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Trauma workers may be at risk of secondary traumatic stress (STS) through indirect exposure to traumatic material, especially if they have experienced personal trauma. This is the first study to ask whether past trauma resolution influences STS and was examined in 64 volunteer crisis workers, a greatly ignored population. Those with nonresolved personal trauma had higher scores on an STS measure than volunteers whose trauma histories were resolved, while the latter showed less STS than the sample as a whole. STS was unrelated to volunteer experience, exposure to victims, or the type of cases found most distressing, indicating that accepted STS risk factors may not apply to volunteers. Findings have resounding implications for the popular view of trauma history as an STS risk factor: this may double as both a significant peril and a protection, depending on whether the past trauma is resolved. [ABSTRACT FROM AUTHOR]

Although sudden death has been linked to posttraumatic stress disorder (PTSD), its role in complicated grief (CG) and sudden death survivors is unknown. This questionnaire study investigated the role of peritraumatic distress in PTSD and CG symptoms in adults (n = 125) an average of 28.37 months (SD = 3.12) after a loved one's sudden death. The Peritraumatic Distress Inventory, Impact of Event Scale-Revised, and Inventory of Complicated Grief were administered to assess symptoms of peritraumatic distress, PTSD, and CG, respectively. Peritraumatic distress was the strongest correlate of both PTSD (β = .42, p < .001) and CG (β = .39, p < .001) symptoms, in a model containing current distress (Hopkins Symptom Checklist-21). Peritraumatic distress may be a key mechanism in the development of both PTSD and CG, therefore suddenly bereaved individuals reporting higher peritraumatic distress may be at risk of both adverse trauma and grief reactions., (Copyright © 2012 International Society for Traumatic Stress Studies.)

The maintenance of photoreceptor cell polarity is compromised by the rhodopsin mutations causing the human disease autosomal dominant retinitis pigmentosa. The severe form mutations occur in the C-terminal sorting signal of rhodopsin, VXPX-COOH. Here, we report that this sorting motif binds specifically to the small GTPase ARF4, a member of the ARF family of membrane budding and protein sorting regulators. The effects of blocking ARF4 action were functionally equivalent to the effects of blocking the rhodopsin C-terminal sorting signal. ARF4 was essential for the generation of post-Golgi carriers targeted to the rod outer segments of retinal photoreceptors. Thus, the severe retinitis pigmentosa alleles that affect the rhodopsin sorting signal interfere with interactions between ARF4 and rhodopsin, leading to aberrant trafficking and initiation of retinal degeneration.

Phosphorylation of activated G-protein-coupled receptors and the subsequent binding of arrestin mark major molecular events of homologous desensitization. In the visual system, interactions between arrestin and the phosphorylated rhodopsin are pivotal for proper termination of visual signals. By using high resolution proton nuclear magnetic resonance spectroscopy of the phosphorylated C terminus of rhodopsin, represented by a synthetic 7-phosphopolypeptide, we show that the arrestin-bound conformation is a well ordered helix-loop structure connected to rhodopsin via a flexible linker. In a model of the rhodopsin-arrestin complex, the phosphates point in the direction of arrestin and form a continuous negatively charged surface, which is stabilized by a number of positively charged lysine and arginine residues of arrestin. Opposite to the mostly extended structure of the unphosphorylated C-terminal domain of rhodopsin, the arrestin-bound C-terminal helix is a compact domain that occupies a central position between the cytoplasmic loops and occludes the key binding sites of transducin. In conjunction with other binding sites, the helix-loop structure provides a mechanism of shielding phosphates in the center of the rhodopsin-arrestin complex and appears critical in guiding arrestin for high affinity binding with rhodopsin.

Rhodopsin, the pigment protein responsible for dim-light vision, is a G protein-coupled receptor that converts light absorption into the activation of a G protein, transducin, to initiate the visual response. We have crystallised detergent-solubilised bovine rhodopsin in the native form and after chemical modifications as needles 10-40 microm in cross-section. The crystals belong to the trigonal space group P3(1), with two molecules of rhodopsin per asymmetric unit, related by a non-crystallographic 2-fold axis parallel with the crystallographic screw axis along c (needle axis). The unit cell dimensions are a=103.8 A, c=76.6 A for native rhodopsin, but vary over a wide range after heavy atom derivatisation, with a between 101.5 A and 113.9 A, and c between 76.6 A and 79.2 A. Rhodopsin molecules are packed with the bundle of transmembrane helices tilted from the c-axis by about 100 degrees . The two molecules in the asymmetric unit form contacts along the entire length of their transmembrane helices 5 in an antiparallel orientation, and they are stacked along the needle axis according to the 3-fold screw symmetry. Hence hydrophobic contacts are prominent at protein interfaces both along and normal to the needle axis. The best crystals of native rhodopsin in this crystal form diffracted X-rays from a microfocused synchrotron source to 2.55 A maximum resolution. We describe steps taken to extend the diffraction limit from about 10 A to 2.6 A.

