Rapid degeneration of rod photoreceptors expressing self-association-deficient arrestin-1 mutant

Arrestin-1 binds light-activated phosphorhodopsin and ensures timely signal shutoff. We show that high transgenic expression of an arrestin-1 mutant with enhanced rhodopsin binding and impaired oligomerization causes apoptotic rod death in mice. Dark rearing does not prevent mutant-induced cell death, ruling out the role of arrestin complexes with light-activated rhodopsin. Similar expression of WT arrestin-1 that robustly oligomerizes, which leads to only modest increase in the monomer concentration, does not affect rod survival. Moreover, WT arrestin-1 co-expressed with the mutant delays retinal degeneration. Thus, arrestin-1 mutant directly affects cell survival via binding partner(s) other than light-activated rhodopsin. Due to impaired self-association of the mutant its high expression dramatically increases the concentration of the monomer. The data suggest that monomeric arrestin-1 is cytotoxic and WT arrestin-1 protects rods by forming mixed oligomers with the mutant and/or competing with it for the binding to non-receptor partners. Thus, arrestin-1 self-association likely serves to keep low concentration of the toxic monomer. The reduction of the concentration of harmful monomer is an earlier unappreciated biological function of protein oligomerization.     

Molecular properties of rhodopsin and rod function

Signal transduction in rod cells begins with photon absorption by rhodopsin and leads to the generation of an electrical response. The response profile is determined by the molecular properties of the phototransduction components. To examine how the molecular properties of rhodopsin correlate with the rod-response profile, we have generated a knock-in mouse with rhodopsin replaced by its E122Q mutant, which exhibits properties different from those of wild-type (WT) rhodopsin. Knock-in mouse rods with E122Q rhodopsin exhibited a photosensitivity about 70% of WT. Correspondingly, their single-photon response had an amplitude about 80% of WT, and a rate of decline from peak about 1.3 times of WT. The overall 30% lower photosensitivity of mutant rods can be explained by a lower pigment photosensitivity (0.9) and the smaller single-photon response (0.8). The slower decline of the response, however, did not correlate with the 10-fold shorter lifetime of the meta-II state of E122Q rhodopsin. This shorter lifetime became evident in the recovery phase of rod cells only when arrestin was absent. Simulation analysis of the photoresponse profile indicated that the slower decline and the smaller amplitude of the single-photon response can both be explained by the shift in the meta-I/meta-II equilibrium of E122Q rhodopsin toward meta-I. The difference in meta-III lifetime between WT and E122Q mutant became obvious in the recovery phase of the dark current after moderate photobleaching of rod cells. Thus, the present study clearly reveals how the molecular properties of rhodopsin affect the amplitude, shape, and kinetics of the rod response.     

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.     

RGS expression rate-limits recovery of rod photoresponses

Signaling through G protein-coupled receptors (GPCRs) underlies many cellular processes, yet it is not known which molecules determine the duration of signaling in intact cells. Two candidates are G protein-coupled receptor kinases (GRKs) and Regulators of G protein signaling (RGSs), deactivation enzymes for GPCRs and G proteins, respectively. Here we investigate whether GRK or RGS governs the overall rate of recovery of the light response in mammalian rod photoreceptors, a model system for studying GPCR signaling. We show that overexpression of rhodopsin kinase (GRK1) increases phosphorylation of the GPCR rhodopsin but has no effect on photoresponse recovery. In contrast, overexpression of the photoreceptor RGS complex (RGS9-1.Gbeta5L.R9AP) dramatically accelerates response recovery. Our results show that G protein deactivation is normally at least 2.5 times slower than rhodopsin deactivation, resolving a long-standing controversy concerning the mechanism underlying the recovery of rod visual transduction.     

