Effect of detergents and lipids on transducin photoactivation by rhodopsin.

Rhodopsin samples, isolated using four different extraction procedures, were used to investigate the photodependent activation of the GTPase activity of transducin. A complete inhibition of transducin light-dependent GTP hydrolytic activity was observed when rhodopsin purified in the presence of 1% digitonin, following rod outer segment (ROS) solubilization with 1% 3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate (CHAPS), was used for its activation [0 pmol of inorganic phosphate (Pi) released/min/pmol of rhodopsin]. Rhodopsin, isolated in the presence of 1% digitonin following ROS solubilization with 1% digitonin, was capable of stimulating slightly transducin GTPase activity, with an initial rate of 1 pmol of GTP hydrolyzed/min/pmol of rhodopsin. However, rhodopsin purified in the presence of 0.2% n-dodecyl-beta-D-maltoside (DM), following ROS solubilization with either 1% CHAPS or 1% DM, stimulated the enzymatic activity of transducin in a light-dependent manner, with an initial rate of 5 pmol of Pi released/min/pmol of rhodopsin. Addition of 0.075% egg phosphatidylcholine (PC) to the four different solubilized rhodopsin samples significantly enhanced light-stimulated GTP hydrolysis by transducin, with initial rates increasing from 0 to 1, 1 to 2, and 5 to 30 pmol of Pi released/min/pmol of rhodopsin, respectively. Furthermore, DM-solubilized rhodopsin induced the hydrolysis of the maximum amount of GTP by transducin at 0.0075% PC, while digitonin-solubilized rhodopsin only stimulated the GTPase activity of transducin to a similar value, when the amount of the photoreceptor protein was increased 4-fold and 0.15% PC was added to the assay mixture. These results suggest that the effective photoactivation of transducin by rhodopsin requires phospholipids, which seem to be differentially eliminated with the detergent extraction procedure utilized during ROS membranes solubilization and photopigment isolation.     

Rhodopsin controls a conformational switch on the transducin gamma subunit

Rhodopsin, a prototypical G protein-coupled receptor, catalyzes the activation of a heterotrimeric G protein, transducin, to initiate a visual signaling cascade in photoreceptor cells. The betagamma subunit complex, especially the C-terminal domain of the transducin gamma subunit, Gtgamma(60-71)farnesyl, plays a pivotal role in allosteric regulation of nucleotide exchange on the transducin alpha subunit by light-activated rhodopsin. We report that this domain is unstructured in the presence of an inactive receptor but forms an amphipathic helix upon rhodopsin activation. A K65E/E66K charge reversal mutant of the gamma subunit has diminished interactions with the receptor and fails to adopt the helical conformation. The identification of this conformational switch provides a mechanism for active GPCR utilization of the betagamma complex in signal transfer to G proteins.     

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.     

Rhodopsin Forms Nanodomains in Rod Outer Segment Disc Membranes of the Cold-Blooded Xenopus laevis

Rhodopsin forms nanoscale domains (i.e., nanodomains) in rod outer segment disc membranes from mammalian species. It is unclear whether rhodopsin arranges in a similar manner in amphibian species, which are often used as a model system to investigate the function of rhodopsin and the structure of photoreceptor cells. Moreover, since samples are routinely prepared at low temperatures, it is unclear whether lipid phase separation effects in the membrane promote the observed nanodomain organization of rhodopsin from mammalian species. Rod outer segment disc membranes prepared from the cold-blooded frog Xenopus laevis were investigated by atomic force microscopy to visualize the organization of rhodopsin in the absence of lipid phase separation effects. Atomic force microscopy revealed that rhodopsin nanodomains form similarly as that observed previously in mammalian membranes. Formation of nanodomains in ROS disc membranes is independent of lipid phase separation and conserved among vertebrates.     

Electrostatic Compensation Restores Trafficking of the Autosomal Recessive Retinitis Pigmentosa E150K Opsin Mutant to the Plasma Membrane

Rhodopsin is the rod photoreceptor G protein-coupled receptor responsible for capturing light. Mutations in the gene encoding this protein can lead to a blinding disease called retinitis pigmentosa, which is inherited frequently in an autosomal dominant manner. The E150K opsin mutant associated with rarely occurring autosomal recessive retinitis pigmentosa localizes to trans-Golgi network membranes rather than to plasma membranes of rod photoreceptor cells. We investigated the molecular mechanisms underlying opsin retention in the Golgi apparatus. Electrostatic calculations reveal that the E150K mutant features an overall accumulation of positive charges between helices H-IV and H-II. Human E150K and several other closely related opsin mutants were then expressed in HEK-293 cells. Spectral characteristics and functional biochemistry of each mutant were analyzed after reconstitution with the cis-retinoid chromophore. UV-visible spectra and rhodopsin/transducin activation assays revealed only minor differences between the purified wild type control and rhodopsin mutants. However, partial restoration of the surface electrostatic charge in the compensatory R69E/E150K double mutant rescues the plasma membrane localization of opsin. These findings emphasize the fundamental importance of electrostatic interactions for appropriate membrane trafficking of opsin and advance our understanding of the pathophysiology of autosomal recessive retinitis pigmentosa due to the E150K mutation.     