Visual arrestin binds to the phosphorylated carboxy-terminal region of rhodopsin to block interactions with transducin and terminate signaling in the rod photoreceptor cells. A synthetic seven-phospho-peptide from the C-terminal region of rhodopsin, Rh(330-348), has been shown to bind arrestin and mimic inhibition of signal transduction. In this study, we examine conformational changes in this synthetic peptide upon binding to arrestin by high-resolution proton nuclear magnetic resonance (NMR). We show that the peptide is completely disordered in solution, but becomes structured upon binding to arrestin. A control, unphosphorylated peptide that fails to bind to arrestin remains highly disordered. Specific NMR distance constraints are used to model the arrestin-bound conformation. The models suggest that the phosphorylated carboxy-terminal region of rhodopsin, Rh(330-348), undergoes significant conformational changes and becomes structured upon binding to arrestin.

Rhodopsin is the best-understood member of the large G protein-coupled receptor (GPCR) superfamily. The G-protein amplification cascade is triggered by poorly understood light-induced conformational changes in rhodopsin that are homologous to changes caused by agonists in other GPCRs. We have applied the "antibody imprint" method to light-activated rhodopsin in native membranes by using nine monoclonal antibodies (mAbs) against aqueous faces of rhodopsin. Epitopes recognized by these mAbs were found by selection from random peptide libraries displayed on phage. A new computer algorithm, FINDMAP, was used to map the epitopes to discontinuous segments of rhodopsin that are distant in the primary sequence but are in close spatial proximity in the structure. The proximity of a segment of the N-terminal and the loop between helices VI and VIII found by FINDMAP is consistent with the X-ray structure of the dark-adapted rhodopsin. Epitopes to the cytoplasmic face segregated into two classes with different predicted spatial proximities of protein segments that correlate with different preferences of the antibodies for stabilizing the metarhodopsin I or metarhodopsin II conformations of light-excited rhodopsin. Epitopes of antibodies that stabilize metarhodopsin II indicate conformational changes from dark-adapted rhodopsin, including rearrangements of the C-terminal tail and altered exposure of the cytoplasmic end of helix VI, a portion of the C-3 loop, and helix VIII. As additional antibodies are subjected to antibody imprinting, this approach should provide increasingly detailed information on the conformation of light-excited rhodopsin and be applicable to structural studies of other challenging protein targets.

Subcellular translocation of phototransduction proteins in response to light has previously been detected by immunocytochemistry. This movement is consistent with the hypothesis that migration is part of a basic cellular mechanism regulating photoreceptor sensitivity. In order to monitor the putative migration of arrestin in response to light, we expressed a functional fusion between the signal transduction protein arrestin and green fluorescent protein (GFP) in rod photoreceptors of transgenic Xenopus laevis. In addition to confirming reports that arrestin is translocated, this alternative approach generated unique observations, raising new questions regarding the nature and time scale of migration. Confocal fluorescence microscopy was performed on fixed frozen retinal sections from tadpoles exposed to three different lighting conditions. A consistent pattern of localization emerged in each case. During early light exposure, arrestin-GFP levels diminished in the inner segments (ISs) and simultaneously increased in the outer segments (OSs), initially at the base and eventually at the distal tips as time progressed. Arrestin-GFP reached the distal tips of the photoreceptors by 45-75 min at which time the ratio of arrestin-GFP fluorescence in the OSs compared to the ISs was maximal. When dark-adaptation was initiated after 45 min of light exposure, arrestin-GFP rapidly re-localized to the ISs and axoneme within 30 min. Curiously, prolonged periods of light exposure also resulted in re-localization of arrestin-GFP. Between 150 and 240 min of light adaptation the arrestin-GFP in the ROS gradually declined until the pattern of arrestin-GFP localization was indistinguishable from that of dark-adapted photoreceptors. This distribution pattern was observed over a wide range of lighting intensity (25-2700 lux). Immunocytochemical analysis of arrestin in wild-type Xenopus retinas gave similar results.

Experimental autoimmune uveitis (EAU) is a disease of the neural retina induced by immunization with retinal antigens, such as interphotoreceptor retinoid-binding protein (IRBP) and arrestin (retinal soluble antigen, S-Ag). EAU serves as a model for human autoimmune uveitic diseases associated with major histocompatibility complex (HLA) genes, in which patients exhibit immunological responses to retinal antigens. Here we report the development of a humanized EAU model in HLA transgenic (TG) mice. HLA-DR3, -DR4, -DQ6, and -DQ8 TG mice were susceptible to IRBP-induced EAU. Importantly, HLA-DR3 TG mice developed severe EAU with S-Ag, to which wild-type mice are highly resistant. Lymphocyte proliferation was blocked by anti-HLA antibodies, confirming that antigen is functionally presented by the human MHC molecules. Disease could be transferred by immune cells with a Th1-like cytokine profile. Antigen-specific T cell repertoire, as manifested by responses to overlapping peptides derived from S-Ag or IRBP, differed from that of wild-type mice. Interestingly, DR3 TG mice, but not wild-type mice, recognized an immunodominant S-Ag epitope between residues 291 and 310 that overlaps with a region of S-Ag recognized by uveitis patients. Thus, EAU in HLA TG mice offers a new model of uveitis that should represent human disease more faithfully than currently existing models.

