[Immunocytochemical localization of opsin in the retina of normal and inherited retinal dystrophic mice].
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PURPOSE: The aim of this project was to investigate the retinal pigment epithelium (RPE) at the molecular level by identification of novel RPE-specific cDNAs that may encode proteins of signal transduction pathways or other proteins that are expressed preferentially in the RPE. METHODS: A bovine RPE cDNA library was constructed in bacteriophage lambda g10 using RPE-enriched poly(A)+ RNA. The library was screened by differential hybridization to bovine RPE and kidney cDNA probes. RESULTS: A member of the hepatahelical receptor family was identified in bovine RPE by molecular cloning. Its deduced amino acid sequence predicts a protein that has 291 amino acid residues and resembles most closely the family of visual pigments. A lysine residue, analogous to the retinaldehyde attachment site in rhodopsin, is conserved in the seventh hydrophobic segment of the novel sequence. Messenger RNA encoding the putative G protein-coupled receptor was detected by in situ hybridization in the RPE, inner nuclear layer, and specific cells of the ganglion cell layer. Immunohistochemical staining of bovine retina showed that the receptor protein is localized in Müller cells, as well as in the RPE. CONCLUSIONS: A novel heptahelical receptor defines a distant evolutionary branch of the visual pigment tree. The selective localization of this putative receptor, its abundance in RPE and retina, and its homology to the visual pigments suggest that the function of this receptor is important in a visual process involving the RPE and Müller cells.
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The gene for the protein moiety of the light-driven chloride pump halorhodopsin (HR), hop gene, was sequenced and the primary structure of the protein derived thereof. The gene has a GC content of 67% and codes for 274 amino acids. A promoter structure, resembling that of the halobacterial 16S rRNA genes, is present and both a terminating stem and a loop sequence is found downstream of the TGA stop codon. A ribosomal binding site is located within the translated region. The HR protein moiety is processed at the amino terminus, as well as the carboxy terminus, yielding a dominant species of calculated Mr 26 961.
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