MII equilibrium on osmotic pressure, we determined that rhodopsin activation entails a bulk influx of 80-100 water molecules into the protein core with a substantial increase in compressibility. A new model is proposed for the functional role of water in GPCR signal transduction, enabling a wet-dry cycling mechanism that amplifies the activation of G-proteins. Our results necessitate a new understanding of GPCR activation, in which the influx of water plays a critical role in establishing the active receptor conformation. [1] N. Leioatts et al. (2014) Biochemistry 53, 376-385.