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We review osmotic stress studies of the G-protein-coupled receptor rhodopsin. Despite the established presence of small amounts of structural water in these receptors, the influence of bulk water on their function remains unknown. Investigations of osmotic stress effects on the GPCR archetype rhodopsin have provided unique data about the role of water in receptor activation. It was discovered that osmolytes shift the rhodopsin equilibrium after photoactivation, either to the active or inactive conformations depending on their molar mass. Experimentally at least 80 water molecules have been found to enter rhodopsin in the transition to the active state. We propose that this influx of water is a necessary condition for receptor activation. If the water movement is blocked, e.g., by large osmolytes or by dehydration, then the receptor does not undergo its functional transition. The results suggest a new model whereby rhodopsin becomes swollen and partially unfolded in the activation mechanism. Water thus acts as a powerful allosteric modulator of functioning for rhodopsin-like receptors. Keywords: G-protein-coupled receptors, membranes, optical spectroscopy, rhodopsin, signal transduction.
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Hydration-mediated G-protein–coupled receptor activation
The Rhodopsin family of G-protein–coupled receptors (GPCRs) comprises the targets of nearly a third of all pharmaceuticals. Despite structural water present in GPCR X-ray structures, the physiological relevance of these solvent molecules to rhodopsin signaling remains unknown. Here, we show experimental results consistent with the idea that rhodopsin activation in lipid membranes is coupled to bulk water movements into the protein. To quantify hydration changes, we measured reversible shifting of the metarhodopsin equilibrium due to osmotic stress using an extensive series of polyethylene glycol (PEG) osmolytes. We discovered clear evidence that light activation entails a large influx of bulk water (∼80–100 molecules) into the protein, giving insight into GPCR activation mechanisms. Various size polymer osmolytes directly control rhodopsin activation, in which large solutes are excluded from rhodopsin and dehydrate the protein, favoring the inactive state. In contrast, small osmolytes initially forward shift the activation equilibrium until a quantifiable saturation point is reached, similar to gain-of-function protein mutations. For the limit of increasing osmolyte size, a universal response of rhodopsin to osmotic stress is observed, suggesting it adopts a dynamic, hydrated sponge-like state upon photoactivation. Our results demand a rethinking of the role of water dynamics in modulating various intermediates in the GPCR energy landscape. We propose that besides bound water, an influx of bulk water plays a necessary role in establishing the active GPCR conformation that mediates signaling.
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- PAR ID:
- 10346523
- Date Published:
- Journal Name:
- Proceedings of the National Academy of Sciences
- Volume:
- 119
- Issue:
- 21
- ISSN:
- 0027-8424
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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We summarize and critically review osmotic stress studies of the G-protein-coupled receptor rhodopsin. Although small amounts of structural water are present in these receptors, the effect of bulk water on their function remains uncertain. Studies of the influences of osmotic stress on the GPCR archetype rhodopsin have given insights into the functional role of water in receptor activation. Experimental work has discovered that osmolytes shift the metarhodopsin equilibrium after photoactivation, either to the active or inactive conformations according to their molar mass. At least 80 water molecules are found to enter rhodopsin in the transition to the photoreceptor active state. We infer that this movement of water is both necessary and sufficient for receptor activation. If the water influx is prevented, e.g., by large polymer osmolytes or by dehydration, then the receptor functional transition is back shifted. These findings imply a new paradigm in which rhodopsin becomes solvent swollen in the activation mechanism. Water thus acts as an allosteric modulator of function for rhodopsin-like receptors in lipid membranes.more » « less
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1073/pnas.2117349119
