Native Disk Membranes Research Articles

Extent of Internal Hydration influence the Activation of GPCR Rhodopsin

Rhodopsin is an archetypical GPCR responsible for dim‐light vision, which adopts a dynamic equilibrium between two metastable intermediates, metarhodopsin‐I (MI) and metarhodopsin‐II (MII), upon absorption of light. MI is an inactive conformational state of this GPCR, while MII is a fully active conformational state. We hypothesized that the MI to MII transition following isomerization of the retinal ligand involves a flood of bulk water into the protein core. To observe this change in the hydration level of the receptor during activation that is typically invisible to structural techniques such as X‐ray crystallography or cryo‐EM, we carried out a set of experiments using an osmotic stress approach. In this approach, the hydration level of rhodopsin was changed by placing the protein in native disk membranes in solutions with varying levels of osmotic pressure. Inert hydrophilic polyethylene glycol (PEG) solutes with different molecular weights and concentrations were used as the osmolytes. The effect of osmotic pressure on conformational dynamics was then quantified by measuring the reversible shifting of the metarhodopsin equilibrium using UV‐Visible spectroscopy. Results from these experiments show an influx of 80–100 water molecules into the rhodopsin interior, forming a partially disordered, solvent‐swollen active MII conformational state during photoactivation, a result supported by atomistic molecular dynamics simulations work [1, 2]. Additionally, we observed that the size of the polymer osmolyte played an important role in the modulation of rhodopsin’s activation: large osmolytes (PEG > 600) back shift the equilibrium to inactive MI state, while small osmolytes (PEG < 600) forward‐shift the equilibrium to MII state. We attribute this size dependence to the degree of osmolyte penetration into the rhodopsin core. Large polymers behave similarly to ideal osmolytes and dehydrate rhodopsin, while smaller polymers wriggle into the rhodopsin interior and stabilize the open MII conformation. These results necessitate a new understanding of GPCR activation in which the hydration level of protein is paramount in governing conformational energy landscapes. [1] U. Chawla et al. (2021) Angew. Chem. Int. Ed. 60, 2288–2295. [2] N. Leioatts et al. (2014) Biochemistry 53, 376−385.

Capturing a rhodopsin receptor signalling cascade across a native membrane

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.

Lipid-Dependent Ensemble of Conformational Substates in Rhodopsin Activation Studied by FTIR and UV-Visible Spectroscopy

G protein-coupled receptors (GPCRs) transmit signals across lipid membranes where rhodopsin is an important prototype. Here we address the role of soft matter in GPCR function for this class of integral membrane proteins [1,2]. Our hypothesis is the process involves an ensemble of activated states affected by membrane protein-lipid bilayer interactions [1]. Rhodopsin was investigated in lipid membranes (native disk membranes, POPC, DOPC) by Fourier transform infrared (FTIR) and UV-visible spectroscopy. Application of different spectroscopic methods together with pH titration at various temperatures allowed us to identify distributions of states in the MI-to-MII activating equilibrium and determine equilibrium constants and thermodynamic parameters of the transitions. Analysis of pH titration curves indicated that more than one activated state exists [3] supporting the concept of rhodopsin as a dynamically activated receptor. Furthermore, temperature changes had a crucial effect on rhodopsin activation enabling coexistence and simultaneous observation of multiple inactive and active states. Variation of the lipid composition indicated the POPC bilayer noticeably shifted the equilibrium to the inactive MI state [1] while the DOPC behavior was similar to native membranes. To improve data reduction, we analyzed the MI-to-MII equilibrium for the whole range of FTIR spectra where changes were observed between the MI and MII states. The results show the distribution of pKa and alkaline end-point values for all temperatures and different lipid compositions, indicating that activation of rhodopsin yields a conformational ensemble (collectively designated as MII state). We hypothesize that collective large-scale helical fluctuations in the metarhodopsin equilibrium are coupled to activation of transducin in visual signaling. [1] M.F. Brown (2017) Annu. Rev. Biophys. 46, 379-410. [2] U. Chawla et al. (2020) Angew. Chem. Int. doi.org/10.1002/anie.202003342. [3] M. Mahalingam et al. (2008) PNAS 105, 17795-17800.

