Capturing a rhodopsin receptor signalling cascade across a native membrane

Abstract

G protein-coupled receptors (GPCRs) are cell-surface receptors that respond to various stimuli to induce signalling pathways across cell membranes. Recent progress has yielded atomic structures of key intermediates1,2 and roles for lipids in signalling3,4. However, capturing signalling events of a wild-type receptor in real time, across a native membrane to its downstream effectors, has remained elusive. Here we probe the archetypal class A GPCR, rhodopsin, directly from fragments of native disc membranes using mass spectrometry. We monitor real-time photoconversion of dark-adapted rhodopsin to opsin, delineating retinal isomerization and hydrolysis steps, and further showing that the reaction is significantly slower in its native membrane than in detergent micelles. Considering the lipids ejected with rhodopsin, we demonstrate that opsin can be regenerated in membranes through photoisomerized retinal–lipid conjugates, and we provide evidence for increased association of rhodopsin with unsaturated long-chain phosphatidylcholine during signalling. Capturing the secondary steps of the signalling cascade, we monitor light activation of transducin (Gt) through loss of GDP to generate an intermediate apo-trimeric G protein, and observe Gαt•GTP subunits interacting with PDE6 to hydrolyse cyclic GMP. We also show how rhodopsin-targeting compounds either stimulate or dampen signalling through rhodopsin–opsin and transducin signalling pathways. Our results not only reveal the effect of native lipids on rhodopsin signalling and regeneration but also enable us to propose a paradigm for GPCR drug discovery in native membrane environments.

Convergent evolution of animal and microbial rhodopsins

“…Furthermore, to directly detect the molecular components of the cascade in real-time and within native rod photoreceptor environment, mass spectroscopy has been established. 65 …”

“…Furthermore, mass spectroscopy technique can be a powerful tool to directly detect the molecular components of the cascade in real-time and within native archaeal and bacterial cells like the previous study using native rod photoreceptor cells. 65 In contrast to animal rhodopsins, physiological roles and signal transduction cascades of most microbial rhodopsins (e.g., outward sodium pumps and inward proton pumps) in host organisms are unexplored. The detailed physiological and transcriptome analysis of transgenic animals has revealed the physiological roles and signal transduction cascades of animal rhodopsins.…”

“…Furthermore, to directly detect the molecular components of the cascade in real-time and within native rod photoreceptor environment, mass spectroscopy has been established. 65 In contrast to the vertebrate visual signal transduction cascade, arthropod and mollusc visual rhodopsins activate Gq by GDP-GTP exchange reaction in rhabdomeric photoreceptor cells. 61 The a subunit of the activated Gq binds to and activates phospholipase C (PLC) that hydrolyzes phosphatidylinositol 4,5-bisphosphatase (PIP 2 ) to 1,4,5-triphosphate (InsP 3 ) and diacylglycerol (DAG).…”

Mass spectrometry of intact membrane proteins: shifting towards a more native-like context

“…Further method developments, such as supercharging or new activation methods, can further improve the analysis of SMALP-solubilised membrane proteins. An exciting detergent-free method has recently emerged that involves the ejection of lipid vesicles derived directly from native membranes [ 54 , 55 ]; this is discussed in more detail in a later section.…”

Kinetics of cone specific G-protein signaling in avian photoreceptor cells

“…Vertebrate phototransduction is a sensory signaling pathway providing the link between a physical stimulus (photon) and a change in membrane potential. The molecular reactions of the biochemical cascade involving the prototypical G protein-coupled receptors (GPCR) rhodopsin or cone opsin, a heterotrimeric G protein (transducin, G t ), a cGMP-specific phosphodiesterase (PDE), and a cyclic nucleotide-gated cation channel (CNG-channel) that is directly controlled by the intracellular cGMP concentration, are understood in quantitative terms ( Pugh and Lamb, 2000 ; Chen et al, 2022 ; Hofmann and Lamb, 2022 ). Similar holds true for the deactivation steps of each of the biochemical reactions in the photoexcitation process.…”