Genomic and cDNA sequences for the mouse cellular retinol binding protein I (mCRBPI) are presented. A specific cis‐acting element responsible for retinoic acid (RA) inducibility of the mCRBPI promoter was identified and characterized. Deletion mapping of a CRBPI promoter‐‐chloramphenicol acetyltransferase reporter gene construct localized this element to a 259 bp restriction fragment located approximately 1 kb upstream from the transcription start‐site. A sequence closely resembling the previously characterized RA response element (RARE) of the RA receptor beta 2 (RAR‐beta 2) promoter, and consisting of a direct repeat of the motif 5′‐GGTCA‐3′ separated by three nucleotides, was found within this restriction fragment. Mutation of these 5′‐GGTCA‐3′ motifs to GGAGC and GGGGC abolished RA‐inducible transcription whereas a mutation to a direct repeat of the GTTCA motif found in the RARE of the RAR‐beta 2 promoter resulted in enhanced inducibility. Oligonucleotides containing the direct repeat of the GGTCA motif were able to confer RA‐dependent transcriptional enhancement to the herpes simplex thymidine kinase promoter, as well as to bind directly all three retinoic acid receptors (RARs) alpha, beta and gamma, as determined by gel retardation/shift assays. The control of CRBPI gene transcription by RA‐RAR complexes interacting with the RARE characterized here may correspond to a feedback mechanism important in regulating retinoid metabolism and action.

The lateral eyes of the horseshoe crab Limulus polyphemus undergo dramatic daily changes in structure and function that lead to enhanced retinal sensitivity and responsiveness to light at night. These changes are controlled by a circadian neural input that alters photoreceptor and pigment cell shape, pigment migration, and phototransduction. Clock input to the eyes also regulates photomechanical movements within photoreceptors, including membrane shedding. The biochemical mechanisms underlying these diverse effects of the clock on the retina are unknown, but a major biochemical consequence of activating clock input to the eyes is a rise in the concentration of cAMP in photoreceptors and the phosphorylation of a 122 kDa visual system-specific protein. We have cloned and sequenced cDNA encoding the clock-regulated 122 kDa phosphoprotein and show here that it is a new member of the myosin III family. We report that Limulus myosin III is similar to other unconventional myosins in that it binds to calmodulin in the absence of Ca 2ϩ ; it is novel in that it is phosphorylated within its myosin globular head, probably by cAMP-dependent protein kinase. The protein is present throughout the photoreceptor, including the region occupied by the photosensitive rhabdom. We propose that the phosphorylation of Limulus myosin III is involved in one or more of the structural and functional changes that occur in Limulus eyes in response to clock input.

In this study, we address the mechanism of visual arrestin release from light-activated rhodopsin using fluorescently labeled arrestin mutants. We find that two mutants, I72C and S251C, when labeled with the small, solvent-sensitive fluorophore monobromobimane, exhibit spectral changes only upon binding light-activated, phosphorylated rhodopsin. Our analysis indicates that these changes are probably due to a burying of the probes at these sites in the rhodopsin-arrestin or phospholipid-arrestin interface. Using a fluorescence approach based on this observation, we demonstrate that arrestin and retinal release are linked and are described by similar activation energies. However, at physiological temperatures, we find that arrestin slows the rate of retinal release ϳ2-fold and abolishes the pH dependence of retinal release. Using fluorescence, EPR, and biochemical approaches, we also find intriguing evidence that arrestin binds to a post-Meta II photodecay product, possibly Meta III. We speculate that arrestin regulates levels of free retinal in the rod cell to help limit the formation of damaging oxidative retinal adducts. Such adducts may contribute to diseases like atrophic age-related macular degeneration (AMD). Thus, arrestin may serve to both attenuate rhodopsin signaling and protect the cell from excessive retinal levels under bright light conditions.The visual photoreceptor rhodopsin is perhaps the best model system for understanding the mechanisms used in Gprotein-coupled receptor (GPCR) 1 signaling, as detailed information exists on the structures and dynamic interactions of the protein constituents (1). Visual activation begins with absorption of light by the 11-cis-retinal chromophore in rhodopsin. The photoactivated form of rhodopsin, Rho* or "Meta II," interacts with and activates the G-protein transducin, which exchanges nucleotide and then diffuses to interact with downstream effectors. Signaling by Rho* is terminated by slow thermal decay and the release of retinal. Alternatively, signaling can be quickly terminated by a process that begins with phosphorylation of rhodopsin's C-terminal tail through the action of rhodopsin kinase (2, 3). The phosphorylated Rho* is then bound by arrestin, which stops signaling by physically occluding the G-protein binding site (4, 5).In the present study, we address how these two inactivation mechanisms are related and, specifically, what governs arrestin release from rhodopsin. Arrestin is known to bind to phosphorylated Meta II, a form in which the photolyzed chromophore all-trans-retinal is still attached to the receptor by a deprotonated Schiff base. Arrestin does not bind phosphorylated opsin, but all-trans retinal added exogenously can stimulate arrestin binding to phosphorylated opsin (6, 7). Early studies showed indirectly that retinal release and arrestin release are probably interrelated events (7,8). However, how these processes are linked or whether arrestin binds other photointermediates of rhodopsin (such as the storage form Meta III) is still unknown...

