In the G protein-coupled receptor rhodopsin, the conserved NPxxY(x) 5,6F motif connects the transmembrane helix VII and the cytoplasmic helix 8. The less geometrically constrained retinal analogue 9-demethyl-retinal prevents efficient transformation of rhodopsin to signaling metarhodopsin (Meta) II after retinal photoisomerization. Here, we demonstrate that Ala replacement mutations within the NPxxY(x) 5,6F domain, which eliminate an interaction between aromatic residues Y306 and F313, allow formation of Meta II despite the presence of 9-demethyl-retinal. Also a disulfide bond linking residues 306 and 313 in the 9-demethylretinal-reconstituted mutant Y306C͞F313C͞C316S prevented Meta II formation, whereas the reduced form of the mutant readily transformed to Meta II after illumination. These observations suggest that the interaction between residues 306 and 313 is disrupted during the Meta I͞Meta II transition. However, this enhancement in Meta II formation is not reflected in the G protein activation, which is dramatically reduced for these mutants, suggesting that changes in the Y306 -F313 interaction also lead to a proper realigning of helix 8 after photoisomerization. The E134Q mutation, located in the second conserved motif, D(E)RY, rescues activity in 9-demethyl-retinal-reconstituted mutants to different degrees, depending on the position of the Ala replacement in the NPxxY(x) 5,6F motif, thus revealing distinct roles for the NP and Y(x) 5,6F portions. Our studies underscore the importance of the NPxxY(x) 5,6F and D(E)RY motifs in providing structural constraints in rhodopsin that rearrange in response to photoisomerization during formation of the G protein-activating Meta II. The dual control of the structural rearrangements secures reliable transformation of quiescent rhodopsin to activating Meta II.GPCR ͉ NPxxY motif R hodopsin is a prototypical receptor from the largest subfamily A of G protein-coupled receptors (GPCRs), which are thought to operate through similar signaling mechanisms (1, 2). The only known crystal structure of a GPCR, that of rhodopsin (3), confirmed the presence of seven transmembrane helices (H). The structure describes the receptor in its inactive ground state with the bound inverse agonist, 11-cis-retinal. The -amino group of K296 in H-VII tethers the chromophore 11-cis-retinal to opsin via a covalent Schiff base (SB) bond (Fig. 1). A salt-bridge between the protonated SB and the counterion, the conserved Glu-113 in H-III, contributes largely to constrain the receptor in the inactive conformation. Additional constraints involve hydrogen bond networks and hydrophobic interactions linking transmembrane helices, sequestration of the Arg residue of the D(E)RY motif in the hydrophobic milieu, and intramolecular interactions within the NPxxY(x) 5,6 F region (3, 4). Absorption of light energy isomerizes the 11-cis-retinylidene chromophore, generating the agonist all-trans-retinylidene in situ. Subsequent thermal relaxation of the retinal-protein complex leads within milliseconds to an e...

Mutations in the retinal pigment epithelium gene encoding RPE65 are a cause of the incurable early-onset recessive human retinal degenerations known as Leber congenital amaurosis. Rpe65-deficient mice, a model of Leber congenital amaurosis, have no rod photopigment and severely impaired rod physiology. We analyzed retinoid flow in this model and then intervened by using oral 9-cis-retinal, attempting to bypass the biochemical block caused by the genetic abnormality. Within 48 h, there was formation of rod photopigment and dramatic improvement in rod physiology, thus demonstrating that mechanismbased pharmacological intervention has the potential to restore vision in otherwise incurable genetic retinal degenerations.