Visual arrestin is converted from a 'basal' state to an 'activated' state by interaction with the phosphorylated C-terminus of photoactivated rhodopsin (R*), but the conformational changes in arrestin that lead to activation are unknown. Small-angle X-ray scattering (SAXS) was used to investigate the solution structure of arrestin and characterize changes attendant upon activation. Wild-type arrestin forms dimers with a dissociation constant of 60 micro m. Small conformational changes, consistent with local movements of loops or the mobile N- or C-termini of arrestin, were observed in the presence of a phosphopeptide corresponding to the C-terminus of rhodopsin, and with an R175Q mutant. Because both the phosphopeptide and the R175Q mutation promote binding to unphosphorylated R*, we conclude that arrestin is activated by subtle conformational changes. Most of the arrestin will be in a dimeric state in vivo. Using the arrestin structure as a guide [Hirsch, J.A., Schubert, C., Gurevich, V.V. & Sigler, P.B. (1999) Cell 97, 257-269], we have identified a model for the arrestin dimer that is consistent with our SAXS data. In this model, dimerization is mediated by the C-terminal domain of arrestin, leaving the N-terminal domains free for interaction with phosphorylated R*.

Lewis rats immunized with myelin basic protein (MBP) developed experimental autoimmune encephalomyelitis (EAE) and associated anterior uveitis (AU). Although several cryptic epitopes of MBP have strong encephalitogenic and uveitogenic properties, the peptide corresponding to the MBP residues 1-20 was uniquely capable of inducing AU without EAE. In this study, we showed that acetylation of the N-terminal amino acid did not produce encephalitogenicity, did not enhance uveitogenicity, and did not improve T cell proliferation in Lewis rats. The cytokine production profile induced by MBP(1-20) immunization was consistent with a Th1 response. In MBP-injected rats and in peptide-injected rats, the frequency of the IFN-gamma-secreting cells in MBP(69-89)-stimulated T cells was significantly higher than the frequency of IFN-gamma-secreting cells in MBP(1-20)-stimulated T cells. However, similar numbers of IFN-gamma-producing specific cells were found in the eyes of MBP(69-89) and MBP(1-20) immunized rats. In these rats, the iris-infiltrating cells consisted of a much higher percentage of CD4(+) T cells expressing L-selectin (CD62L) than did those cells found in the spinal cord. The results demonstrate that MBP(1-20) is immunogenic and uveitogenic, although it induced only weak proliferation and weak Th1 reaction. The fact that T cells with the same specificity have different effects on target organs suggested that, in the eye and spinal cord, a distinct mechanism might mediate the recruitment of cells to these organs.

Visual arrestin inactivates the phototransduction cascade by specifically binding to light-activated phosphorylated rhodopsin. This study describes the combined use of insertional mutagenesis and immunochemical approaches to probe the structural determinants of arrestin function. Recombinant arrestins with insertions of a 10-amino acid c-Myc tag (EQKLISEEDL) were expressed in yeast and characterized. When the tag was placed on the C terminus after amino acid 399, between amino acids 99 and 100 or between residues 162 and 163, binding to rhodopsin was found to be very similar to that of wild-type arrestin. Two stable mutants with Myc insertions in the 68-78 loop were also generated. Binding to rhodopsin was markedly decreased for one (72myc73) and completely abolished for the other (77myc78). Limited proteolysis assays using trypsin in the absence or presence of heparin were performed on all mutants and confirmed their overall conformational integrity. Rhodopsin binding to either 162myc163 or 72myc73 arrestins in solution was completely inhibited in the presence of less than a 2-fold molar excess of anti-Myc antibody relative to arrestin. In contrast, the antibody did not block the interaction of the 399myc or 99myc100 arrestins with rhodopsin. These results indicate that an interactive surface for rhodopsin is located on or near the concave region of the N-domain of arrestin.

Purpose: To determine whether substitution of the potential phosphorylation sites of bovine rhodopsin's carboxyl-terminal region with the acidic residues aspartic acid, glutamic acid, or cysteic acid promotes the activation of arrestin., Methods: Three peptide analogues of the 19-residue carboxyl-terminal region of rhodopsin (330-348) were synthesized: the fully phosphorylated peptide (7P-peptide), the peptide with all potential phosphorylation sites substituted with glutamic acid (7E-peptide), and the peptide with the phosphorylation sites substituted with cysteic acid (7Cya-peptide). The peptides were tested in assays in which the 7P-peptide had previously been shown to have an effect. Rhodopsin with glutamic acid (Etail) or aspartic acid (Dtail) substituted for the phosphorylation sites in rhodopsin were constructed and expressed in COS-7 cells and tested in an in vitro assay., Results: Earlier work has demonstrated that the 7P-peptide activates arrestin, showing induction of arrestin binding to light-activated unphosphorylated rhodopsin, inhibition of the light-induced phosphodiesterase (PDE) activity in rod outer segments (ROS) with excess arrestin, increase in the initial rapid proteolysis of arrestin by trypsin, and enhanced reactivity of one of arrestin's sulfhydryl groups with inhibition of the reactivity of another. None of these effects was observed in the presence of 7E-peptide or 7Cya-peptide. The 7Cya-peptide inhibited the PDE activity in ROS, but the same effect was observed both in the presence and the absence of excess arrestin. Because none of the other effects was observed with the 7Cya-peptide, the authors conclude that the 7Cya-peptide does not activate arrestin, but acts, probably nonspecifically, through some other part of the transduction system. Considerable arrestin-mediated rhodopsin inactivation was observed with both the Etail and the Dtail mutant, although these substitutions did not yield rhodopsins that were equivalent to phosphorylated rhodopsin., Conclusions: These results, taken together, suggest that the negative charge due to phosphates in the carboxyl-terminal region of rhodopsin are required for the full activation of arrestin and that acidic amino acids (carboxyl and sulfonic) do not mimic the negative charge of phosphorylated residues.

