Search

Opsins are G protein-coupled receptors (GPCRs) that have evolved to detect light stimuli and initiate intracellular signaling cascades. Their role as signal transducers is critical to light perception across the animal kingdom. Opsins covalently bind to the chromophore 11-cis retinal, which isomerizes to the all-trans isomer upon photon absorption, causing conformational changes that result in receptor activation. Monostable opsins, responsible for vision in vertebrates, release the chromophore after activation and must bind another retinal molecule to remain functional. In contrast, bistable opsins, responsible for non-visual light perception in vertebrates and for vision in invertebrates, absorb a second photon in the active state to return the chromophore and protein to the inactive state. Structures of bistable opsins in the activated state have proven elusive, limiting our understanding of how they function as bidirectional photoswitches. Here we present active state structures of a bistable opsin, jumping spider rhodopsin isoform-1 (JSR1), in complex with its downstream signaling partners, the G i and G q heterotrimers. These structures elucidate key differences in the activation mechanisms between monostable and bistable opsins, offering essential insights for the rational engineering of bistable opsins into diverse optogenetic tools to control G protein signaling pathways., (© 2024. The Author(s).)

β-arrestins mediate regulatory processes for over 800 different G protein-coupled receptors (GPCRs) by adopting specific conformations that result from the geometry of the GPCR-β-arrestin complex. However, whether β-arrestin1 and 2 respond differently for binding to the same GPCR is still unknown. Employing GRK knockout cells and β-arrestins lacking the finger-loop-region, we show that the two isoforms prefer to associate with the active parathyroid hormone 1 receptor (PTH1R) in different complex configurations ("hanging" and "core"). Furthermore, the utilisation of advanced NanoLuc/FlAsH-based biosensors reveals distinct conformational signatures of β-arrestin1 and 2 when bound to active PTH1R (P-R*). Moreover, we assess β-arrestin conformational changes that are induced specifically by proximal and distal C-terminal phosphorylation and in the absence of GPCR kinases (GRKs) (R*). Here, we show differences between conformational changes that are induced by P-R* or R* receptor states and further disclose the impact of site-specific GPCR phosphorylation on arrestin-coupling and function., (© 2022. The Author(s).)

Cellular functions of arrestins are determined in part by the pattern of phosphorylation on the G protein-coupled receptors (GPCRs) to which arrestins bind. Despite high-resolution structural data of arrestins bound to phosphorylated receptor C-termini, the functional role of each phosphorylation site remains obscure. Here, we employ a library of synthetic phosphopeptide analogues of the GPCR rhodopsin C-terminus and determine the ability of these peptides to bind and activate arrestins using a variety of biochemical and biophysical methods. We further characterize how these peptides modulate the conformation of arrestin-1 by nuclear magnetic resonance (NMR). Our results indicate different functional classes of phosphorylation sites: 'key sites' required for arrestin binding and activation, an 'inhibitory site' that abrogates arrestin binding, and 'modulator sites' that influence the global conformation of arrestin. These functional motifs allow a better understanding of how different GPCR phosphorylation patterns might control how arrestin functions in the cell.