Cryo-EM structure of the native rhodopsin dimer in nanodiscs

Imaging of rod photoreceptor outer-segment disc membranes by atomic force microscopy and cryo-electron tomography has revealed that the visual pigment rhodopsin, a prototypical class A G protein-coupled receptor (GPCR), can organize as rows of dimers. GPCR dimerization and oligomerization offer possibilities for allosteric regulation of GPCR activity, but the detailed structures and mechanism remain elusive. In this investigation, we made use of the high rhodopsin density in the native disc membranes and of a bifunctional cross-linker that preserves the native rhodopsin arrangement by covalently tethering rhodopsins via Lys residue side chains. We purified cross-linked rhodopsin dimers and reconstituted them into nanodiscs for cryo-EM analysis. We present cryo-EM structures of the cross-linked rhodopsin dimer as well as a rhodopsin dimer reconstituted into nanodiscs from purified monomers. We demonstrate the presence of a preferential 2-fold symmetrical dimerization interface mediated by transmembrane helix 1 and the cytoplasmic helix 8 of rhodopsin. We confirmed this dimer interface by double electron-electron resonance measurements of spin-labeled rhodopsin. We propose that this interface and the arrangement of two protomers is a prerequisite for the formation of the observed rows of dimers. We anticipate that the approach outlined here could be extended to other GPCRs or membrane receptors to better understand specific receptor dimerization mechanisms.

Exploring the Rhodopsin Dimer Interface in Live Cells

In low-light environments, the G protein-coupled receptor (GPCR), rhodopsin, is responsible for detection of light within the retina. Several studies have shown evidence for dimeric, or oligomeric, conformations of rhodopsin within native disc membranes. However, while many oligomeric packing structures have been proposed, the exact dimerization interface of rhodopsin has yet to be firmly establish. Here we investigate rhodopsin dimerization in live cells with fluorescence correlation spectroscopy (FCS) and pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). One of the proposed dimerization interfaces is between TM4/5 of rhodopsin. Recently we found evidence for a similar interface in red cone opsin. Based on those previous findings, we have made point mutations along rhodopsin's TM 5, and found significant disruption of the dimerization affinity compared to the wild-type construct. Our results provide new chemical details for the previously proposed packing structure between TM 4 and 5 of rhodopsin. Moreover, our findings further support the idea of conserved dimerization interface motifs shared among class A GPCRs. Such a discovery may provide more general conceptualization of self-association among one of the largest classes of transmembrane receptors, which is essential for properly gauging the potential relationship between dimerization and function.

Conformational equilibria of light-activated rhodopsin in nanodiscs

Conformational equilibria of G-protein-coupled receptors (GPCRs) are intimately involved in intracellular signaling. Here conformational substates of the GPCR rhodopsin are investigated in micelles of dodecyl maltoside (DDM) and in phospholipid nanodiscs by monitoring the spatial positions of transmembrane helices 6 and 7 at the cytoplasmic surface using site-directed spin labeling and double electron-electron resonance spectroscopy. The photoactivated receptor in DDM is dominated by one conformation with weak pH dependence. In nanodiscs, however, an ensemble of pH-dependent conformational substates is observed, even at pH 6.0 where the MIIbH+ form defined by proton uptake and optical spectroscopic methods is reported to be the sole species present in native disk membranes. In nanodiscs, the ensemble of substates in the photoactivated receptor spontaneously decays to that characteristic of the inactive state with a lifetime of ∼16 min at 20 °C. Importantly, transducin binding to the activated receptor selects a subset of the ensemble in which multiple substates are apparently retained. The results indicate that in a native-like lipid environment rhodopsin activation is not analogous to a simple binary switch between two defined conformations, but the activated receptor is in equilibrium between multiple conformers that in principle could recognize different binding partners.

Formation and Decay of the Arrestin·Rhodopsin Complex in Native Disc Membranes

In the G protein-coupled receptor rhodopsin, light-induced cis/trans isomerization of the retinal ligand triggers a series of distinct receptor states culminating in the active Metarhodopsin II (Meta II) state, which binds and activates the G protein transducin (Gt). Long before Meta II decays into the aporeceptor opsin and free all-trans-retinal, its signaling is quenched by receptor phosphorylation and binding of the protein arrestin-1, which blocks further access of Gt to Meta II. Although recent crystal structures of arrestin indicate how it might look in a precomplex with the phosphorylated receptor, the transition into the high affinity complex is not understood. Here we applied Fourier transform infrared spectroscopy to monitor the interaction of arrestin-1 and phosphorylated rhodopsin in native disc membranes. By isolating the unique infrared signature of arrestin binding, we directly observed the structural alterations in both reaction partners. In the high affinity complex, rhodopsin adopts a structure similar to Gt-bound Meta II. In arrestin, a modest loss of β-sheet structure indicates an increase in flexibility but is inconsistent with a large scale structural change. During Meta II decay, the arrestin-rhodopsin stoichiometry shifts from 1:1 to 1:2. Arrestin stabilizes half of the receptor population in a specific Meta II protein conformation, whereas the other half decays to inactive opsin. Altogether these results illustrate the distinct binding modes used by arrestin to interact with different functional forms of the receptor.