The retinoid cycle is a recycling system that replenishes the 11-cis-retinal chromophore of rhodopsin and cone pigments. Photoreceptor-specific retinol dehydrogenase (prRDH) catalyzes reduction of all-trans-retinal to all-trans-retinol and is thought to be a key enzyme in the retinoid cycle. We disrupted mouse prRDH (human gene symbol RDH8) gene expression by targeted recombination and generated a homozygous prRDH knock-out (prRDH؊/؊) mouse. Histological analysis and electron microscopy of retinas from 6-to 8-week-old prRDH؊/؊ mice revealed no structural differences of the photoreceptors or inner retina. For brief light exposure, absence of prRDH did not affect the rate of 11-cis-retinal regeneration or the decay of Meta II, the activated form of rhodopsin. Absence of prRDH, however, caused significant accumulation of all-trans-retinal following exposure to bright lights and delayed recovery of rod function as measured by electroretinograms and single cell recordings. Retention of all-trans-retinal resulted in slight overproduction of A2E, a condensation product of all-trans-retinal and phosphatidylethanolamine. We conclude that prRDH is an enzyme that catalyzes reduction of all-trans-retinal in the rod outer segment, most noticeably at higher light intensities and prolonged illumination, but is not an essential enzyme of the retinoid cycle.Reduction and oxidation of retinoids are key reactions of the retinoid cycle (visual cycle), which is critical for the production of the chromophore of rhodopsin, 11-cis-retinal (1, 2). When light strikes the visual pigments (rhodopsin and cone opsins) in photoreceptors, it causes the 11-cis-retinylidene chromophore to isomerize to its all-trans configuration, before all-trans-retinal is released from the binding site of the pigments (3) (see Scheme 1). The NADPH-dependent reduction of all-trans-retinal in photoreceptor outer segments is the first step in the regeneration of bleached visual pigment. The reduction occurs directly on the cytoplasmic surface of outer segment disk membranes. Once all-trans-retinal escapes into the internal disk space, it is pumped out to the cytosol by a photoreceptorspecific ATP-binding transporter (4 -8). Several all-trans-retinol dehydrogenases (RDHs) 1 from the photoreceptor cells have been identified. First, Haeseleer et al. (9) cloned a cone-specific enzyme from the short-chain dehydrogenase/reductase (SDR) family with properties that suggest participation in the retinoid cycle. Next, Rattner and colleagues (10) reported the identification of a novel member of the SDR family, photoreceptor RDH (prRDH or RDH8), that localized to photoreceptors and possessed enzymatic properties closely matching those previously reported for RDH activity in ROS. The authors suggested that prRDH is the enzyme responsible for the reduction of all-trans-retinal to all-trans-retinol within the photoreceptor outer segment. The sequence homology among SDRs is typically low (20 -40%), but the structural homology is high and most protein folds are conserved (11). prR...

The visual process is initiated by the photoisomerization of 11-cis-retinal to all-trans-retinal. For sustained vision the 11-cis-chromophore must be regenerated from all-trans-retinal. This requires RPE65, a dominant retinal pigment epithelium protein. Disruption of the RPE65 gene results in massive accumulation of alltrans-retinyl esters in the retinal pigment epithelium, lack of 11-cis-retinal and therefore rhodopsin, and ultimately blindness. We reported previously (Van Hooser, J. P., Aleman, T. S., He, Y. G., Cideciyan, A. V., Kuksa, V., Pittler, S. J., Stone, E. M., Jacobson, S. G., and Palczewski, K. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8623-8628) that in Rpe65؊/؊ mice, oral administration of 9-cis-retinal generated isorhodopsin, a rod photopigment, and restored light sensitivity to the electroretinogram. Here, we provide evidence that early intervention by 9-cis-retinal administration significantly attenuated retinal ester accumulation and supported rod retinal function for more than 6 months post-treatment. In single cell recordings rod light sensitivity was shown to be a function of the amount of regenerated isorhodopsin; high doses restored rod responses with normal sensitivity and kinetics. Highly attenuated residual rod function was observed in untreated Rpe65؊/؊ mice. This rod function is likely a consequence of low efficiency production of 11-cis-retinal by photo-conversion of alltrans-retinal in the retina as demonstrated by retinoid analysis. These studies show that pharmacological intervention produces long lasting preservation of visual function in dark-reared Rpe65؊/؊ mice and may be a useful therapeutic strategy in recovering vision in humans diagnosed with Leber congenital amaurosis caused by mutations in the RPE65 gene, an inherited group of early onset blinding and retinal degenerations. Leber congenital amaurosis (LCA)1 is a group of conditions that cause blindness or severe visual impairment from birth. All show both rod and cone dysfunction, a negligible (not recordable) electroretinogram (ERG), and nystagmus. They result in early onset retinal dystrophy (1), which over time may be accompanied by pigmentary changes in the retina, hence "amaurosis" (Greek for darken). LCA is caused by defects in at least five different genes that disrupt a variety of different cellular functions (2-6).In ϳ12% of all LCA cases the gene for a 65-kDa protein (RPE65) of retinal pigment epithelium cells (RPE) is disabled (7,8). RPE65 is heavily expressed in RPE cells, where it plays an essential role in the retinoid cycle. This is a set of tightly interconnected events that involve both photoreceptors and RPE cells. The photoisomerization of the visual pigment chromophore (11-cis-retinal) produces all-trans-retinal, which is reduced in the photoreceptor, transferred to the RPE, converted back to 11-cis-retinal, and then transferred back to the photoreceptor to regenerate the original visual pigment (reviewed in Ref. 9). The precise function of RPE65 in retinoid processing is unknown.Genetically enginee...

