Rhodopsin Oligomerization Research Articles

Physiological changes in bilayer thickness induced by cholesterol control GPCR rhodopsin function

We monitored the effect on function of the G-protein-coupled receptor (GPCR) rhodopsin from small, stepwise changes in bilayer thickness induced by cholesterol. Over a range of phosphatidylcholine bilayers with hydrophobic thickness from ≈21 Å to 38 Å, the metarhodopsin-I (MI)/metarhodopsin-II (MII) equilibrium was monitored with UV-visible spectroscopy while ordering of hydrocarbon chains was probed by 2H-NMR. Addition of cholesterol shifted equilibrium toward MII for bilayers thinner than the average length of hydrophobic transmembrane helices (27 Å) and to MI for thicker bilayers, while small bilayer thickness changes within the range of the protein hydrophobic thickness drastically up- or downregulated MII formation. The cholesterol-induced shifts toward MII for thinner membranes correlated with the cholesterol-induced increase of bilayer hydrophobic thickness measured by NMR, consistent with continuum elastic modeling. The energetic penalty of adding cholesterol to thick bilayers caused rhodopsin oligomerization and a shift toward MI. In membranes of physiological thickness, changes in bilayer mechanical properties induced by cholesterol potentiated the interplay between bilayer and protein thickness resulting in large swings of the MI-MII equilibrium. In membrane containing cholesterol, elastic deformations near the protein are a dominant energetic contribution to the functional equilibrium of the model GPCR rhodopsin.

Identification of small-molecule allosteric modulators that act as enhancers/disrupters of rhodopsin oligomerization

The elongated cilia of the outer segment of rod and cone photoreceptor cells can contain concentrations of visual pigments of up to 5 mM. The rod visual pigments, G protein–coupled receptors called rhodopsins, have a propensity to self-aggregate, a property conserved among many G protein–coupled receptors. However, the effect of rhodopsin oligomerization on G protein signaling in native cells is less clear. Here, we address this gap in knowledge by studying rod phototransduction. As the rod outer segment is known to adjust its size proportionally to overexpression or reduction of rhodopsin expression, genetic perturbation of rhodopsin cannot be used to resolve this question. Therefore, we turned to high-throughput screening of a diverse library of 50,000 small molecules and used a novel assay for the detection of rhodopsin dimerization. This screen identified nine small molecules that either disrupted or enhanced rhodopsin dimer contacts in vitro. In a subsequent cell-free binding study, we found that all nine compounds decreased intrinsic fluorescence without affecting the overall UV-visible spectrum of rhodopsin, supporting their actions as allosteric modulators. Furthermore, ex vivo electrophysiological recordings revealed that a disruptive, hit compound #7 significantly slowed down the light response kinetics of intact rods, whereas compound #1, an enhancing hit candidate, did not substantially affect the photoresponse kinetics but did cause a significant reduction in light sensitivity. This study provides a monitoring tool for future investigation of the rhodopsin signaling cascade and reports the discovery of new allosteric modulators of rhodopsin dimerization that can also alter rod photoreceptor physiology.

Rhodopsin Oligomerization in Synthetic Lipid Bilayers and Native Cellular Membranes as Studied by DEER of a Spin-labeled Retinal Analog

Oligomerization of integral membrane proteins is often the final step of protein folding. This final step is likely to be affected by a number of factors including biophysical interactions with lipids and other proteins and so is the oligomers’ stability and their further organization into clusters/superlattices. Thus, the protein oligomers observed in different membrane mimetics including synthetic liposomes could be different from those formed in native membranes. Here we employed DEER spectroscopy to measure a distribution of long range (ca., 2-7 nm) constraints between specific protein sidechains labeled with paramagnetic tags. Spin-labeled derivative of retinal (4-[2,2,5,5-tetramethyl-1- pyrrolidinyloxy-3-carboxyl]-retinal) was synthesized and added to apo-rhodospsin for a tight non-covalent binding similar to the native retinal. Such an approach eliminated the need for covalent modification of proteins with conventional nitroxides. Further, the retinal binding is a useful probe for oligomerization of functional rhodopsins as the protein is known to lose the retinal upon denaturing. Thus, non-functional rhodopsins would be EPR-silent. Experimental DEER signals from such spin-labeled rhodopsin revealed significant differences in protein oligomerization in synthetic vs. native membranes. Specifically, while oligomers with well-defined distances were found to form in DMPC-DMPA, some shorter distances were determined to be present in the native cellular membranes. The latter distances were attributed to smaller oligomers (dimers, trimers, etc.) that could be intermediates to larger oligomers or incomplete oligomers which further oligomerization was terminated by the membrane defects and/or other protein components. The work at NCSU was supported by U.S. DOE Contract DE-FG02-02ER15354.