Lewis rats immunized with myelin basic protein (MBP) develop experimental autoimmune encephalomyelitis (EAE) and associated anterior uveitis (AU), which can relapse without recurring of EAE. In this study, we analyzed the repertoire of MBP epitopes that play a role in acute and recurrent AU by injection of MBP synthetic peptides. In addition to the encephalitogenic epitopes 69-89 and 87-99, several cryptic epitopes were found to be strongly uveitogenic in Lewis rats upon immunization with synthetic peptides, including 100-120, 121-140 and 142-167. However, the peptide corresponding to the MBP residues 1-20 was uniquely capable of inducing AU without EAE. Immunization with intact MBP was not essential for the induction of the recurrence of AU. The responses of T cells from lymph nodes and spleens showed a dominant response to the original disease-induced epitope with responses to secondary epitopes. In conclusion, the analysis of pathogenic determinants important for the induction of uveitis provides further evidence that MBP-specific T cells also contribute to the pathogenesis of anterior uveitis. Moreover, this also suggests that a distinct immunoregulatory mechanism exists in the eye and spinal cord because of the uniqueness of the epitope 1-20 in AU but not EAE, and the capability of MBP-specific T cells of inducing AU without EAE.

Lewis rats immunized with myelin basic protein (MBP) develop experimental autoimmune encephalomyelitis (EAE) and associated anterior uveitis (AU). Rats recover and become resistant to further reinduction of EAE. We investigated whether the resistance to reinduction of EAE was associated with the resistance to AU in LEW rats reinjected with MBP. We demonstrated that while rats remained resistant to EAE, they become susceptible to uveitis after recovery, and suffered a second episode of disease. The susceptibility to reinduced disease was associated with the recognition of new MBP epitopes. In contrast to the initial episode of AU, TCR Vbeta8.2 predominance was not observed in the iris/ciliary body. Our results suggest that T cells specific for MBP, which are rapidly reactivated when re-exposed to antigen, are sufficient to induce clinical uveitis in LEW rats. This process may involve a shifting of T cell specificity from the major encephalitogenic peptide utilizing the Vbeta8.2 receptor to a more diverse cell repertoire.

The sulfhydryl groups of the three cysteines in bovine arrestin react with DTNB very slowly (over a period of several hours). In the presence of the synthetic phosphopeptide comprising the fully phosphorylated carboxyl-terminal 19 amino acids of bovine rhodopsin, the reactivity of one of the sulfhydryls was enhanced while that of another was greatly reduced. Since this synthetic peptide was shown to activate arrestin with respect to its binding to unphosphorylated, light-activated rhodopsin, the reactivity of the sulfhydryl groups of a constitutively active R175Q arrestin mutant was examined. All three of the sulfhydryl groups of the mutant arrestin R175Q reacted rapidly with DTNB, but not as rapidly as with SDS-denatured arrestin. The arrestin mutant R175Q bound to light-activated, unphosphorylated rhodopsin in ROS disk membranes. The arrestin mutant R175Q also inhibited the light-activated PDE activity with an IC50 of 1.3 microM under the experimental conditions that were used. These data indicate that each of these forms of arrestin is a different conformation. The activated conformation of arrestin that binds to phosphorylated rhodopsin in vivo may be yet another conformation. We conclude that arrestin is a flexible molecule that is able to attain several different conformations, all of which are able to attain the activated functional state of arrestin.

Purpose: The presence of anti-rhodopsin antibodies in patients with normal pressure glaucoma (NPG) has been previously demonstrated with western blot analysis and enzyme-linked immunosorbent assay. To learn more about the characteristics, origin, and possible significance of these antibodies, the epitopic specificity of the anti-rhodopsin antibodies was examined in four NPG patients., Methods: Antibodies in patient sera were assayed by western blot analysis against purified bovine rhodopsin. Peptides derived from particular segments of the rhodopsin sequence were tested for activity in competing for rhodopsin-antibody binding., Results: Of a series of nine peptides that constitute most of the hydrophilic regions of rhodopsin, only one, consisting of the C-terminal 25 amino acids, prevented binding of the patient antibodies to rhodopsin. Higher resolution mapping using a set of dodecamers of overlapping sequences from the C-terminal region demonstrated that antibody binding is completely dependent on the last two amino acids. Removing the C-terminal alanine alone, or amidating the C terminus carboxyl group, also eliminated antibody binding., Conclusions: Because four of four patient antibodies examined exhibited the identical epitopic specificity, it is likely that a common mechanism underlies their generation. This may indicate that molecular mimicry has occurred, because several pathogens contain similar C-terminal sequences. Although they may serve as diagnostic markers, and provide evidence that there is an autoimmune component in some patients with glaucoma, the role, if any, that these antibodies play in the pathogenesis of the disease remains unknown.

