Activation of arrestin: requirement of phosphorylation as the negative charge on residues in synthetic peptides from the carboxyl-terminal region of rhodopsin.

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.

Importance of cryptic myelin basic protein epitopes in the pathogenicity of acute and recurrent anterior uveitis associated with EAE.

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.

Epitope recognition and T cell receptors in recurrent autoimmune anterior uveitis in Lewis rats immunized with myelin basic protein.

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.

Sulfhydryl reactivity demonstrates different conformational states for arrestin, arrestin activated by a synthetic phosphopeptide, and constitutively active arrestin.

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.

Epitope mapping of anti-rhodopsin antibodies from patients with normal pressure glaucoma.

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.

Identification of regions of arrestin that bind to rhodopsin.

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.

Identification of a guanylyl cyclase-activating protein-binding site within the catalytic domain of retinal guanylyl cyclase 1.

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.

Effects of phosphorylation on the structure of the G-protein receptor rhodopsin.

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.

Regulation of sorting and post-Golgi trafficking of rhodopsin by its C-terminal sequence QVS(A)PA.

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.

Rhodopsins from three frog and toad species: sequences and functional comparisons.

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.

Arrangement of rhodopsin transmembrane alpha-helices.

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.

Short wavelength-sensitive opsins from the Saharan silver and carpenter ants.

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.

Functional reconstitution of photoreceptor guanylate cyclase with native and mutant forms of guanylate cyclase-activating protein 1.

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.

Interval mapping of quantitative trait loci controlling humoral immunity to exogenous antigens: evidence that non-MHC immune response genes may also influence susceptibility to autoimmunity.

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.

Ant opsins: sequences from the Saharan silver ant and the carpenter ant.

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.

The occurrence of serum autoantibodies against enolase in cancer-associated retinopathy.

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.