Rhodopsin Oligomerization and Aggregation.

Rhodopsin is the light receptor in photoreceptor cells of the retina and a prototypical G protein-coupled receptor. Two types of quaternary structures can be adopted by rhodopsin. If rhodopsin folds and attains a proper tertiary structure, it can then form oligomers and nanodomains within the photoreceptor cell membrane. In contrast, if rhodopsin misfolds, it cannot progress through the biosynthetic pathway and instead will form aggregates that can cause retinal degenerative disease. In this review, emerging views are highlighted on the supramolecular organization of rhodopsin within the membrane of photoreceptor cells and the aggregation of rhodopsin that can lead to retinal degeneration.

Investigation of Photoinduced Oligomerization of Rhodopsin by Native Mass Spectrometry

Investigation of Photoinduced Oligomerization of Rhodopsin by Native Mass Spectrometry

Rhodopsin/Lipid Hydrophobic Matching—Rhodopsin Oligomerization and Function

Lipid composition of the membrane and rhodopsin packing density strongly modulate the early steps of the visual response of photoreceptor membranes. In this study, lipid-order and bovine rhodopsin function in proteoliposomes composed of the sn-1 chain perdeuterated lipids 14:0d27-14:1-PC, 16:0d31-16:1-PC, 18:0d35-18:1-PC, or 20:0d39-20:1-PC at rhodopsin/lipid molar ratios from 1:70 to 1:1000 (mol/mol) were investigated. Clear evidence for matching of hydrophobic regions on rhodopsin transmembrane helices and hydrophobic thickness of lipid bilayers was observed from 2H nuclear magnetic resonance order parameter measurements at low rhodopsin concentrations. Thin bilayers stretched to match the length of transmembrane helices observed as increase of sn-1 chain order, while thicker bilayers were compressed near the protein. A quantitative analysis of lipid-order parameter changes suggested that the protein adjusts its conformation to bilayer hydrophobic thickness as well, which confirmed our earlier circular-dichroism measurements. Changes in lipid order parameters upon rhodopsin incorporation vanished for bilayers with a hydrophobic thickness of 27 ± 1 Å, suggesting that this is the bilayer thickness at which rhodopsin packs in bilayers at the lowest membrane perturbation. The lipid-order parameter studies also indicated that a hydrophobic mismatch between rhodopsin and lipids triggers rhodopsin oligomerization with increasing rhodopsin concentrations. Both hydrophobic mismatch and rhodopsin oligomerization result in substantial shifts of the equilibrium between the photointermediates metarhodopsin I and metarhodopsin II; increasing bilayer thickness favors formation of metarhodopsin II while oligomerization favors metarhodopsin I. The results highlight the importance of hydrophobic matching for rhodopsin structure, oligomerization, and function.

Rhodopsin Crowding in Model Lipid Bilayers - Functional Implications

In the rod outer segment disks of the retina, rhodopsin is densely packed in phospholipid bilayers with a high content of polyunsaturated acyl chains. It has been hotly debated if oligomerizationof rhodopsin is a critical step for efficient activation of G-protein. Here, we investigated the effect of rhodopsin density in synthetic membranes on the interaction with its cognate G-protein transducin (Gt). Experiments were conducted at rhodopsin:lipid ratios ranging from 1:4000 to 1:70 with sn1-stearoyl sn2-oleoyl phosphatidylcholine (18:0-18:1 PC) model bilayers at ambient temperature.The amount of metarhodopsin-II (MII) formed after photoactivation was determined by UV-visible spectroscopy. Guanine nucleotide exchange rate measured using labeled GTPγS was used to monitor Gt affinity for activated rhodopsin, the rate of rhodopsin catalyzed Gt activation, and the decay rate of the active photointermediate.At low rhodopsin density (1:1000 and below), MII concentration was the highest and independent of rhodopsin concentration. Increasing rhodopsin packing density correlated with a shift in the metarhodopsin-I (MI)/MII equilibrium towards MI. After photoactivation, rhodopsin decayed at two different rates (t1/2 ∼ 3 min and > 60 min) and the proportion of the fast decaying photointermediate decreased with increasing rhodopsin density. Finally, MII-catalyzed GDP-GTPγS nucleotide exchange rates for Gt were crucially affected by rhodopsin density. The enzymatic power of rhodopsin (Vmax/Km for Gt) was higher by 2 orders of magnitude at low rhodopsin density as compared to high rhodopsin density.Our results suggest that, in model membranes, rhodopsin exists as an equilibrium between at least two populations: monomeric at low rhodopsin density with rapid decay and high catalytic efficiency and oligomeric at high rhodopsin density with slow decay and inefficient catalysis of nucleotide exchange.