Structure-based biophysical analysis of the interaction of rhodopsin with G protein and arrestin.

In this chapter, we describe a set of complementary techniques that we use to study the activation of rhodopsin, a G protein-coupled receptor (GPCR), and its functional interactions with G protein and arrestin. The protein reagents used for these studies come from native disc membranes or heterologous expression, and G protein and arrestin are often replaced with less complex synthetic peptides derived from key interaction sites of these binding partners (BPs). We first report on our approach to protein X-ray crystallography and describe how protein crystals from native membranes are obtained. The crystal structures provide invaluable resolution, but other techniques are required to assess the dynamic equilibria characteristic for active GPCRs. The simplest approach is "Extra Meta II," which uses UV/Vis absorption spectroscopy to monitor the equilibrium of photoactivated states. Site-specific information about the BPs (e.g., arrestin) is added by fluorescence techniques employing mutants labeled with reporter groups. All functional changes in both the receptor and interacting proteins or peptides are seen with highest precision using Fourier transform infrared (FTIR) difference spectroscopy. In our approach, the lack of site-specific information in FTIR is overcome by parallel molecular dynamics simulations, which are employed to interpret the results and to extend the timescale down to the range of conformational substates.

High-resolution atomic force microscopy imaging of rhodopsin in rod outer segment disk membranes.

Atomic force microscopy (AFM) is a powerful imaging technique that allows recording topographical information of membrane proteins under near-physiological conditions. Remarkable results have been obtained on membrane proteins that were reconstituted into lipid bilayers. High-resolution AFM imaging of native disk membranes from vertebrate rod outer segments has unveiled the higher-order oligomeric state of the G protein-coupled receptor rhodopsin, which is highly expressed in disk membranes. Based on AFM imaging, it has been demonstrated that rhodopsin assembles in rows of dimers and paracrystals and that the rhodopsin dimer is the fundamental building block of higher-order structures.

Characterization of Human, Mouse, and Frog Rhodopsin Microdomains Within Native Disc Membranes

Rhodopsin is the light-activated receptor located in the disc membranes of rod photoreceptor cells in the retina and initiates vision via the phototransduction signaling cascade. There are divergent views on rhodopsin's quaternary organization within native disc membranes. The classical view posits that rhodopsin molecules function as freely diffusing monomers. However, recent evidence suggests that rhodopsin oligomerizes and forms higher order structures within the membrane. An accurate description of signaling events in phototransduction and of associated disease mechanisms is reliant on a comprehensive understanding of how rhodopsin is organized within native disc membranes. The aim of the current study was to determine and quantify the physiological arrangement of several vertebrate rhodopsins within their native disc membranes using atomic force microscopy (AFM). AFM is a microscopic method that allows for the imaging of membrane proteins in their native environment under physiological conditions. Disc membranes, with 90% of the total protein content comprised of rhodopsin, were isolated from human, mouse, and frog ocular tissue. AFM images of single-bilayer disc membranes revealed that these vertebrate disc membranes have similar topographies. Topographic features in these images indicate that rhodopsin is organized into microdomains and that the formation of these microdomains is not an effect of a low temperature environment. The microdomains formed by rhodopsin from each species were quantified and comparatively analyzed. By characterizing these microdomains, a baseline has been established for the organization of rhodopsin in human, mouse, and frog disc membranes. These characterizations of microdomain rhodopsin organization may serve as a guide in future investigations which will need to address the importance and role of such an organization on phototransduction and diseased states.