In vertebrate retinal photoreceptors, photoisomerization of opsin-bound visual chromophore 11-cis-retinal to all-trans-retinal triggers phototransduction events. Regeneration of the chromophore is a critical step in restoring photoreceptors to their dark-adapted state. This regeneration process, called the retinoid cycle, takes place in the photoreceptor outer segments and in the retinal pigmented epithelium (RPE). We have suggested that the regeneration of the chromophore might occur through a retinyl carbocation intermediate. Here, we provide evidence that isomerization is inhibited by positively charged retinoids, which could act as transition state analogs of the isomerization process. We demonstrate that retinylamine (Ret-NH2) potently and selectively inhibits the isomerization step of the retinoid cycle in vitro and in vivo. Ret-NH2 binds a protein(s) in the RPE microsomes, but it does not bind RPE65, a protein implicated in the isomerization reaction. Although Ret-NH2 inhibits the regeneration of visual chromophore in rods and, in turn, severely attenuates rod responses, it has a much smaller effect on cone function in mice. Ret-NH2 interacts only at micromolar concentrations with retinoic acid receptor, does not activate retinoid-X receptor, and is not a substrate for CYP26s, the retinoic acid-metabolizing cytochrome P450 enzymes. Ret-NH2 can be a significant investigational tool to study the mechanism of regeneration of visual chromophore.

Protein conformational disorders, which include certain types of retinitis pigmentosa, are a set of inherited human diseases in which mutant proteins are misfolded and often aggregated. Many opsin mutants associated with retinitis pigmentosa, the most common being P23H, are misfolded and retained within the cell. Here, we describe a pharmacological chaperone, 11-cis-7-ring retinal, that quantitatively induces the in vivo folding of P23H-opsin. The rescued protein forms pigment, acquires mature glycosylation, and is transported to the cell surface. Additionally, we determined the temperature stability of the rescued protein as well as the reactivity of the retinal-opsin Schiff base to hydroxylamine. Our study unveils novel properties of P23H-opsin and its interaction with the chromophore. These properties suggest that 11-cis-7-ring retinal may be a useful therapeutic agent for the rescue of P23H-opsin and the prevention of retinal degeneration.

In the retinal rod and cone photoreceptors, light photoactivates rhodopsin or cone visual pigments by converting 11-cis-retinal to all-trans-retinal, the process that ultimately results in phototransduction and visual sensation. The production of 11-cis-retinal in adjacent retinal pigment epithelial (RPE) cells is a fundamental process that allows regeneration of the vertebrate visual system. Here, we present evidence that all-trans-retinol is unstable in the presence of H(+) and rearranges to anhydroretinol through a carbocation intermediate, which can be trapped by alcohols to form retro-retinyl ethers. This ability of all-trans-retinol to form a carbocation could be relevant for isomerization. The calculated activation energy of isomerization of all-trans-retinyl carbocation to the 11-cis-isomer was only approximately 18 kcal/mol, as compared to approximately 36 kcal/mol for all-trans-retinol. This activation energy is similar to approximately 17 kcal/mol obtained experimentally for the isomerization reaction in RPE microsomes. Mass spectrometric (MS) analysis of isotopically labeled retinoids showed that isomerization proceeds via alkyl cleavage mechanism, but the product of isomerization depended on the specificity of the retinoid-binding protein(s) as evidenced by the production of 13-cis-retinol in the presence of cellular retinoid-binding protein (CRBP). To test the influence of an electron-withdrawing group on the polyene chain, which would inhibit carbocation formation, 11-fluoro-all-trans-retinol was used in the isomerization assay and was shown to be inactive. Together, these results strengthen the idea that the isomerization reaction is driven by mass action and may occur via carbocation intermediate.

Retinoids carry out essential functions in vertebrate development and vision. Many of the retinoid processing enzymes remain to be identified at the molecular level. To expand the knowledge of retinoid biochemistry in vertebrates, we studied the enzymes involved in plant metabolism of carotenoids, a related group of compounds. We identified a family of vertebrate enzymes that share significant similarity and a putative phytoene desaturase domain with a recently described plant carotenoid isomerase (CRTISO), which isomerizes prolycopene to all-trans-lycopene. Comparison of heterologously expressed mouse and plant enzymes indicates that unlike plant CRTISO, the CRTISO-related mouse enzyme is inactive toward prolycopene. Instead, the CRTISO-related mouse enzyme is a retinol saturase carrying out the saturation of the 13-14 double bond of all-trans-retinol to produce all-trans-13,14-dihydroretinol. The product of mouse retinol saturase (RetSat) has a shifted UV absorbance maximum, max ‫؍‬ 290 nm, compared with the parent compound, all-trans-retinol ( max ‫؍‬ 325 nm), and its MS analysis (m/z ‫؍‬ 288) indicates saturation of a double bond. The product was further identified as all-trans-13,14-dihydroretinol, since its characteristics were identical to those of a synthetic standard. Mouse RetSat is membrane-associated and expressed in many tissues, with the highest levels in liver, kidney, and intestine. All-trans-13,14-dihydroretinol was also detected in several tissues of animals maintained on a normal diet. Thus, saturation of all-transretinol to all-trans-13,14-dihydroretinol by RetSat produces a new metabolite of yet unknown biological function.Retinoids are essential for many important biological functions, such as development, immunity, cellular differentiation, and vision of vertebrates. Retinoids encompassing both natural derivatives of all-trans-retinol and their synthetic analogues exert their functions through several active compounds. Esterification of retinol by lecithin-retinol acyltransferase (LRAT)