Arrestin facilitates phototransduction inactivation through binding to photoactivated and phosphorylated rhodopsin (RP). However, the specific portions of arrestin that bind to RP are not known. In this study, two different approaches were used to determine the regions of arrestin that bind to rhodopsin: panning of phage-displayed arrestin fragments against RP and cGMP phosphodiesterase (PDE) activity inhibition using synthetic arrestin peptides spanning the entire arrestin protein. Phage display indicated the predominant region of binding was contained within amino acids 90-140. A portion of this region (residues 95-140) expressed as a fusion protein with glutathione S-transferase is capable of binding to rhodopsin regardless of the activation or phosphorylation state of the receptor. Within this region, the synthetic peptide of residues 109-130 was shown to completely inhibit the binding of arrestin to rhodopsin with an IC50 of 1.1 mM. The relatively high IC50 of this competition suggests that this portion of the molecule may be only one of several regions of binding between arrestin and RP. A survey of synthetic arrestin peptides in the PDE assay indicated that the two most effective inhibitors of PDE activity were peptides of residues 111-130 and 101-120. These results indicate that at least one of the principal regions of binding between arrestin and RP is contained within the region of residues 109-130.

Regulation of cAMP and cGMP production is a fundamental step in a broad range of signal transduction systems, including phototransduction. To identify regions within photoreceptor guanylyl cyclase 1 (GC1) that interact with GC-activating proteins (GCAPs), we synthesized the intracellular fragment of GC1, residues 491-1110, as a set of 15 amino acid long, partially overlapping peptides on the surface of individual pins arranged in a microtiter plate format. This pin assay identified 8 peptides derived from different regions of the GC1 intracellular domain that bind GCAPs. Peptide variants containing these sequences were synthesized as free peptides and tested for their ability to inhibit GC1 stimulation by GCAPs. A free peptide,968GTFRMRHMPEVPVRIRIG, from the catalytic domain of GC1 was the strongest inhibitor of GCAP1/GCAP2-mediated activation. In native GC1, this polypeptide fragment is likely to form a loop between alpha-helix 3 and beta-strand 4. When this region in GC1 was replaced by the corresponding sequence of GCAP-insensitive GC type A, GCAPs did not stimulate the GC1 mutant. The corresponding loops in related adenylyl cyclase (AC) are involved in the activating and inhibiting interactions with Gs alpha and Gi alpha, respectively. Thus, despite interacting with different activating proteins, both AC and GC activity may be modulated through their respective regions within catalytic domains.

Upon activation by light, rhodopsin is subject to phosphorylation by rhodopsin kinase at serine and threonine residues in the carboxyl terminal region of the protein. A 19 amino acid peptide that corresponds to the carboxyl terminal end of rhodopsin (residues 330-348) and contains these phosphorylation sites was synthesized. The structure of this peptide was determined using two-dimensional proton NMR. The structure of this peptide was similar to that determined for this region in peptides corresponding to the carboxyl 33 and 43 amino acids of rhodopsin. The effect of phosphorylation on the structure of the carboxyl terminal domain of rhodopsin was determined by solving the solution structures of the peptide containing residues 330-348 with phosphorylation at one (residue 343), three (residues 343, 338, and 334) and seven residues (residues 334, 335, 336, 338, 340, 342, 343). These data indicate that the major structural change occurs upon phosphorylation of the first residue, and that an additional structural change occurs with seven phosphates.

Several mutations that cause severe forms of the human disease autosomal dominant retinitis pigmentosa cluster in the C-terminal region of rhodopsin. Recent studies have implicated the C-terminal domain of rhodopsin in its trafficking on specialized post-Golgi membranes to the rod outer segment of the photoreceptor cell. Here we used synthetic peptides as competitive inhibitors of rhodopsin trafficking in the frog retinal cell-free system to delineate the potential regulatory sequence within the C terminus of rhodopsin and model the effects of severe retinitis pigmentosa alleles on rhodopsin sorting. The rhodopsin C-terminal sequence QVS(A)PA is highly conserved among different species. Peptides that correspond to the C terminus of bovine (amino acids 324-348) and frog (amino acids 330-354) rhodopsin inhibited post-Golgi trafficking by 50% and 60%, respectively, and arrested newly synthesized rhodopsin in the trans-Golgi network. Peptides corresponding to the cytoplasmic loops of rhodopsin and other control peptides had no effect. When three naturally occurring mutations: Q344ter (lacking the last five amino acids QVAPA), V345M, and P347S were introduced into the frog C-terminal peptide, the inhibitory activity of the peptides was no longer detectable. These observations suggest that the amino acids QVS(A)PA comprise a signal that is recognized by specific factors in the trans-Golgi network. A lack of recognition of this sequence, because of mutations in the last five amino acids causing autosomal dominant retinitis pigmentosa, most likely results in abnormal post-Golgi membrane formation and in an aberrant subcellular localization of rhodopsin.