Constitutively active rhodopsin mutants causing night blindness are effectively phosphorylated by GRKs but differ in arrestin-1 binding

The effects of activating mutations associated with night blindness on the stoichiometry of rhodopsin interactions with G protein-coupled receptor kinase 1 (GRK1) and arrestin-1 have not been reported. Here we show that the monomeric form of WT rhodopsin and its constitutively active mutants M257Y, G90D, and T94I, reconstituted into HDL particles are effectively phosphorylated by GRK1, as well as two more ubiquitously expressed subtypes, GRK2 and GRK5. All versions of arrestin-1 tested (WT, pre-activated, and constitutively monomeric mutants) bind to monomeric rhodopsin and show the same selectivity for different functional forms of rhodopsin as in native disc membranes. Rhodopsin phosphorylation by GRK1 and GRK2 promotes arrestin-1 binding to a comparable extent, whereas similar phosphorylation by GRK5 is less effective, suggesting that not all phosphorylation sites on rhodopsin are equivalent in promoting arrestin-1 binding. The binding of WT arrestin-1 to phospho-opsin is comparable to the binding to its preferred target, P-Rh*, suggesting that in photoreceptors arrestin-1 only dissociates after opsin regeneration with 11-cis-retinal, which converts phospho-opsin into inactive phospho-rhodopsin that has lower affinity for arrestin-1. Reduced binding of arrestin-1 to the phospho-opsin form of G90D mutant likely contributes to night blindness caused by this mutation in humans.

Supramolecular Architecture of Rhodopsin in Native Photoreceptors Revealed by Cryo-Electron Tomography

Vision begins with the absorption of photons by rhodopsin, the visual pigment in photoreceptors. Rhodopsin belongs to the family of G protein-coupled receptors (GPCRs). Some family members form functional dimers or oligomers. Whether rhodopsin forms oligomers and whether these oligomers are functionally relevant is controversial 1-3. We study rhodopsin organization in vitreous ultrathin cryo-sections of intact, dark-adapted photoreceptors by cryo-electron tomography. This technique ascertains that rhodopsin is preserved in a close-to-native state. Briefly: retina is fixed by high-pressure freezing, ultra-thin sectioned and visualized by cryo-electron tomography. In the reconstructed and processed tomograms the organization of rhodopsin molecules becomes visible. We identify three levels of hierarchical supramolecular organization. Rhodopsin forms dimers; the dimers form rows; and rows come in special pairs like rail tracks. Rows are aligned parallel to the disk incisure. A row is comprised of at least 10 dimers and is about 50 nm in length. The distance between rows within a track is 5 nm; and tracks are separated by 15 nm. We propose that rhodopsin tracks provide a template that organizes the spatio-temporal interaction of preassembled signalling components on the disk surface. Aligned rows of immobile rhodopsin renders photoreceptors highly dichroic and might provide the structural basis for detection of polarized light. We envisage that rhodopsin-like type A GPCRs, which are highly homologous, also entertain a supramolecular organization.References1 Chabre, M., Cone, R. & Saibil, H. R. Biophysics - is rhodopsin dimeric in native rods? Nature 426, 30-31 (2003).2 Chabre, M. & le Maire, M. Monomeric G-protein-coupled receptor as a functional unit. Biochemistry 44, 9395-94O3 (2005).3 Fotiadis, D. et al. Atomic-force microscopy: rhodopsin dimers in native disc membranes. Nature 421, 127-128 (2003).

Membrane Bilayer Environment Influences Thermodynamics of Rhodopsin Membrane Protein-Lipid Interactions

The interaction between lipid bilayers and membrane proteins plays an important role in the functionality of GPCRs such as rhodopsin. During rhodopsin photoactivation, the photoreactive 11-cis retinylidene chromophore is isomerized to an all-trans state attaining an equilibrium between the MetaI and MetaII states. Lipid bilayer interaction with rhodopsin during its photoactivation process is explained using the flexible surface model (FSM). The FSM describes the elastic coupling of membrane lipids to integral membrane proteins through a balance of curvature and hydrophobic forces in lipid-protein interactions [1]. The FSM predicts a shift of the MetaI-MetaII equilibrium towards the activated MetaII state for unsaturated phosphatidylcholines with an increase in acyl chain length [1] or temperature. using UV-visible spectroscopy, we test the FSM by evaluating how the MetaI-MetaII equilibrium and thermodynamic parameters are influenced by a change in membrane environment. Absorption wavelength maxima at 485 nm and 380 nm correspond to the MetaI and MetaII states (inactive and active MetaII-H+), respectively. Accurate results are obtained by using the Rayleigh-Gans approximation to Mie scattering to compensate for light scattering in the UV-visible spectra. We determine effects of the type of head group, lipid to protein ratio, acyl chain length, and temperature on the MetaI-MetaII equilibrium by comparing pH titration curves for reconstituted rhodopsin in different lipid bilayers to those for native disk membranes [2,3]. The thermodynamic parameters are obtained for this equilibrium by fitting a modified phenomenological Henderson-Hasselbalch function [2] to the pH titration curves. These thermodynamic parameters illustrate how free energy drives the structural changes in rhodopsin upon photoactivation and can be used to model other GPCRs.[1] A.V. Botelho et al. (2006) BJ91, 4464-4477.[2] M. Mahalingam et al. (2008) PNAS105, 17795-17800.[3] E. Zaitseva et al. (2010) JACS132, 4815-4821.

