Sponge Model of G-Protein Binding and Unbinding in Membranes

Abstract

Rhodopsin, a prototypical G-protein coupled receptor (GPCR), functions as a sensitive photon detector in retinal rod cells. Signal transduction by rhodopsin is achieved through the binding and release of the transducin G-protein. Despite the numerous recent structures of complexes with effector proteins, the mechanism underlying G-protein release from an activated GPCR remains elusive [1]. Here we propose that rhodopsin hydration/dehydration cycling facilitates transducin binding and release. The dissociation constant (K d ) between photoactivated rhodopsin and the high-affinity C-terminal analog of transducin (G alpha CT) was measured in response to osmotic stress. The active metarhodopsin-II (MII) fraction of rhodopsin was obtained using UV-visible spectrophotometry, and osmotic stress was generated using various molecular weight polyethylene glycol (PEG) polymers. Preliminary data indicate that large molecular weight polymers, such as PEG1500, are unable to enter the protein interior, and dehydrate the rhodopsin. The opposite effect is observed for small molecular weight polymers, which enter the protein core and have a hydrating effect. Large molecular weight polymers reduce the binding affinity for G alpha CT to rhodopsin, while small molecular weight polymers increase this binding affinity. Our results indicate that rhodopsin hydration is necessary for transducin binding. We therefore propose the sponge model for GPCR signaling, in which an influx of water during rhodopsin activation is necessary to stabilize the active MII conformation and to facilitate transducin binding. Formation of the high-affinity rhodopsintranducin complex displaces water from the protein interior, destabilizing the receptor. Nucleotide exchange further dehydrates the receptor, causing it to return to the inactive MI conformation, thus releasing transducin in the process. This wet/dry cycle can repeat many times for one photoexcitation event, explaining how many molecules of transducin can be activated by one molecule of rhodopsin. [1] U. Chawla, et al. (2016) Angew. Chem. Int. Ed. 55, 588-592.