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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(More)
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(More)
Rhodopsin kinase was purified to near homogeneity by affinity binding to light-exposed rod cell outer segment membranes, followed by DEAE-cellulose and hydroxyapatite chromatography. This resulted in a 1055-fold purification of highly active rhodopsin kinase with an overall recovery of 19%. Rhodopsin kinase is a single polypeptide chain with Mr =(More)
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(More)
We have produced monoclonal antibodies directed against defined amino acid sequences of rhodopsin by using peptides for hybridoma selection, rather than for immunization. Mice were immunized with rhodopsin, and hybridoma culture supernatants were assayed by ELISA to detect anti-rhodopsin antibodies. These antibodies were further tested by competition ELISA(More)
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,(More)
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(More)
The binding sites for four monoclonal antibodies, rho 1D4, rho 3C2, rho 3A6, and rho 1C5, have been localized within the C-terminal region of bovine rhodopsin: Asp18'-Glu-Ala16'-Ser-Thr-Thr-Val12'-Ser-Lys-Thr-Gl u8'-Thr-Ser-Gln-Val4'-Ala-Pr o -Ala1'. Antibody binding sites were localized by using synthetic C-terminal peptides in conjunction with solid-phase(More)
A dipeptide containing the binding site for retinal in bovine rhodopsin has been isolated and its sequence determined. Rhodopsin containing [11-3H]retinal was prepared in chromatographically pure form, and the [3H]retinal was reductively linked to its binding site on opsin by using borane--dimethylamine. The [3H]retinylopsin in octyl glucoside was(More)
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(More)