Monomeric Rhodopsin Is Sufficient for Normal Rhodopsin Kinase (GRK1) Phosphorylation and Arrestin-1 Binding

G-protein-coupled receptor (GPCR) oligomerization has been observed in a wide variety of experimental contexts, but the functional significance of this phenomenon at different stages of the life cycle of class A GPCRs remains to be elucidated. Rhodopsin (Rh), a prototypical class A GPCR of visual transduction, is also capable of forming dimers and higher order oligomers. The recent demonstration that Rh monomer is sufficient to activate its cognate G protein, transducin, prompted us to test whether the same monomeric state is sufficient for rhodopsin phosphorylation and arrestin-1 binding. Here we show that monomeric active rhodopsin is phosphorylated by rhodopsin kinase (GRK1) as efficiently as rhodopsin in the native disc membrane. Monomeric phosphorylated light-activated Rh (P-Rh*) in nanodiscs binds arrestin-1 essentially as well as P-Rh* in native disc membranes. We also measured the affinity of arrestin-1 for P-Rh* in nanodiscs using a fluorescence-based assay and found that arrestin-1 interacts with monomeric P-Rh* with low nanomolar affinity and 1:1 stoichiometry, as previously determined in native disc membranes. Thus, similar to transducin activation, rhodopsin phosphorylation by GRK1 and high affinity arrestin-1 binding only requires a rhodopsin monomer.

Conservation of Molecular Interactions Stabilizing Bovine and Mouse Rhodopsin

Rhodopsin is the light receptor that initiates phototransduction in rod photoreceptor cells. The structure and function of rhodopsin are tightly linked to molecular interactions that stabilize and determine the receptor's functional state. Single-molecule force spectroscopy (SMFS) was used to localize and quantify molecular interactions that structurally stabilize bovine and mouse rhodopsin from native disk membranes of rod photoreceptor cells. The mechanical unfolding of bovine and mouse rhodopsin revealed nine major unfolding intermediates, each intermediate defining a structurally stable segment in the receptor. These stable structural segments had similar localization and occurrence in both bovine and mouse samples. For each structural segment, parameters describing their unfolding energy barrier were determined by dynamic SMFS. No major differences were observed between bovine and mouse rhodopsin, thereby implying that the structures of both rhodopsins are largely stabilized by similar molecular interactions.

Monomeric rhodopsin in nanodiscs is phosphorylated by rhodopsin kinase and binds arrestin with high affinity in the presence of negatively charged lipids

The role of dimerization of class A GPCRs is hotly debated. We show that monomeric rhodopsin in nanodiscs is phosphorylated by rhodopsin kinase (GRK1) and binds arrestin1 as well as in native disc membranes. The efficiency of rhodopsin phosphorylation is the same in phosphatidylcholine (PC) nanodiscs containing 0–50% phosphatidylserine (PS). In contrast, arrestin1 binding to P‐Rh* in pure PC is low. Binding increases with the proportion of PS and plateaus at ~10‐fold higher level at ~30% PS. To investigate the role of PS in the arrestin1‐P‐Rh* interaction, we used structurally different “enhanced” arrestin1 mutants that bind P‐Rh* and Rh* with comparable affinity. Truncated arrestin1(1–378) that has no C‐tail and 3A mutant where the interaction anchoring the C‐tail to the body of the molecule is destabilized were significantly less dependent on PS, demonstrating >50% of maximum binding in pure PC. In contrast, polar core mutants with the same phenotype, R175E and D296R, depend on PS almost as much as WT arrestin1. Thus, monomeric receptor is normally phosphorylated and binds arrestin. Negatively charged lipids promote interaction by “pushing” the arrestin C‐tail away from the receptor‐binding surface. This is the first indication of the function of multiple negative charges present in the C‐tail of all arrestin subtypes. NIH grants EY011500 (VVG), GM033775 (SGS), HL071818 (JJT), and DFG Sfb740 and ER 294/1‐1 (OPE)