Rhodopsin and phototransduction: a model system for G protein-linked receptors.

Rhodopsin is the photoreceptor protein in rod cells of the vertebrate retina and the first member of the class of G protein-coupled receptors for which the amino acid sequence was determined. Rhodopsin is available in greater quantities than any other receptor of its class and therefore has been studied biochemically and biophysically by methods difficult or impossible to apply to its fellow receptors. Such studies support a model in which rhodopsin consists of seven transmembrane helices that form a binding pocket for its ligand, 11-cis retinal. Insights into the structure and function of rhodopsin serve as a model for understanding the structure and function of other members of the receptor class. Rhodopsin undergoes a change in conformation upon photoexcitation and activates a G protein, transducin, and is phosphorylated by a receptor-specific kinase, rhodopsin kinase. The phosphorylated photoactivated rhodopsin is bound by arrestin, thereby terminating activity of the receptor in the signal transduction process. These auxiliary proteins that function with rhodopsin on rod cells serve as models for understanding how other members of the receptor family may function in conjunction with other G proteins, kinases, and arrestin-like proteins.     

11-cis- and all-trans-retinols can activate rod opsin: rational design of the visual cycle

Rhodopsin is the photosensitive pigment in the rod photoreceptor cell. Upon absorption of a photon, the covalently bound 11- cis-retinal isomerizes to the all- trans form, enabling rhodopsin to activate transducin, its G protein. All -trans-retinal is then released from the protein and reduced to all -trans-retinol. It is subsequently transported to the retinal pigment epithelium where it is converted to 11- cis-retinol and oxidized to 11- cis-retinal before it is transported back to the photoreceptor to regenerate rhodopsin and complete the visual cycle. In this study, we have measured the effects of all -trans- and 11- cis-retinals and -retinols on the opsin's ability to activate transducin to ascertain their potentials for activating the signaling cascade. Only 11- cis-retinal acts as an inverse agonist to the opsin. All -trans-retinal, all -trans-retinol, and 11- cis-retinol are all agonists with all -trans-retinal being the most potent agonist and all -trans-retinol being the least potent. Taken as a whole, our study is consistent with the hypothesis that the steps in the visual cycle are optimized such that the rod can serve as a highly sensitive dim light receptor. All -trans-retinal is immediately reduced in the photoreceptor to prevent back reactions and to weaken its effectiveness as an agonist before it is transported out of the cell; oxidation of 11- cis-retinol occurs in the retinal pigment epithelium and not the rod photoreceptor cell because 11- cis-retinol can act as an agonist and activate the signaling cascade if it were to bind an opsin, effectively adapting the cell to light.     

Fourier transform infrared study of photoreceptor membrane. I. Group assignments based on rhodopsin delipidation and reconstitution

Fourier transform infrared spectroscopy has been used to study the structure of bovine photoreceptor membrane. Rhodopsin appears to contain an extensive alpha-helical structure which is arranged predominantly perpendicular to the membrane plane. Spectra of delipidated rhodopsin and rhodopsin membranes reconstituted from dioleyl-phosphatidylcholine were compared with native photoreceptor membrane from rod outer segments in order to facilitate peak assignments. It is concluded that spectroscopic peaks characteristic of several protein and lipid groups can be assigned. We also find delipidation leads to alteration of the rhodopsin structure which is restored upon reconstitution. Membranes both suspended in ²H₂O and dehydrated were compared in order to detect possible conformational differences. Dehydration does not appear to grossly alter rhodopsin structure, although it may affect delipidated rhodopsin.     

