Paul A. Hargrave

Arrangement of rhodopsin transmembrane α-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 α-helices was obtained from a 9å 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.

The Structure of Bovine Rhodopsin

We have isolated 16 peptides from a cyanogen bromide digest of rhodopsin. These cyanogen bromide peptides account for the complete composition of the protein. Methionine-containing peptides from other chemical and enzymatic digests of rhodopsin have allowed us to place the cyanogen bromide peptides in order, yielding the sequence of the protein. We have completed the sequence of most of the cyanogen bromide peptides. This information, in conjunction with that from other laboratories, forms the basis for our prediction of the secondary structure of the protein and how it may be arranged in the disk membrane.

The structure of rhodopsin and the rod outer segment disk membrane

Abstract Rhodopsin is the photoreceptor protein of rod cells in the vertebrate retina. Physical studies of the photoreceptor membrane provide a low-resolution structure and show that rhodopsin has a large fraction of its mass embedded in the lipid bilayer. The complete amino acid sequence of bovine rhodopsin has recently been obtained and this, combined with other information, leads to testable predictions of the basic structural features of vertebrate rhodopsin.

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 rhodopsins cytoplasmic regions. We selected for antibodies against predominantly linear determinants (as distinct from complex assembled determinants) and have isolated antibodies that recognize rhodopsins 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 rhodopsins 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.

Rhodopsin's protein and carbohydrate structure: Selected aspects

A topographic model for rhodopsin has been constructed based upon evaluation of rhodopsins sequence by a secondary structure prediction algorithm as well as chemical and enzymatic modification of rhodopsin in the membrane [Hargrave et al. (1983) Biophys. Struct. Mech. 9, 235-244]. The non-uniform distribution of several amino acids in the primary structure and within the topographic model is discussed. The seven predicted helices were evaluated and each helix was found to have one surface which is much more hydrophobic than the other. Stereoscopic views of a three dimensional model with a functional color-coding scheme incorporating these features are presented. The amino acid sequence of rhodopsin has been compared to other proteins in the Dayhoff Protein Data Bank. No obvious relationship to any other protein sequenced was found. High resolution proton magnetic resonance spectroscopy was used to reinvestigate the structure and relative proportions of rhodopsins major and minor oligosaccharide chains. One major (Man3GlcNAc3) and two minor (Man4GlcNAc3 and Man5GlcNAc3) were observed.

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 rhodopsins sequence is exposed at the internal membrane surface of the rod cell disc membrane. Rhodopsins 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.

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.

A humanized model of experimental autoimmune uveitis in HLA class II transgenic mice

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.

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 be important in regulating their phosphorylation by casein kinase II.

Arrestin migrates in photoreceptors in response to light: a study of arrestin localization using an arrestin-GFP fusion protein in transgenic frogs.

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.