Protonation Switches in GPCR Activation: Physiologically Significant Rhodopsin Photointermediates

Rhodopsin is a paradigm for GPCRs, yet unlike other class A members, its bound chromophore's UV/vis absorbance provides excellent time-resolved information about GPCR activation steps. At least three species equilibrate on the millisecond time scale after rhodopsin photoexcitation in a membrane lipid environment. The first equilibrium is pH-independent and involves Meta I480, the visible absorbing, protonated Schiff base (PSB) species, and Meta IIa, the UV absorbing, deprotonated SB species. The second equilibrium, involving spectrally silent proton uptake by Meta IIa to produce Meta IIb, accounts for the fact that low pH causes anomalous disappearance of the protonated SB species, Meta I480. The equilibria affect production of the G protein-activating species R∗ and are of great interest. However they must be studied promptly because inactivation steps follow, and long illumination increases secondary photolysis of photoproducts. We used time-resolved absorbance measurements of bovine rhodopsin on the microsecond-to-hundred millisecond time scale to study kinetics of lumirhodopsin decay and the effect of membrane environment on the first equilibrium constant, K1, and on the pKa of the second equilibrium. Reconstituted membranes of rhodopsin with POPC, DOPC, or a mixture of DOPC and DOPE were studied at 30°C. We also extended previous 20°C studies of the pH dependence of the equilibria in the native disk membranes, to determine how increased temperature affects lumirhodopsin decay through the purely transient 380 nm absorbing species, Meta I380, into the final equilibrium mixture. Meta I380 has recently attracted substantial interest, since time-resolved circular dichroism measurements on the microsecond timescale suggest the chromophore has a different conformation than in later 380-nm photointermediates. Our results suggest SB deprotonation precedes other activating changes in the protein. Significant details are now emerging that give new insights into rhodopsin activation and complement FTIR and spin-label approaches.

2SE1415 視細胞円板膜上でのロドプシンとトランスデューシンの1分子イメージング(2SE ロドプシンからの多様な展開を見る,第48回日本生物物理学会年会)

2SE1415 視細胞円板膜上でのロドプシンとトランスデューシンの1分子イメージング(2SE ロドプシンからの多様な展開を見る,第48回日本生物物理学会年会)

Calorimetric Studies of Bovine Rod Outer Segment Disk Membranes Support a Monomeric Unit for Both Rhodopsin and Opsin

The photoreceptor rhodopsin is a G-protein coupled receptor that has recently been proposed to exist as a dimer or higher order oligomer, in contrast to the previously described monomer, in retinal rod outer segment disk membranes. Rhodopsin exhibits considerably greater thermal stability than opsin (the bleached form of the receptor), which is reflected in an ∼15°C difference in the thermal denaturation temperatures (Tm) of rhodopsin and opsin as measured by differential scanning calorimetry. Here we use differential scanning calorimetry to investigate the effect of partial bleaching of disk membranes on the Tm of rhodopsin and of opsin in native disk membranes, as well as in cross-linked disk membranes in which rhodopsin dimers are known to be present. The Tms of rhodopsin and opsin are expected to be perturbed if mixed oligomers are present. The Tm remained constant for rhodopsin and opsin in native disks regardless of the level of bleaching. In contrast, the Tm of cross-linked rhodopsin in disk membranes was dependent on the extent of bleaching. The energy of activation for denaturation of rhodopsin and cross-linked rhodopsin was calculated. Cross-linking rhodopsin significantly decreased the energy of activation. We conclude that in native disk membranes, rhodopsin behaves predominantly as a monomer.

G Protein–Coupled Receptor Rhodopsin

The rhodopsin crystal structure provides a structural basis for understanding the function of this and other G protein-coupled receptors (GPCRs). The major structural motifs observed for rhodopsin are expected to carry over to other GPCRs, and the mechanism of transformation of the receptor from inactive to active forms is thus likely conserved. Moreover, the high expression level of rhodopsin in the retina, its specific localization in the internal disks of the photoreceptor structures [termed rod outer segments (ROS)], and the lack of other highly abundant membrane proteins allow rhodopsin to be examined in the native disk membranes by a number of methods. The results of these investigations provide evidence of the propensity of rhodopsin and, most likely, other GPCRs to dimerize, a property that may be pertinent to their function.