Site of G protein binding to rhodopsin mapped with synthetic peptides from the alpha subunit.

The interaction between receptors and guanine nucleotide binding (G) proteins leads to G protein activation and subsequent regulation of effector enzymes. The molecular basis of receptor-G protein interaction has been examined by using the ability of the G protein from rods (transducin) to cause a conformational change in rhodopsin as an assay. Synthetic peptides corresponding to two regions near the carboxyl terminus of the G protein alpha subunit, Glu311-Val328 and Ile340-Phe350, compete with G protein for interaction with rhodopsin. Amino acid substitution studies show that Cys321 is required for this effect. Ile340-Phe350 and a modified peptide, acetyl-Glu311-Lys329-amide, mimic G protein effects on rhodopsin conformation, showing that these peptides bind to and stabilize the activated conformation of rhodopsin.

The amino-terminal tryptic peptide of bovine rhodopsin. A glycopeptide containing two sites of oligosaccharide attachment.

A glycopeptide (T1) composed of 16 amino acids has been isolated from a tryptic digest of bovine rhodopsin. Its sequence is Met-Asn(CHO)-Gly-Thr-Glu-Gly-Pro-Asn-Phe-Tyr-Val-Pro-Phe-Ser-Asn(CHO)-Lys. Both rhodopsin and peptide T1 are blocked at their amino terminals. When a method specific for isolating amino-terminal tryptic peptides from proteins is applied to rhodopsin, peptide T1 is demonstrated to be the amino-terminal peptide of rhodopsin. Peptide T1 contains two sites at which carbohydrate is attached, whereas rhodopsin was previously thought to contain only a single such site.

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.

The deduced amino-acid sequence of opsin from rabbit rod photoreceptors.

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).

X-ray crystal structure of sangivamycin, a potent inhibitor of protein kinases.

The X-ray crystal structure of sangivamycin, a potent nucleoside inhibitor of protein kinases, has been determined. Sangivamycin crystallizes from water with its purine ring in a conformation anti to its ribose sugar. Such an anti conformation has been detected in solution for sangivamycin and other potent protein kinase inhibitors and appears to correlate with inhibitor potency [(1990) Biochemistry (in press)]. An intramolecular hydrogen bond between purine ring substituents is detected in the X-ray structure and may be an important structural feature of sangivamycin related to its degree of inhibition of rhodopsin kinase and of protein kinases C and A.

Synthetic phosphopeptides are substrates for casein kinase II.

Casein kinase II is a protein serine/threonine kinase that exhibits a preference for acidic substrates. Previous studies have demonstrated that a glutamic acid 3 amino acids C-terminal (+3) to a serine or threonine is required for phosphorylation. To examine the ability of phosphoserine and phosphothreonine residues to serve as specificity determinants for casein kinase II, phosphopeptides containing either of these phosphoamino acids in the +3 position were synthesized and tested as substrates. Phosphopeptides containing phosphoserine in the +3 position were readily phosphorylated. In contrast, corresponding phosphothreonine-containing peptides were very poorly phosphorylated. These results imply that prior phosphorylation of substrate proteins on serine, but not threonine residues, may II.

Synthetic phosphopeptide from rhodopsin sequence induces retinal arrestin binding to photoactivated unphosphorylated rhodopsin.

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.

Genetic control of antibody response to bovine rhodopsin in mice: epitope mapping of rhodopsin structure.

Inbred strains of mice of independent haplotype were immunized with bovine rhodopsin. All mice tested except SJL developed significant titers of specific antibodies 21 days after a single immunization. Anti-rhodopsin antibody level differed among conventional inbred strains. Comparison of the immune response to rhodopsin of congenic mice on two different genetic backgrounds showed that animals with an A background typically produced higher levels of specific antibody than mice with a B10 background. Titer of specific antibodies in antisera of mice of the same H-2 haplotype but different Igh haplotype differed; e.g. for H-2d haplotype, NZB (Ighn) generated the highest level of antibody with BALB/c (Igha), DBA/2 (Ighc), and B10.D2 (Ighb) strains giving successively lower responses. The location of immunodominant regions of bovine rhodopsin was investigated in primary sera among strains of mice. Sera were tested for their binding of anti-rhodopsin antibodies to synthetic peptides covering the entire primary structure of rhodopsin. From direct binding studies with hydrophilic rhodopsin peptides, the majority of the antigenic binding sites were localized in the sequence of the amino terminus, the II-III loop and the carboxyl terminus. Binding to these antigenic peptides was not strain restricted. Application of the overlapping synthetic peptide strategy of Geysen enabled refinement of these epitopes and determination of an additional major epitope in the hydrophobic sequence 304-310.

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.

Role of anti-recoverin autoantibodies in cancer-associated retinopathy.

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.

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.

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.