Membrane Curvature Elastic Stress Strongly Modulates Metarhodopsin II Formation

We studied the influence of curvature elastic stress in lipid monolayers induced by hydrophobic mismatch between lipid bilayer thickness and the hydrophobic length of rhodopsin transmembrane helices on the metarhodopsin I (MI)/metarhodopsin II (MII) equilibrium. Experiments were conducted at the low rhodopsin/lipid ratio of 1/1,000 to suppress rhodopsin oligomerization. Elastic stress was generated by reconstituting dark-adapted rhodopsin into a series of phosphatidylcholine (PC) bilayers with acyl chains of 14-20 carbons in length and cholesterol content varied from 0 to 30 mol %. 2H NMR was used to monitor the adjustment of the length of hydrocarbon chains to the protein and to characterize monolayer curvature at the protein-lipid interface. We found that the average length of hydrophobic regions on rhodopsin transmembrane helices is 2.6 ± 0.1 nm. Thinner bilayers stretch and bend with negative monolayer curvature to match the length of hydrophobic helices, while thicker bilayers get thinner with positive monolayer curvature near the protein. Shifts in the MI/MII equilibrium depended on the sign of monolayer curvature: negative curvature favored MI while positive curvature favored MII. The results suggest that MII formation generates negative curvature in monolayers near the protein, thus raising curvature stress for thin bilayers but releasing stress for thicker bilayers. Addition of cholesterol increases bilayer hydrophobic thickness, but otherwise showed identical behavior: bilayers with a hydrophobic thickness less than 2.6 nm favored MI while thicker bilayers favored MII. In case of severe hydrophobic mismatch between rhodopsin and bilayers, the behavior was complicated by rhodopsin oligomerization that seems to favor MI. The sensitivity of the MI/MII equilibrium to negative curvature elastic stress was observed earlier in experiments with phosphatidylethanolamines by us and the Brown laboratory. Negative curvature stress upon MII formation is critical for understanding shifts in the MI/MII equilibrium.

Digitonin and sodium dodecylsulfate-solubilized frog rho-dopsin: Behavior under native and denaturing polyacrylamide gel electrophoresis

Rhodopsin oligomerization and dissociation in vivo and under experimental conditions is an important topic both for a basic understanding of photoreceptor structure-function but also as a potential eye disease mechanism. In this study, to estimate a state rhodopsin after solubilization with mild and harsh detergents, we applied the native (blue native-PAGE, BN- PAGE) and denaturing electrophoresis (blue-urea- PAGE, BU-PAGE; blue-SDS-PAGE, BSDS-PAGE and SDS- PAGE). After blue BN-PAGE and BSDS- PAGE, rhodopsin and opsin, respectively, were presented in gels a major band of dimer with slight contents of higher oligomers without any traces of monomer, thus testifying in favor dimer-heteromeric state of frog rhodopsin in the photoreceptor membrane. Despite all oligomer bands gave positive staining with the rhodopsin-specific monoclonal antibodies (mAb), subsequent SDS-PAGE in combination with electroelution in denaturing conditions showed that stained bands are not homogenous and besides of opsin oli-gomers contain a small admixture of proteins with unknown function. Unfolding of opsin oligomers by solubilization in SDS, as compared with folded opsin in digitonin, induces their transition to a more compact conformation. It was manifested in a more rapid migration of opsin oligomers toward to anode. Cooling of digitonin/SDS mixed extracts at 4?C for 24 hours led to a partial reverse transition of unfolded opsin dimer to initial folded conformation, thus de- monstrating the entropic nature of this transition. Opsin monomer can be observed in the gels only after harsh dissociation of oligomers under BU-PAGE or SDS-PAGE. The electro elution of the individual opsin oligomers with denaturing buffer followed by SDS-PAGE resulted in dissociation of dimer to monomers. However, unexpectedly, the trimer was dissociated to a prevailing dimer and a small portion of monomer. The products dissociation of both opsin tetramer and pentamer are difficult to determine precisely, but they are neither monomer nor dimer. Dissociation data show that the degree of opsin oli- gomerization by unknown reasons affects the pattern of dissociation of its aggregates. Obtained in this paper data indicate a need for further detailed study the obscure mechanisms of aggregation-dissociation of rhodopsin.