The frequency of thermal 'dark events' in the membrane current of rhodopsin rods of the bullfrog, Rana catesbeiana, is considerably lower than observed in rods of two toad species, even though all three rhodopsins have approximately the same absorbance characteristics. In order to map amino acid substitutions possibly associated with thermal stability in the genus Rana, the cDNA's coding for the rhodopsins of Bufo bufo, B. marinus and R. temporaria were sequenced and the conceptually translated protein sequences aligned to the previously sequenced rhodopsins of R. catesbeiana, R. pipiens and Xenopus laevis. Across the six anuran species studied, there are sixteen non-conserved substitutions and six changes that include gain or loss of a hydroxyl group. Serine or threonine at position 220 is unique to the three Rana species, phenylalanine at position 270 is unique to all three Ranas and to X. laevis, and phenylalanine at position 274 is unique to both species of the genus Bufo. This investigation produces a list of substitutions that are candidates for future studies of thermal stability. In addition, a number of amino acids are identified that apparently do not influence absorbance characteristics, at least not cumulatively., (Copyright 1998 Academic Press Limited.)

Rhodopsins, the photoreceptors in rod cells, are G-protein-coupled receptors with seven hydrophobic segments containing characteristic conserved sequence patterns that define a large family. Members of the family are expected to share a conserved transmembrane structure. Direct evidence for the arrangement of seven alpha-helices was obtained from a 9A projection map of bovine rhodopsin. Structural constraints inferred from a comparison of G-protein-coupled receptor sequences were used to assign the seven hydrophobic stretches in the sequence to features in the projection map. A low-resolution three-dimensional structure of bovine rhodopsin and two projection structures of frog rhodopsin confirmed the position of the three least tilted helices, 4, 6 and 7. A more elongated peak of density for helix 5 indicated that it is tilted or bent, but helices 1, 2 and 3 were not resolved. Here we have used electron micrographs of frozen-hydrated two-dimensional frog rhodopsin crystals to determine the structure of frog rhodopsin. Seven rods of density in the map are used to estimate tilt angles for the seven helices. Density visible on the extracellular side of the membrane suggests a folded domain. Density extends from helix 6 on the intracellular side, and a short connection between helices 1 and 2, and possibly a part of the carboxy terminus, are visible.

We have previously cloned the opsins coding for the long-wavelength visual pigments from the Saharan silver ant and carpenter ant. Here we report two new cDNA clones isolated from cDNA libraries which also code for opsin proteins. These cDNAs code for deduced proteins with 369 amino acids which are 91% identical to each other, but only 38% identical to the previously cloned opsins. Phyletic comparisons suggest that these opsins are likely the ultraviolet sensitive visual pigments, a conclusion that is supported by the presence of a phenylalanine at the counterion position in the third transmembrane segment.

In rod and cone photoreceptor cells, activation of particulate guanylate cyclase (retGC1) is mediated by a Ca2+-binding protein termed GCAP1, that detects changes in [Ca2+]free. In this study, we show that N-acylated GCAP1 restored Ca2+ sensitivity of native and recombinant photoreceptor retGC1. ATP increased the affinity of retGC1 for GCAP1 and accelerated catalysis. Using peptides derived from the GCAP1 sequence, we found that at least three regions, encompassing the N-terminus, the EF-1 motif, and the EF-3 motif, were likely involved in the interaction with retGC1. Mutation of 2Gly to Ala (GCAP1-G2A), which abolished myristoylation and a 25 amino acid truncation at the N-terminus (delta25-GCAP1) reduced retGC1-stimulating activity dramatically, while deletion of 10 amino acids (delta10-GCAP1) reduced the specific activity by only approximately 60% and modified the Ca2+ sensitivity. At 10(-6) M [Ca2+]free, in conditions that inactivated native GCAP1, retGC1 showed significant activity in the presence of delta10-GCAP1. Native and all three mutant forms of GCAP1 had similar affinities for Ca2+ as demonstrated by gel filtration and the changes in tryptophan fluorescence. All mutants bound to ROS membranes in a Ca2+-independent manner, except delta25-GCAP1, which was mostly soluble. These findings suggest that the N-terminal region is important in tethering of GCAP1 to the ROS membranes.

IgG Ab titers elicited to bovine rhodopsin in CFA differ 8- to 10-fold between H2s identical inbred strains A.SW/snJ (high responder) and SJL/snJ (low responder). This variation in IgG Ab titer resulted from a dramatic difference in the rise in Ab titer occurring during the maturation of the T-dependent humoral immune response. To determine the positions of non-MHC genes controlling this quantitative variation in T-dependent humoral immune responsiveness, 206 reciprocal (A.SW/snJ x SJL/snJ)F2 female progeny were immunized and assayed for anti-rhodopsin responsiveness. The genomes of these progeny were screened with 115 polymorphic simple sequence repeat markers covering >90% of the mouse genome. interval mapping analysis localized the positions of these non-MHC immune response genes to genomic intervals on chromosomes 1, 5, and 13. Interestingly, these three intervals coincide exactly with three intervals recently shown to contain genes contributing to susceptibility to systemic lupus erythematosus and/or the production of autoimmune anti-dsDNA Abs. These results suggest that some genes affecting levels of humoral immune responsiveness to exogenous Ag may also play a role in genetic susceptibility to humoral autoimmune diseases. Analyses of the modes of inheritance demonstrated that high responder alleles were inherited from both parental genomes, indicative of epistatic interactions among genes influencing humoral immune responsiveness.