Light activation of one rhodopsin molecule causes the phosphorylation of hundreds of others. A reaction observed in electropermeabilized frog rod outer segments exposed to dim illumination

A rhodopsin phosphorylation reaction that occurs with high-gain is observed if measurements are made in electropermeabilized frog rod outer segments (ROS) stimulated by a dim flash of light in the operating range of the photoreceptor. Flashes of light exciting 1000 or fewer of the 3 x 10(9) rhodopsins present/ROS results in the incorporation of 1400 phosphates from ATP into the rhodopsin pool for each excited rhodopsin (Rho*). This amplification decreases with increasing light intensity, falling most sharply after each disk has absorbed one photon. The high-gain reaction is lost if the ROS are broken into vesicles by shearing, leaving a low-gain rhodopsin phosphorylation characterized in previous studies using brighter illumination. The high-gain but not the low-gain phosphorylation appears to be regulated by G-protein and by calcium levels in the range over which intracellular calcium changes when rod photoreceptors are illuminated. Kinetic measurements made on the phosphorylation observed at higher light intensities shows that it initially occurs rapidly enough for a role in terminating the photoresponse. The high-gain phosphorylation observed at lower light intensities may play a global role in regulating light-adaptation of the rod photoreceptor, and its existence suggests that a search for a similar high-gain modification in systems using the homologous beta-adrenergic or muscarinic acetylcholine receptors might be rewarding.     

Immunochemical evidence for the light-regulated modulation of phosphatidylinositol-4,5-bisphosphate in rat photoreceptor cells

Immunocytochemical localization of phosphatidylinositol-4,5-bisphosphate (PIP2) in the rat rod photoreceptor outer segments (OS) was investigated with rabbit antiPIP2 antibodies. The OS of the light-adapted rat eye showed little or no staining, whereas the OS of the dark-adapted eye were intensely stained for PIP2. The immunoreactivity of photoreceptor PIP2 in the eye exposed to a brief flash of light was markedly reduced. However, subsequent dark-adaptation of the flash-bleached eye resulted in a rapid recovery of PIP2 immunoreactivity; dark-adaptation for 5 min was sufficient for recovery to the fully dark-adapted level. In dark-adapted eyes exposed to graded light intensities, the PIP2 immunostaining varied with light levels and was correlated with unbleached rhodopsin concentrations. These results suggest that PIP2 in the rat photoreceptor cells is rapidly hydrolyzed upon light exposure and rapidly synthesized in the dark and that the decrease of PIP2 level is triggered by photic bleaching of rhodopsin.     

Light-induced changes in GTP and ATP in frog rod photoreceptors. Comparison with recovery of dark current and light sensitivity during dark adaptation

Light decreases GTP and ATP levels in purified suspensions of physiologically active frog rod outer segments still attached to their inner segment ellipsoids (OS-IS). (a) The GTP decrease is slower in OS-IS (t1/2 = 40 s) than in isolated outer segments (t1/2 = 7 s), which suggests there is more effective buffering in OS-IS. (b) The GTP decrease becomes detectable only at intensities greater than those required to saturate the photoresponse. As the intensity of a continuous light is increased over 4 log units, GTP levels decrease linearly with log intensity by as much as 60%. GTP is reduced to steady intermediate levels during extended illumination of intermediate intensity. (c) At levels of illumination bleaching greater than 0.003% of the rhodopsin, a decrease in ATP levels becomes detectable. (d) Following a flash, GTP levels fall and then rise with a recovery time dependent on the intensity of the flash. (e) After both 0.2 and 2% flash bleaches, the recovery of GTP levels parallels the recovery of light sensitivity, which is slower than the recovery of the dark current. This raises the possibility of a link between GTP levels and light sensitivity.     