Unknown Mechanisms Regulating the GPCR Signal Cascade in Vertebrate Photoreceptors

The process of phototransduction – the conversion of a light signal into an electrical response occurring in vertebrate photoreceptors – is the best studied and characterized example of a GPCR signal cascade. The literature contains the view that the basic mechanisms of phototransduction are already known and that data obtained from photoreceptors can be applied to other GPCR signaling systems. In this review, we compare the contemporary model of phototransduction with data from studies of other GPCR signal cascades with the aim of identifying similarities and differences between them. Working mainly from physiological and biophysical data, we show that the current scheme of phototransduction lacks important regulatory mechanisms, whose biochemical substrates remain unknown. This provides, for example, rapid control of the lifetime of the active state of G-protein (transducin) during light adaptation. There are also slow (several minutes) processes modifying components of the signal cascade which regulate the rate of deactivation of activated receptor (rhodopsin) and transducin. By analogy with other GPCR signal cascades, the existence of several other signaling pathways from light-activated rhodopsin can be suggested; these may use different second messengers (not only cGMP but also, for example, cAMP). We also show that rhodopsin oligomerization may occur in photoreceptors and that this process may provide physiological control of amplification in the signal cascade. The missing mechanisms are by no means unimportant and may support regulation of photoreceptor sensitivity over a range from one to two orders of magnitude.

Rhodopsin - Rhodopsin Oligomerization in Model Lipid Bilayers - Functional Implications

We studied rhodopsin oligomerization as a function of rhodopsin concentration and lipid composition and related oligomerization to shifts in rhodopsin function. In the rod outer segment disks of the retina, rhodopsin is densely packed in phospholipid bilayers with a high content of polyunsaturated acyl chains. In model membranes, increasing rhodopsin packing density was linked to a shift in the metarhodopsin-I (MI)/metarhodopsin-II (MII) equilibrium towards MI as well as to lower rates of MII formation. We reconstituted rhodopsin into various phosphatidylcholine bilayers at rhodopsin/lipid ratios ranging from 1:1,000 to 1:70 and followed rhodopsin oligomerization by cross linking of rhodopsin and changes in lipid-rhodopsin interactions by 2H NMR. The amount of MII formed after photoactivation was determined by UV/vis spectroscopy and the rate of transducin activation studied with a GTPγS-assay. At low rhodopsin concentrations (1/300 and lower) rhodopsin appears to be predominantly monomeric. At rhodopsin/lipid ratios higher than 1/300, the level of oligomerization increases in a highly cooperative fashion with concentration such that at physiological concentrations rhodopsin is mostly oligomeric. Protein function correlated tightly with rhodopsin oligomerization. Data on the influence of bilayer properties on the monomer - oligomer transition of rhodopsin and the rate of transducin activation will be presented.

Damage of photoreceptor membrane lipids and proteins induced by photosensitized generation of singlet oxygen.

It has been shown that illumination of rod outer segment suspension in the presence of photosensitizers (methylene blue lambda greater than or equal to 620 nm; retinal 370 less than or equal to lambda less than or equal to 390 nm) results in chemical modification of the lipid and protein components of the photo-receptor membranes. This modification can be registered by accumulation of lipid peroxidation (LPO) products as well as oligomerization of rhodopsin and a decrease of rhodopsin thermal stability. These effects are prevented by 'O2-quenchers and free radical scavengers. It has been found that the electric activity (ERG) of isolated frog retina is inhibited due to photosensitized generation of 'O2 which can be overcome by preliminary addition of 'O2-quenchers and free radical scavengers to the incubation medium. The LPO products are accumulated in the retinae of rats exposed to high intensity light in vivo. It is concluded that 'O2 and LPO are involved in light-induced damage of the retina.