Differential Rhodopsin Regeneration in Photoreceptor Membranes is Correlated with Variations in Membrane Properties

Rhodopsin, the major transmembrane protein in both the plasma membrane and the disk membranes of photoreceptor rod outer segments (ROS) forms the apo-protein opsin upon the absorption of light. In vivo the regeneration of rhodopsin is necessary for subsequent receptor activation and for adaptation, in vitro this regeneration can be followed after the addition of 11-cis retinal. In this study we investigated the ability of bleached rhodopsin to regenerate in the compositionally different membrane environments found in photoreceptor rod cells. When 11-cis retinal was added to bleached ROS plasma membrane preparations, rhodopsin did not regenerate within the same time course or to the same extent as bleached rhodopsin in disk membranes. Over 80% of the rhodopsin in newly formed disks regenerated within 90 minutes while only 40% regenerated in older disks. Since disk membrane cholesterol content increases as disks are displaced from the base to the apical tip of the outer segment, we looked at the affect of membrane cholesterol content on the regeneration process. Enrichment or depletion of disk membrane cholesterol did not alter the % rhodopsin that regenerated. Bulk membrane properties measured with a sterol analog, cholestatrienol and a fatty acid analog, cis parinaric acid, showed a more ordered, less fluid, lipid environment within plasma membrane relative to the disks. Collectively these results show that the same membrane receptor, rhodopsin, functions differently as monitored by regeneration in the different lipid environments within photoreceptor rod cells. These differences may be due to the bulk properties of the various membranes.     

The Activation Pathway of Human Rhodopsin in Comparison to Bovine Rhodopsin

Rhodopsin, the photoreceptor of rod cells, absorbs light to mediate the first step of vision by activating the G protein transducin (G_t). Several human diseases, such as retinitis pigmentosa or congenital night blindness, are linked to rhodopsin malfunctions. Most of the corresponding in vivo studies and structure-function analyses (e.g. based on protein x-ray crystallography or spectroscopy) have been carried out on murine or bovine rhodopsin. Because these rhodopsins differ at several amino acid positions from human rhodopsin, we conducted a comprehensive spectroscopic characterization of human rhodopsin in combination with molecular dynamics simulations. We show by FTIR and UV-visible difference spectroscopy that the light-induced transformations of the early photointermediates are very similar. Significant differences between the pigments appear with formation of the still inactive Meta I state and the transition to active Meta II. However, the conformation of Meta II and its activity toward the G protein are essentially the same, presumably reflecting the evolutionary pressure under which the active state has developed. Altogether, our results show that although the basic activation pathways of human and bovine rhodopsin are similar, structural deviations exist in the inactive conformation and during receptor activation, even between closely related rhodopsins. These differences between the well studied bovine or murine rhodopsins and human rhodopsin have to be taken into account when the influence of point mutations on the activation pathway of human rhodopsin are investigated using the bovine or murine rhodopsin template sequences.     

Interaction of small G proteins with photoexcited rhodopsin

Bovine rod outer segment (ROS) membranes contain in addition to the heterotrimeric G protein transducin, several small GTP-binding proteins (23-27 kDa). Furthermore, these membranes contain two substrate proteins (about 22 and 24 kDa) for botulinum C3 ADP-ribosyltransferase known to ADP-ribosylate small G proteins in any mammalian cell type studied so far. Most interestingly, [32P]ADP-ribosylation of ROS membrane small G proteins by C3 is regulated by light and guanine nucleotides in a manner similar to pertussis toxin-catalyzed [32P]ADP-ribosylation of the alpha-subunit of transducin. These findings suggest that not only the heterotrimeric G protein transducin but also the C3 substrate small G proteins present in ROS membranes interact with photoexcited rhodopsin and thus contribute to its signalling action.     

Hydrophobic amino acids at the cytoplasmic ends of helices 3 and 6 of rhodopsin conjointly modulate transducin activation

Rhodopsin is the visual photoreceptor responsible for dim light vision. This receptor is located in the rod cell of the retina and is a prototypical member of the G-protein-coupled receptor superfamily. The structural details underlying the molecular recognition event in transducin activation by photoactivated rhodopsin are of key interest to unravel the molecular mechanism of signal transduction in the retina. We constructed and expressed rhodopsin mutants in the second and third cytoplasmic domains of rhodopsin – where the natural amino acids were substituted by the human M3 acetylcholine muscarinic receptor homologous residues – in order to determine their potential involvement in G-protein recognition. These mutants showed normal chromophore formation and a similar photobleaching behavior than WT rhodopsin, but decreased thermal stability in the dark state. The single mutant V138^3.53 and the multiple mutant containing V227^5.62 and a combination of mutations at the cytoplasmic end of transmembrane helix 6 caused a reduction in transducin activation upon rhodopsin photoactivation. Furthermore, combination of mutants at the second and third cytoplasmic domains revealed a cooperative role, and partially restored transducin activation. The results indicate that hydrophobic interactions by V138^3.53, V227^5.62, V250^6.33, V254^6.37 and I255^6.38 are critical for receptor activation and/or efficient rhodopsin–transducin interaction.     