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ABSTRACTcDNA clones encoding opsins from the lateral eyes and median ocelli of the horseshoe crab, Limulus polyphemus, were isolated from cDNA libraries. The opsin cDNAs obtained from the lateral eye and ocellar libraries code for deduced proteins with 376 amino acids. The two cDNAs are 96% identical at the nucleic acid level, differing primarily at the 3' untranslated region, and are apparently the products of two separate genes. (4)(5)(6) and the inactivation of rhodopsin (7,8). Even though all visual pigments known in the animal kingdom have the same basic composition of opsin and a vitamin A-derived chromophore, there are differences in some characteristics of the visual pigments between species, such as spectral absorbance, kinetics, or interaction with subsequent enzymes in the cascade. Since the chromophore is almost universally the same, changes in the structure ofthe opsin moiety must predominantly account for the differences observed in the various rhodopsins. Analysis of the primary structure of visual pigments from different species continues to define and illuminate the amino acid domains that are involved in these differences. In this study, we present the amino acid sequences for opsin from the lateral eye and median ocelli of the horseshoe crab. § Limulus is only the second class of arthropod from which the visual pigment has been sequenced. Further, to our knowledge this research is the first study of the primary sequence of any of the visualThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. system proteins in the horseshoe crab, despite the animal's longstanding significance in vision studies. MATERIALS AND METHODSThe basic strategy for identifying and sequencing the cDNA for Limulus opsin was to generate cDNA libraries from Limulus lateral eyes and ocelli, amplify a portion of the opsin cDNA by polymerase chain reaction (PCR) using degenerate oligonucleotide primers, and obtain the full-length cDNA by screening the library for opsin clones by using the amplified cDNA as a homologous probe.RNA

The results show that disruption of the cytoskeletal network in rod photoreceptors has specific effects on the translocation of arrestin and transducin. These effects suggest that the light-driven translocation of visual proteins at least partially relies on an active motor-driven mechanism for complete movement of arrestin and transducin.

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The photoreceptors of the horseshoe crab Limulus polyphemus are classical preparations for studies of the photoresponse and its modulation by circadian clocks. An extensive literature details their physiology and ultrastructure, but relatively little is known about their biochemical organization largely because of a lack of antibodies specific for Limulus photoreceptor proteins. We developed antibodies directed against Limulus opsin, visual arrestin, and myosin III, and we have used them to examine the distributions of these proteins in the Limulus visual system. We also used a commercial antibody to examine the distribution of calmodulin in Limulus photoreceptors. Fixed frozen sections of lateral eye were examined with conventional fluorescence microscopy; ventral photoreceptors were studied with confocal microscopy. Opsin, visual arrestin, myosin III, and calmodulin are all concentrated at the photosensitive rhabdomeral membrane, which is consistent with their participation in the photoresponse. Opsin and visual arrestin, but not myosin III or calmodulin, are also concentrated in extra-rhabdomeral vesicles thought to contain internalized rhabdomeral membrane. In addition, visual arrestin and myosin III were found widely distributed in the cytosol of photoreceptors, suggesting that they have functions in addition to their roles in phototransduction. Our results both clarify and raise new questions about the functions of opsin, visual arrestin, myosin III, and calmodulin in photoreceptors and set the stage for future studies of the impact of light and clock signals on the structure and function of photoreceptors.