Robust self-association is a common feature of mammalian visual arrestin-1

Arrestin-1 binds light-activated phosphorhodopsin and ensures rapid signal termination. Its deficiency in humans and mice results in prolonged signaling and rod degeneration. However, most of the biochemical studies were performed on bovine arrestin-1, which was shown to self-associate forming dimers and tetramers, although only the monomer binds rhodopsin. It is unclear whether self-association is a property of arrestin-1 in all mammals or a specific feature of bovine protein. To address this issue, we compared self-association parameters of purified human and mouse arrestin-1 with those of its bovine counterpart using multiangle light scattering. We found that mouse and human arrestin-1 also robustly self-associate, existing in a monomer-dimer-tetramer equilibrium. Interestingly, the combination of dimerization and tetramerization constants in these three species is strikingly different. While tetramerization of bovine arrestin-1 is highly cooperative (K(D,dim)(4) > K(D,tet)), K(D,dim) ～ K(D,tet) in the mouse form and K(D,dim) ? K(D,tet) in the human form. Importantly, in all three species at very high physiological concentrations of arrestin-1 in rod photoreceptors, most of it is predicted to exist in oligomeric form, with a relatively low concentration of the free monomer. Thus, it appears that maintenance of low levels of the active monomer is the biological role of arrestin-1 self-association.     

Rapid and reproducible deactivation of rhodopsin requires multiple phosphorylation sites

Efficient single-photon detection by retinal rod photoreceptors requires timely and reproducible deactivation of rhodopsin. Like other G protein-coupled receptors, rhodopsin contains multiple sites for phosphorylation at its COOH-terminal domain. Transgenic and electrophysiological methods were used to functionally dissect the role of the multiple phosphorylation sites during deactivation of rhodopsin in intact mouse rods. Mutant rhodopsins bearing zero, one (S338), or two (S334/S338) phosphorylation sites generated single-photon responses with greatly prolonged, exponentially distributed durations. Responses from rods expressing mutant rhodopsins bearing more than two phosphorylation sites declined along smooth, reproducible time courses; the rate of recovery increased with increasing numbers of phosphorylation sites. We conclude that multiple phosphorylation of rhodopsin is necessary for rapid and reproducible deactivation.     

Rhodopsin mutant P23H destabilizes rod photoreceptor disk membranes

Mutations in rhodopsin cause retinitis pigmentosa in humans and retinal degeneration in a multitude of other animals. We utilized high-resolution live imaging of the large rod photoreceptors from transgenic frogs (Xenopus) to compare the properties of fluorescently tagged rhodopsin, Rho-EGFP, and Rho(P23H)-EGFP. The mutant was abnormally distributed both in the inner and outer segments (OS), accumulating in the OS to a concentration of ～0.1% compared to endogenous opsin. Rho(P23H)-EGFP formed dense fluorescent foci, with concentrations of mutant protein up to ten times higher than other regions. Wild-type transgenic Rho-EGFP did not concentrate in OS foci when co-expressed in the same rod with Rho(P23H)-EGFP. Outer segment regions containing fluorescent foci were refractory to fluorescence recovery after photobleaching, while foci in the inner segment exhibited recovery kinetics similar to OS regions without foci and Rho-EGFP. The Rho(P23H)-EGFP foci were often in older, more distal OS disks. Electron micrographs of OS revealed abnormal disk membranes, with the regular disk bilayers broken into vesiculotubular structures. Furthermore, we observed similar OS disturbances in transgenic mice expressing Rho(P23H), suggesting such structures are a general consequence of mutant expression. Together these results show that mutant opsin disrupts OS disks, destabilizing the outer segment possibly via the formation of aggregates. This may render rods susceptible to mechanical injury or compromise OS function, contributing to photoreceptor loss.     

Light causes phosphorylation of nonactivated visual pigments in intact mouse rod photoreceptor cells