Direct observation of the complex formation of GDP-bound transducin with the rhodopsin intermediate having a visible absorption maximum in rod outer segment membranes

Rhodopsin is a photoreceptive protein that is present in rod photoreceptor cells, inducing a GDP-GTP exchange reaction on the retinal G-protein transducin (Gt) upon light absorption. This exchange reaction proceeds through at least three steps, which include the binding of photoactivated rhodopsin to GDP-bound Gt, the dissociation of GDP from the rhodopsin-Gt complex, and the binding of GTP to the nucleotide-unbound Gt. These steps have been thought to occur after the formation of the rhodopsin intermediate, meta-II; however, the extra formation of meta-II, which reflects the formation of a complex with Gt, was inhibited in the presence of excess GDP. Here, we use a newly developed CCD spectrophotometer to show that a meta-II precursor, meta-Ib, which has an absorption maximum at visible region, can bind to Gt in its GDP-bound form in urea-washed bovine rod outer segment membranes. The affinity of meta-Ib for GDP-bound Gt is about two times less than that of meta-II for GDP-unbound Gt, indicating that the extra formation of meta-II is observed at equilibrium even in the presence of the meta-Ib-Gt complex. This is the first identification of a complex that includes the GDP-bound form of G protein. Our results strongly suggest that the protein conformational change of the rhodopsin intermediate after binding to Gt is important for the induction of the nucleotide release from the alpha-subunit of Gt.     

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.     

Interaction of rhodopsin with the G-protein,transducin

Rhodopsin, upon activation by light, transduces the photon signal by activation of the G-protein, transducin. The well-studied rhodopsin/transducin system serves as a model for the understanding of signal transduction by the large class of G-protein-coupled receptors. The interactive form of rhodopsin, R*, is conformationally similar or identical to rhodopsin's photolysis intermediate Metarhodopsin II (MII). Formation of MII requires deprotonation of rhodopsin's protonated Schiff base which appears to facilitate some opening of the rhodopsin structure. This allows a change in conformation at rhodopsin's cytoplasmic surface that provides binding sites for transducin. Rhodopsin's 2nd, 3rd and putative 4th cytoplasmic loops bind transducin at sites including transducin's 5 kDa carboxyl-terminal region. Site-specific mutagenesis of rhodopsin is being used to distinguish sites on rhodopsin's surface that are important in binding transducin from those that function in activating transducin. These observations are consistent with and extend studies on the action of other G-protein-coupled receptors and their interactions with their respective G proteins.     

Light- and guanosine 5'-3-O-(thio)triphosphate-sensitive localization of a G protein and its effector on detergent-resistant membrane rafts in rod photoreceptor outer segments

Detergent-resistant membrane microdomains in the plasma membrane, known as lipid rafts, have been implicated in various cellular processes. We report here that a low-density Triton X-100-insoluble membrane (detergent-resistant membrane; DRM) fraction is present in bovine rod photoreceptor outer segments (ROS). In dark-adapted ROS, transducin and most of cGMP-phosphodiesterase (PDE) were detergent-soluble. When ROS membranes were exposed to light, however, a large portion of transducin localized in the DRM fraction. Furthermore, on addition of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) to light-bleached ROS, transducin became detergent-soluble again. PDE was not recruited to the DRM fraction after light stimulus alone, but simultaneous stimulation by light and GTPgammaS induced a massive translocation of all PDE subunits to the DRM. A cholesterol-removing reagent, methyl-beta-cyclodextrin, selectively but partially solubilized PDE from the DRM, suggesting that cholesterol contributes, at least in part, to the association of PDE with the DRM. By contrast, transducin was not extracted by the depletion of cholesterol. These data suggest that transducin and PDE are likely to perform their functions in phototransduction by changing their localization between two distinct lipid phases, rafts and surrounding fluid membrane, on disc membranes in an activation-dependent manner.     