Rod cell-specific antigens in retinoblastoma.

We investigated rhodopsin immunoreactivity in five well-differentiated retinoblastomas using a panel of monoclonal antibodies directed against specific antigenic sites in the amino- and carboxyl-terminal portions of rhodopsin. All five monoclonal antibodies bound to the rod cell outer segment of nontumorous retina in all 10% formaldehyde solution-fixed, paraffin-embedded tissue sections. A characteristic "halo" cell surface staining pattern was observed in four (80%) of five tumors treated with two monoclonal antibodies, B6-30 (rhodopsin amino-terminal specific) and K16-107 (rhodopsin carboxyl-terminal specific). In each case, the staining pattern was limited to well-differentiated areas of the tumor containing Flexner-Wintersteiner rosettes or fleurettes. One tumor was not stained by any monoclonal antibody, whereas all monoclonal antibodies stained the rod cell outer segments of nontumorous retina. Our studies indicate that selected retinoblastomas may be differentiated along a rod photoreceptorlike cell lineage.

The partial primary structure of bovine rhodopsin and its topography in the retinal rod cell disc membrane.

The amino-terminal 39 amino acids of bovine rhodopsin have the sequence where both carbohydrate attachment sites (CHO) contain GlcNAc(3)Man(3). This region of rhodopsin's sequence is exposed at the internal membrane surface of the rod cell disc membrane. Rhodopsin's carboxyl-terminal 40 amino acids have the sequence where amino acid 1? is the carboxyl-terminal amino acid of rhodopsin. Serines and threonines in the sequence 6? ? 15? are phosphorylated by rhodopsin kinase in a light-dependent reaction. Trypsin can digest native rhodopsin, in the disc membrane at and thermolysin can hydrolyze bonds , and . Limited proteolysis by thermolysin at a site internal in the molecule has been exploited in order to prepare rhodopsin as two large fragments, F1 and F2. Cysteine(33)?, is highly reactive in the dark and is modified by N-ethylmaleimide and several alkylating agents. The carboxyl-terminal region 1?-39? reacts with the membrane-impermeable nitrene from N-(4-azido-2-nitrophenyl)-2-aminoethyl sulfonate and is therefore exposed at the external (cytoplasmic) surface of the disc membrane. 1-azldopyrene, a hydrophobic nitrene precursor, is being used to map those regions of the rhodopsin sequence which are located in a hydrophobic environment.

Molecular biology of the visual pigments.

With the identification and structural characterization of several visual pigments has come a new era of investigation. The above comparisons of amino acids sequences predict specific functional domains that may be tested to tell us how visual pigments function to absorb light and transform this "signal" to trigger a neural response. The details of how rod and cone pigments differ are now known for human pigments. The striking similarities between vertebrate and invertebrate pigments are remarkable for pigments that have been subject to divergence for over 500 million years. There are yet challenges ahead of us. The true tertiary structure of visual pigments must be obtained from a 3-dimensional crystal structure. The predictions for functional domains of interaction with the GTP binding protein must be confirmed or redefined. A rigorous definition of the chromophore environment and the properties that control the wavelength of absorption of 11-cis retinal chromophore are certainly still on the drawing boards. Specific genetic alteration through in vitro mutagenesis promises much insight, but the technology for expressing these membrane proteins in functional form has yet to be achieved. We may expect, however, these problems will be addressed and in the next few years facts should replace what are now speculations. Finally, it is a delightful observation that nature has capitalized on a general biochemical mechanism for control of second messengers in the cytoplasm of cells. Protein structural data deduced from genetic information now document the hypothesis that the structure and function of receptors for the catecholamines and that of visual pigments are similar. The receptors for serotonin, leukotrienes, prostaglandins, histamine and acetylcholine (muscarinic) are expected to belong to this same family. The lessons learned about visual pigments can be applied broadly to a general set of membrane receptors.

Molecular, enzymatic and functional properties of rhodopsin kinase from rat pineal gland.

Rhodopsin kinase activity from rat pineal gland and from rat retina are indistinguishable, based upon determination of a variety of enzymatic and molecular properties. Both activities are independent of calcium, cyclic nucleotides, and calmodulin. Both are activated by spermine and inhibited by adenosine and some rhodopsin kinase specific adenosine derivatives such as sangivamycin. The Km's for rhodopsin, ATP, and GTP are indistinguishable for the protein kinase in extracts from the retina and from the pineal gland. The apparent molecular weight of the kinase from both sources, as determined by gel filtration and autoradiography of the 32P-labeled autophosphorylated kinase, is about 70 kDa. Rhodopsin kinase activity from pineal binds in a light-dependent manner to rhodopsin in rod outer segments as does the enzyme from retina. Monoclonal antibodies against bovine rhodopsin were used in an immunochemical study that identified a rhodopsin-immunoreactive protein in rat pineal gland and retina. Using an ELISA we demonstrated the presence of a rhodopsin-immunoreactive protein in rat pineal gland equivalent to 0.075 pmol rhodopsin per gland. Frog pineal organ (Rana catesbiana) contains 33 times more of this rhodopsin-like protein than does rat pineal gland.