Phosphorylation of G-protein-coupled receptors (GPCRs) is a required step in signal deactivation. Rhodopsin, a prototypical GPCR, exhibits high gain phosphorylation in vitro whereby a hundred-fold molar excess of phosphates are incorporated into the rhodopsin pool per molecule of activated rhodopsin. The extent by which high gain phosphorylation occurs in the intact mammalian photoreceptor cell, and the molecular mechanism underlying this reaction in vivo, is not known. Trans-phosphorylation is a mechanism proposed for high gain phosphorylation, whereby rhodopsin kinase, upon phosphorylating the activated receptor, continues to phosphorylate nearby nonactivated rhodopsin. We used two different transgenic mouse models to test whether trans-phosphorylation occurs in the intact photoreceptor cell. The first transgenic model expressed a murine cone pigment, S-opsin, together with the endogenous rhodopsin in the rod cell. We showed that selective stimulation of rhodopsin also led to phosphorylation of S-opsin. The second mouse model expressed the constitutively active human opsin mutant K296E. K296E, in the arrestin-/- background, also led to phosphorylation of endogenous mouse rhodopsin in the dark-adapted retina. Both mouse models provide strong support of trans-phosphorylation as an underlying mechanism of high gain phosphorylation, and provide evidence that a substantial fraction of nonactivated visual pigments becomes phosphorylated through this mechanism. Because activated, phosphorylated receptors exhibit decreased catalytic activity, our results suggest that dephosphorylation would be an important step in the full recovery of visual sensitivity during dark adaptation. These results may also have implications for other GPCR signaling pathways.     

A variant of arrestin-1 binds rod outer segment membranes in a light-independent manner

A 50-kDa-polypeptide band peripherally bound to retinal rod outer segment (ROS) membranes was purified by anion-exchange chromatography. When the 50-kDa protein was compared with purified arrestin-1, it was observed that: (1) both proteins comigrated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and were recognized by either anti-50-kDa protein polyclonal antibodies or anti-arrestin-1 monoclonal antibodies; (2) protein fragments and peptide fingerprint maps obtained following limited and complete proteolysis with specific proteases were very similar for both molecules; and (3) several chromatographically-purified tryptic peptides from the 50-kDa protein possessed the same amino acid composition as tryptic peptides deduced from the reported arrestin-1 primary structure. Consequently, arrestin-1 and the purified 50-kDa protein must correspond to variants of the same molecule. However, in contrast to arrestin-1 that associated to the ROS membranes only in the presence of light and ATP, the 50-kDa protein interacted with the ROS membranes in a light-independent manner, either in the presence or absence of ATP. These results clearly established that phosphorylated and illuminated rhodopsin is not the membrane anchor for this variant of arrestin-1.     

Molecular cloning and characterization of rhodopsin in a teleost (Plecoglossus altivelis,Osmeridae)

Amplified fragments encoding exon-4 of opsin cDNAs were cloned from the retina of landlocked ayu (Plecoglossus altivelis), and sequenced. On the basis of the sequence homology to previously characterized fish visual pigments, one clone was identified as rod opsin (AYU-Rh), and two clones as green (AYU-G1, -G2), one as red (AYU-R) and two as ultraviolet (AYU-UV1, -UV2) cone opsins. The 335-amino acid sequence deduced from the full-length cDNA of AYU-Rh included residues highly conserved in vertebrate rhodopsins and showed the greatest degree (88%) of similarity with salmon rhodopsin. Southern blotting analysis indicated that ayu possess two rhodopsin genes, one encoding visual rhodopsin (AYU-Rh) and the other non-visual extra-ocular rhodopsin (AYU-ExoRh). RT-PCR experiments revealed that AYU-Rh was expressed in the retina and AYU-ExoRh in the pineal gland. In situ hybridization experiments showed that the mRNA of AYU-Rh was localized only in rod cells not in cone cells. Lake and river type landlocked ayu having different amounts of retinal and 3-hydroxyretinal in their retinas expressed a rhodopsin (AYU-Rh) of identical amino acid sequence.     

Surfaces of rod photoreceptor disk membranes: light-activated enzymes

The light-activated GTP-binding protein (GBP) in toad rod outer segments has been located on the cytoplasmic surface (CS) of rod disk membranes by correlating biochemical results with images of quick- frozen, freeze-fractured, and deep-etched rod outer segments. This has been accomplished by selectively removing and replacing the 8-12-nm particles that are found on the CS of disk membranes, exactly in parallel with the GBP. In contrast, the large particles are not correlated with another major disk enzyme, the light-activated cGMP phosphodiesterase. We have been unable to visualize this protein. The surface density of large particles, one particle per eleven rhodopsins in isolated rod outer segments and one particle per nine rhodopsins in intact retina, correlates well with previous biochemical estimates of GBP numbers based on enzyme activity. After the identification of the large particles, we tested the effects of light on the density of particles on the surface of disk membranes in intact retinas. Retinas quick-frozen at various intervals after a bright flash of light show a modest increase (approximately 30%) in particle density by 10 s after the flash but no increase before 1 s. The number of particles on the disk membrane returns to dark levels between 1 and 10 min after the flash. The 1-s latency in the change of particle binding would appear to rule out this process as a mechanism for initiating phototransduction in the rod.     