Covalent Bond between Ligand and Receptor Required for Efficient Activation in Rhodopsin

Rhodopsin is an extensively studied member of the G protein-coupled receptors (GPCRs). Although rhodopsin shares many features with the other GPCRs, it exhibits unique features as a photoreceptor molecule. A hallmark in the molecular structure of rhodopsin is the covalently bound chromophore that regulates the activity of the receptor acting as an agonist or inverse agonist. Here we show the pivotal role of the covalent bond between the retinal chromophore and the lysine residue at position 296 in the activation pathway of bovine rhodopsin, by use of a rhodopsin mutant K296G reconstituted with retinylidene Schiff bases. Our results show that photoreceptive functions of rhodopsin, such as regiospecific photoisomerization of the ligand, and its quantum yield were not affected by the absence of the covalent bond, whereas the activation mechanism triggered by photoisomerization of the retinal was severely affected. Furthermore, our results show that an active state similar to the Meta-II intermediate of wild-type rhodopsin did not form in the bleaching process of this mutant, although it exhibited relatively weak G protein activity after light irradiation because of an increased basal activity of the receptor. We propose that the covalent bond is required for transmitting structural changes from the photoisomerized agonist to the receptor and that the covalent bond forcibly keeps the low affinity agonist in the receptor, resulting in a more efficient G protein activation.     

Receptor-dependent G-protein activation in lipidic cubic phase

The primary step in cellular signaling by G-protein-coupled receptors (GPCRs) is the interaction of the agonist-activated transmembrane receptor with an intracellular G-protein. Understanding the underlying molecular mechanisms requires the structural determination of receptor G-protein complexes that are not yet achieved. The crystal structure of the bovine photoreceptor rhodopsin, a prototypical GPCR, was solved recently and the structures of different states of engineered G-proteins were reported. Posttranslational hydrophobic modifications of G-proteins are in most cases removed for crystallization but play functional roles for interactions among G-protein subunits with receptors, as well as membranes. Bovine rhodopsin is reconstituted into lipidic cubic phases to assess their potential for crystallization of receptor G-protein complexes under conditions that may preserve the structural and functional roles of hydrophobic protein modifications. Three-dimensional bilayers of a bicontinuous lipidic cubic phase are successfully employed for crystallization of membrane and soluble proteins. UV-visible absorption and attenuated total reflection Fourier transform IR difference spectroscopy reveal that light activation of cubic phase reconstituted rhodopsin results in the generation of a metarhodopsin II-like state. Via diffusion along aqueous channels, transducin couples efficiently to this photoproduct as evidenced by the nucleotide-dependent increase of transducin fluorescence. Thus, rhodopsin transducin interactions do not crucially depend on the presence of sn1 and sn2 acyl chains, phospholipid head groups, or membrane planarity. Because lipidic cubic phases preserve the essential functional and structural properties of native rhodopsin and transducin, they appear suitable for the detergent-free crystallization of receptor G-protein complexes carrying a normal pattern of hydrophobic modifications.     

Rhodopsin is spatially heterogeneously distributed in rod outer segment disk membranes

The visual photoreception takes place in the retina, where specialized rod and cone photoreceptor cells are located. The rod outer segments contain a stack of 500-2,000 sealed membrane disks. Rhodopsin is the visual pigment located in rod outer segment disks, it is a member of the G-protein-coupled receptor (GPCR) superfamily, an important group of membrane proteins responsible for the majority of physiological responses to stimuli such as light, hormones, peptides, etc. Alongside rhodopsin, peripherin/Rom proteins located in the disk rims are thought to be responsible for disk morphology. Here we describe the supramolecular structure of rod outer segment disk membranes and the spatial organization of rhodopsin and peripherin/Rom molecules. Using atomic force microscopy operated in physiological buffer solution, we found that rhodopsin is loosely packed in the central region of the disks, in average about 26?000 molecules covering approximately one third of the disk surface. Peripherin/Rom proteins form dense assemblies in the rim region. A protein-free lipid bilayer girdle separates the rhodopsin and peripherin/Rom domains. The described supramolecular assembly of rhodospin, peripherin/Rom and lipids in native rod outer segment disks is consistent with the functional requirements of photoreception.     

Purification of squid rhodopsin and reassembly into lipid bilayers

Rhodopsin from squid photoreceptor membranes was solubilized in octyl glucoside and purified to a single band on SDS-polyacrylamide gels of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 46 000. Purified rhodopsin was recombined with phospholipids to form vesicles by detergent dialysis. Spectroscopic analysis of the rhodopsin-lipid vesicles showed that the interconversion between acid and basic metarhodopsin had a $\text{p}\text{K}$ of 8. Furthermore, rhodopsin in the vesicles could be photoregenerated from metarhodopsin in solutions of either neutral or alkaline pH. These two spectroscopic properties are comparable to those for rhodopsin in photoreceptor membranes. The results indicate that the native conformation of rhodopsin is preserved during purification and after recombination with phospholipids into vesicles. This preparation is, therefore, an active starting point for functional reconstitution studies.