Anti-rhodopsin monoclonal antibodies of defined specificity: characterization and application.

A panel of anti-bovine rhodopsin monoclonal antibodies (MAbs) of defined site-specificity has been prepared and used for functional and topographic studies of rhodopsins. In order to select these antibodies, hybridoma supernatants that contained anti-rhodopsin antibodies have been screened by enzyme-linked immunosorbent assay (ELISA) in the presence of synthetic peptides from rhodopsin's cytoplasmic regions. We selected for antibodies against predominantly linear determinants (as distinct from complex assembled determinants) and have isolated antibodies that recognize rhodopsin's amino terminus, its carboxyl terminus, as well as the hydrophilic helix-connecting regions 61-75, 96-115, 118-203, 230-252 and 310-321. Detailed specificities have been further determined by using a series of overlapping peptides and chemically modified rhodopsins as competitors. A group of seven antibodies with epitopes clustered within the amino terminal region of rhodopsin and a group of 15 antibodies with epitopes within the carboxyl terminal region are described. These MAbs have high affinities for rhodopsin with Kas in the range of 10(8)-10(10) M-1. Some MAbs specific for the carboxyl and amino terminal regions were used to compare these bovine rhodopsin sequences to those of different vertebrates. The MAbs cross-reacted with the different species tested to different extents indicating that there is some similarity in the sequences of these regions. However, some differences in the sequences were indicated by a reduced or absent cross-reactivity with some MAbs. In membrane topographic studies the MAbs showed both the presence and the accessibility of rhodopsin sequences 330-348, 310-321 and 230-252 on the cytoplasmic surface of the disk membrane. Similarly, sequences 1-20 and 188-203 were shown to reside on the lumenal surface of the disk and to be accessible to a macromolecular (antibody) probe. Antibodies directed against rhodopsin's carboxyl terminal sequence did not bind well to highly phosphorylated rhodopsin. Similarly, these antibodies as well as those against the V-VI loop inhibited phosphorylation of rhodopsin. Antibody A11-82P, specific for phosphorylated rhodopsin, recognized rhodopsin containing two or more phosphates and inhibited its further phosphorylation.

A rapid sensitive radioimmunoassay for rhodopsin: development and application.

Antisera have been raised against unbleached chromatographically pure bovine rhodopsin. A rapid sensitive radioimmunoassay (RIA) has been developed for rhodopsin, employing [125I]rhodopsin labeled using the Bolton-Hunter reagent. Protein A-bearing Staphylococcus aureus cells are used to precipitate the immune complex. Subpicomolar amounts of rhodopsin can be determined when the RIA is performed in the dark using any of nine different detergents tested. When the RIA is performed using bleached rhodopsin, the results are dependent upon the detergent in which the assay is performed. Bleached rhodopsin is most immunologically similar to unbleached rhodopsin in the mildest detergents and less similar in harsher detergents. One and a half times more bleached rhodopsin is required to compete to the same extent as unbleached rhodopsin when the RIA is performed in digitonin. Results for other detergents are: sucrose monoester, 5.0; CHAPS, 64; sodium cholate, 91; octylglucoside, 250; Triton X-100, 430; Emulphogene, 570; Ammonyx LO approximately 14,000; CTAB, unmeasureable. Other species of rhodopsin were tested as competitive in the RIA. Pig rhodopsin is 1/100th as effective a competitor as bovine rhodopsin, rat 1/200th, and frog 1/800th.

A monoclonal antibody that binds to photoreceptors in the turtle retina.

We have raised monoclonal antibodies to photoreceptor cells in the retina of the turtle (Pseudemys scripta elegans). One of these antibodies, 15-18 (an IgG1), was studied by immunoelectron microscopy using colloidal gold, and found to bind to the outer segments of all rods and some single cones, but did not stain turtle double cones. Immunoblotting and immunoprecipitation show that antibody 15-18 binds to an antigen of apparent Mr approximately 34,5000 which is probably turtle opsin. Antibody 15-18 binds visual pigments from several species, including bovine opsin. In order to determine the antigenic site bound by 15-18 in bovine opsin, synthetic peptides were used as competitors in an enzyme-linked immunoassay (ELISA). The antigenic site is located in the surface loop connecting rhodopsin helices IV-V, in the sequence 190-197. Antibody 15-18 binds to the external surface of rod cell outer segments, thus providing direct evidence for the predicted orientation of rhodopsin in the plasma membrane.