The visual system of the alligator

The eye tissues and liver of the alligator contain vitamin A₁ alone. The retina contains rhodopsin, typical in absorption spectrum (λ_max 500 mµ); but synthesized in solution from neo-b retinene and opsin much more rapidly than are the frog, mammalian, or chicken rhodopsins previously examined. In this regard alligator rhodopsin resembles the rhodopsins and porphyropsins of fishes, all of which so far investigated are synthesized rapidly in solution. The rates of synthesis in vitro of frog and alligator rhodopsins are matched closely by the rates of rod dark adaptation in living frogs and alligators, measured electrophysiologically at the same temperature. Alligator rods dark-adapt, and alligator rhodopsin is synthesized in solution, at rates characteristically associated with cones and cone pigments in frogs, mammals, and birds.     

Binding of more than one retinoid to visual opsins

Visual opsins bind 11-cis retinal at an orthosteric site to form rhodopsins but increasing evidence suggests that at least some are capable of binding an additional retinoid(s) at a separate, allosteric site(s). Microspectrophotometric measurements on isolated, dark-adapted, salamander photoreceptors indicated that the truncated retinal analog, β-ionone, partitioned into the membranes of green-sensitive rods; however, in blue-sensitive rod outer segments, there was an enhanced uptake of four or more β-ionones per rhodopsin. X-ray crystallography revealed binding of one β-ionone to bovine green-sensitive rod rhodopsin. Cocrystallization only succeeded with extremely high concentrations of β-ionone and binding did not alter the structure of rhodopsin from the inactive state. Salamander green-sensitive rod rhodopsin is also expected to bind β-ionone at sufficiently high concentrations because the binding site is present on its surface. Therefore, both blue- and green-sensitive rod rhodopsins have at least one allosteric binding site for retinoid, but β-ionone binds to the latter type of rhodopsin with low affinity and low efficacy.     

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.     

Binding of rhodopsin and rhodopsin analogues to transducin,rhodopsin kinase and arrestin-1