cDNA clones encoding opsins from compound eyes of carpenter ant, Camponotus abdominalis, and Saharan silver ant, Cataglyphis bombycina, were isolated from cDNA libraries. The opsin cDNAs from each species code for deduced proteins with 378 amino acids which are 92% identical. Of the 30 amino acid differences between the two proteins, 13 are non-conservative. Eight of these non-conservative substitutions are within the membrane spanning domain. The presence of a potential Schiff-base counterion in helix III in both species suggests that these opsins are the protein moiety of the visible range pigments. When compared to all known opsins, these opsins are most similar to the opsin from preying mantis (76% identity at the amino acid level). Phyletic comparisons group the two ant opsins with the other arthropod long wavelength opsins.

Cancer-associated retinopathy (CAR) is an uncommon paraneoplastic disease in which degeneration of the retina occurs as a remote effect of cancer in a distant part of the body. Immunoreactivity of sera from CAR patients and controls have been analyzed. Immunostaining of human retinal proteins showed that a soluble protein of Mr approximately 46 kDa (p46) is labeled by antibodies from several CAR patients with various types of cancer (lung, breast, bladder, prostate, salivary gland, and gastrointestinal tract cancer and chronic lymphocytic leukemia). These sera did not show reactivity with the 23-kDa protein previously associated with CAR. To identify and further characterize p46, the retinal protein was purified to homogeneity by anion-exchange chromatography and preparative gel electrophoresis. Protein sequence analysis of the peptides from p46 revealed a high homology with human enolase, an important glycolytic enzyme. Although enolase has been previously identified as a product of several types of tumors, and enolase activity has been detected in the sera of some cancer patients, the existence of autoantibodies directed to enolase has not been described. This is the first report of the presence of serum antibodies to retinal enolase in the patients with cancer and the CAR syndrome. When antibodies of specific isotypes (IgG, IgM, and IgA) were measured, IgG1 isotype was dominant. The significance of these antibodies for the disease process is under investigation.

Rhodopsin is the G protein-coupled receptor that upon light activation triggers the visual transduction cascade. Rod cell outer segment disc membranes were isolated from dark-adapted frog retinas and were extracted with Tween detergents to obtain two-dimensional rhodopsin crystals for electron crystallography. When Tween 80 was used, tubular structures with a p2 lattice (a = 32 A, b = 83 A, gamma = 91 degrees) were formed. The use of a Tween 80/Tween 20 mixture favored the formation of larger p22(1)2(1) lattices (a = 40 A, b = 146 A, gamma = 90 degrees). Micrographs from frozen hydrated frog rhodopsin crystals were processed, and projection structures to 7-A resolution for the p22(1)2(1) form and to 6-A resolution for the p2 form were calculated. The maps of frog rhodopsin in both crystal forms are very similar to the 9-A map obtained previously for bovine rhodopsin and show that the arrangement of the helices is the same. In a tentative topographic model, helices 4, 6, and 7 are nearly perpendicular to the plane of the membrane. In the higher-resolution projection maps of frog rhodopsin, helix 5 looks more tilted than it appeared previously. The quality of the two frog rhodopsin crystals suggests that they would be suitable to obtain a three-dimensional structure in which all helices would be resolved.

Members of the arrestin protein family are known to participate in the inactivation of rhodopsin and other heptahelical receptors. Arrestins bind to the activated and phosphorylated state of these receptors, consequently blocking the ability of the receptors to activate the guanine-nucleotide-binding protein (G protein). We have determined the sequences of four retinal arrestins from two species of frog, Rana catesbeiana and Rana pipiens. Using polymerase chain reaction on reverse-transcribed mRNA isolated from single photoreceptor cells, we show that two of these arrestins are from rod photoreceptors and two rod photoreceptors and two are from cone photoreceptors. Comparison of these arrestins with the twenty known arrestin sequences identifies three regions of the protein that are well conserved across all phylogenetic groups. These regions may function in the binding of the arrestin to the heptahelical receptors. In addition, the Rana arrestins contain a uniquely acidic C-terminal sequence.

We sequenced selected peptides of alligator rhodopsin that accounted for about half of the total protein. These sequences were confirmed when the total primary structure of alligator rhodopsin was deduced from the cDNA sequence. Differences in the amino-terminal region, compared to that of bovine rhodopsin, account for failure of cross-reactivity of several anti-bovine rhodopsin monoclonal antibodies. Differences in the carboxyl-terminal region give rise to limited antibody cross-reactivity and may also account for a slightly reduced ability of alligator rhodopsin to be phosphorylated by bovine rhodopsin kinase. Alligator rhodopsin regenerates much faster than bovine rhodopsin. The pseudo-first-order rate constant for alligator rhodopsin regeneration is approximately 25 times that of bovine. Phylogenetic analysis of 17 rhodopsin sequences indicates that the alligator is more closely related to the chicken than to the other species examined.

