Decay of an active GPCR: Conformational dynamics govern agonist rebinding and persistence of an active, yet empty, receptor state

Significance G protein-coupled receptors (GPCRs) represent a major pharmaceutical drug target. However, one exception has been the visual photoreceptor rhodopsin, long considered “different” due to its covalently bound, light-sensitive retinal ligands. Here we demonstrate that, in contrast to prior assumptions, release of the agonist all-trans retinal (ATR) is not an irreversible process. Instead, during decay of the active species, ATR can rebind any rhodopsin remaining in an active-like conformation, and this active-like state can transiently persist even after agonist dissociation. These insights demonstrate rhodopsin behaves like other diffusible ligand-binding GPCRs and raise the possibility of treating rhodopsin by pharmaceutical agents. Here, we describe two insights into the role of receptor conformational dynamics during agonist release (all-trans retinal, ATR) from the visual G protein-coupled receptor (GPCR) rhodopsin. First, we show that, after light activation, ATR can continually release and rebind to any receptor remaining in an active-like conformation. As with other GPCRs, we observe that this equilibrium can be shifted by either promoting the active-like population or increasing the agonist concentration. Second, we find that during decay of the signaling state an active-like, yet empty, receptor conformation can transiently persist after retinal release, before the receptor ultimately collapses into an inactive conformation. The latter conclusion is based on time-resolved, site-directed fluorescence labeling experiments that show a small, but reproducible, lag between the retinal leaving the protein and return of transmembrane helix 6 (TM6) to the inactive conformation, as determined from tryptophan-induced quenching studies. Accelerating Schiff base hydrolysis and subsequent ATR dissociation, either by addition of hydroxylamine or introduction of mutations, further increased the time lag between ATR release and TM6 movement. These observations show that rhodopsin can bind its agonist in equilibrium like a traditional GPCR, provide evidence that an active GPCR conformation can persist even after agonist release, and raise the possibility of targeting this key photoreceptor protein by traditional pharmaceutical-based treatments.