AIM: To investigate the interaction of reconstituted rhodopsin, 9-cis-retinal-rhodopsin and 13-cis-retinal-rhodopsin with transducin, rhodopsin kinase and arrestin-1. METHODS: Rod outer segments(ROS) were isolated from bovine retinas. Following bleaching of ROS membranes with hydroxylamine, rhodopsin and rhodopsin analogues were generated with the different retinal isomers and the concentration of the reconstituted pigments was calculated from their UV/visible absorption spectra. Transducin and arrestin-1 were purified to homogeneity by column chromatography, and an enriched-fraction of rhodopsin kinase was obtainedby extracting freshly prepared ROS in the dark. The guanine nucleotide binding activity of transducin was determined by Millipore filtration using β,γ-imido-(3H)-guanosine 5'-triphosphate. Recognition of the reconstituted pigments by rhodopsin kinase was determined by autoradiography following incubation of ROS membranes containing the various regenerated pigments with partially purified rhodopsin kinase in the presence of(γ-32P) ATP. Binding of arrestin-1 to the various pigments in ROS membranes was determined by a sedimentation assay analyzed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis. RESULTS: Reconstituted rhodopsin and rhodopsin analogues containing 9-cis-retinal and 13-cis-retinal rendered an absorption spectrum showing a maximum peak at 498 nm, 486 nm and about 467 nm, respectively, in the dark; which was shifted to 380 nm, 404 nm and about 425 nm, respectively, after illumination. The percentage of reconstitution of rhodopsin and the rhodopsin analogues containing 9-cis-retinal and 13-cis-retinal was estimated to be 88%, 81% and 24%, respectively. Although only residual activation of transducin was observed in the dark when reconstituted rhodopsin and 9-cis-retinal-rhodopsin was used, the rhodopsin analogue containing the 13-cis isomer of retinal was capable of activating transducin independently of light. Moreover, only a basal amount of the reconstituted rhodopsin and 9-cis-retinal-rhodopsin was phosphorylated by rhodopsin kinase in the dark, whereas the pigment containing the 13-cis-retinal was highly phosphorylated by rhodopsin kinase even in the dark. In addition, arrestin-1 was incubated with rhodopsin, 9-cis-retinal-rhodopsin or 13-cis-retinal-rhodopsin. Experiments were performed using both phosphorylated and non-phosphorylated regenerated pigments. Basal amounts of arrestin-1 interacted with rhodopsin, 9-cis-retinal-rhodopsin and 13-cis-retinal-rhodopsin under dark and light conditions. Residual arrestin-1 was also recognized by the phosphorylated rhodopsin and phosphorylated 9-cis-retinal-rhodopsin in the dark. However, arrestin-1 was recognized by phosphorylated 13-cis-retinal-rhodopsin in the dark. As expected, all reformed pigments were capable of activating transducin and being phosphorylated by rhodopsin kinase in a lightdependent manner. Additionally, all reconstituted photolyzed and phosphorylated pigments were capable of interacting with arrestin-1. CONCLUSION: In the dark, the rhodopsin analogue containing the 13-cis isomer of retinal appears to fold in a pseudo-active conformation that mimics the active photointermediate of rhodopsin.     

Light-induced intramolecular charge movements in microbial rhodopsins in intact E. coli cells.

Microbial rhodopsins undergo cyclic photochemical reactions (photocycles) in which proton transfers and conformational changes result in charge displacements during transitions between photocycle intermediates. We report a new photoelectric method to monitor charge movements during rhodopsin photocycling with fast kinetic resolution in suspensions of intact E. coli cells. The method monitors electrical currents resulting from asymmetric photoexcitation of microbial rhodopsins by a unilateral laser flash, and kinetically resolves intramolecular charge movements. We investigated E. coli-expressed proton-transporting rhodopsins, specifically green- and blue-absorbing proteorhodopsins (GPR and BPR, respectively) from uncultivated marine plankton, and sensory rhodopsins, namely receptors from Natronomonas pharaonis and Anabaena (Nostoc) sp. PCC7120. Kinetic components of the currents correlate with photochemical transformations of the pigments, and the integrated current measures net transport by the proton-pumping rhodopsins. The photoelectric measurements distinguish between known light-driven transporters and photosensors, and reveal differences in proton transfer reactions in the two tested proton pumps. Screening of nine newly identified proteorhodopsins reveals two with GPR-type charge movements, five with BPR-type, and two with the characteristics of the sensory rhodopsins. The approach developed in the present work provides a direct, rapid and informative method for studying electrogenic events in rhodopsin photocycles and also gives a clue to functions of newly found microbial rhodopsins in nature.     

High-pH form of bovine rhodopsin.

Since the 1930s, the spectrum of vertebrate rhodopsin has been considered to be independent of pH (Lythgoe, R.J. 1937. J. Physiol. 89:331-358; Wald, G. 1938. J. Gen. Physiol. 21:795-832). Here I report that the spectrum of bovine rhodopsin is pH dependent. At pHs greater than 9.0, there is a shift to shorter wavelengths of its 500-nm absorption band. This shift is accounted for by the existence of a high pH form of bovine rhodopsin, with absorption maximum at 494 nm and a slightly lower extinction coefficient. The high-pH form results from the low-pH form by the deprotonation of a single group with a pK of approximately 10.2 for rhodopsin in rod disk membranes in 4.0 M KCl. The shift is observed for sheep and chicken rhodopsins, but not for frog, toad, and octopus rhodopsins. This indicates a specific amino acid difference between these rhodopsins that is potentially relevant for the mechanism of color regulation.