The amino acid (aa) sequence of rabbit opsin from rod photoreceptor cells was determined by direct aa sequencing and conceptual translation from the cDNA. The cDNA (1198 bp) containing the complete coding region encodes a 348-aa opsin protein. Of the 16 rod cell opsins that are known, rabbit opsin is most similar to human opsin (96.3% identity at the aa level).

A synthetic heptaphosphopeptide comprising the fully phosphorylated carboxyl terminal phosphorylation region of bovine rhodopsin, residues 330-348, was found to induce a conformational change in bovine arrestin. This caused an alteration of the pattern of limited proteolysis of arrestin similar to that induced by binding phosphorylated rhodopsin or heparin. Unlike heparin, the phosphopeptide also induced light-activated binding of arrestin to both unphosphorylated rhodopsin in disk membranes as well as to endoproteinase Asp-N-treated rhodopsin (des 330-348). These findings suggest that one function of phosphorylation of rhodopsin is to activate arrestin which can then bind to other regions of the surface of the photoactivated rhodopsin.

Inactivation of photolyzed rhodopsin requires phosphorylation of the receptor and binding of the 48-kDa regulatory protein arrestin. We recently isolated a novel form of arrestin, termed p44, that is truncated at the COOH terminus (Palczewski, K., Buczylko, J., Ohguro, H., Annan, R. S., Carr, S. A., Crabb, J. W., Kaplan, M. W., Johnson, R. S., and Walsh, K. A. (1994) Protein Sci. 3, 319-329) and strongly inhibits Gt activation by non-phosphorylated rhodopsin. p44 is identical to arrestin except at the COOH terminus, where the 35 amino acids of arrestin are replaced by a single alanine residue. p44 is identified as a splice variant of arrestin based on the identical cDNA sequence of p44 with arrestin (except the 3' non-coding regions), the presence of an exon/intron junction at the Ser369 codon, and identical Southern hybridization patterns generated by the 3' non-coding portion of arrestin and p44. Immunocytochemistry reveals that p44 is localized in the photoreceptor outer segment, whereas arrestin is present throughout the cell. This specificity of localization to the outer segment is consistent with a role of p44 in the phototransduction cascade.

Multipin solid-phase peptide synthesis is widely used for epitope mapping of monoclonal and polyclonal antibodies. However, neither the chemical yield nor the homogeneity of products currently match those of solid-phase synthesis of peptides on resins. In order to improve synthesis parameters, we have repeated the standard procedure and introduced modifications during synthesis of model heptapeptides and peptides from the sequence of rhodopsin and other proteins. Good incorporation of amino acids using the multipin peptide synthesis system can now be obtained in less synthesis time and with less costly reagents.

Purpose: To examine the retina and test the serum of a patient with cancer-associated retinopathy syndrome who was diagnosed with small cell carcinoma of the lung and experienced unexpected visual loss., Methods: Proteins from normal human retina were extracted, separated by one- and two-dimensional gel electrophoresis, transferred to PVDF membrane, and used for immunostaining. Antibody specificity was determined by use of solid-phase peptides in a solid-phase immunoassay., Results: Histologic examination of the retina showed loss of the photoreceptor cell layer. This finding correlated with the results of clinical (loss of vision) and electrophysiologic (abnormal electroretinograph [ERG]) tests. The patient's serum antibodies specifically recognized recoverin, a protein predominantly found in retinal photoreceptor cells. The patient's serum also labeled some higher molecular weight proteins present in normal lung and other normal tissues, as well as in lung cell carcinoma cell lines. The only other tissue in which immunoreactivity against p23 could be found was the optic nerve. Our data revealed a lack of cross-reactivity between specific anti-recoverin antibodies and lung proteins. The results indicate that the patient serum contains more than one type of antibody activity. The autoantibodies were tested for fine immunospecificity by use of solid-phase peptides in a solid-phase immunoassay. Patient's antibodies reacted with a major determinant located in the recoverin sequence 62-68 (PKAYAQH) and with several minor ones., Conclusion: Based on the fact that the recoverin appears to be distributed in several different cell types, we suggest that this protein may be present in cancer cells and may play a role in the pathogenesis of some cancer-associated retinopathies.

Rhodopsin kinase catalyzes the incorporation of up to seven phosphates into the carboxyl terminal region of freshly bleached rhodopsin. In order to study the mechanism of this reaction, we have separated different phosphorylated species of rhodopsin using Mono P FPLC chromatofocusing chromatography. The purity of the isolated species of rhodopsin was determined by isoelectric focusing. Separation yielded two forms of monophosphorylated and two diphosphorylated species of rhodopsin. Other species, containing up to five phosphates, were not fully separated. The phosphorylated forms of rhodopsin were characterized by competition enzyme-linked immunosorbent assay and immunoblotting using anti-rhodopsin site-specific monoclonal antibodies. A combination of the above methods allowed quantitative determination of the formation of different phosphorylated species of rhodopsin. A computer model for the consecutive time course of rhodopsin phosphorylation was developed and employed to characterize this reaction. Our data suggest that the rate of incorporation of the first phosphates into rhodopsin is slower than the rate of formation of more highly phosphorylated species. These data are supported by results showing that some monophosphorylated synthetic peptides are phosphorylated significantly faster than control unphosphorylated peptides.