Localization of HRG4, a photoreceptor protein homologous to Unc-119, in ribbon synapse.
OBJECTIVE
To characterize further HRG4, a novel photoreceptor protein recently identified by subtractive cDNA cloning, by sequence analysis and immunolocalization.


METHODS
The rat homolog of HRG4, RRG4 was expressed and used to prepare an antibody. The antibody was used in Western blot analysis, and immunofluorescent localization at the light and electron microscopic levels of HRG4-RRG4 protein. The HRG4-RRG4 sequence was also analyzed for homologies.


RESULTS
HRG4-RRG4 showed 57% homology with unc-119, a Caenorhabditis elegans neuroprotein causing defects in locomotion, feeding, and chemosensation when mutated. By Western blot analysis, the HRG4-RRG4 protein was demonstrable only in retina and was soluble in nature. Immunofluorescence microscopic study of human and rat retinas, using the HRG4-RRG4 antibody, and other rod and cone photoreceptor-specific antibodies showed that the HRG4-RRG4 protein is localized in the outer plexiform layer of the retina in the synaptic termini of rod and cone photoreceptors. Electron microscopic immunolocalization showed the protein in the cytoplasm and on the presynaptic membranes of the photoreceptor synapses.


CONCLUSIONS
The homology to unc-119 and localization to the photoreceptor synapse are suggestive of a function for HRG4-RRG4 in photoreceptor neurotransmission. HRG4 is the first photoreceptor-enriched synaptic protein to be reported, suggesting that its function may be unique to the specialized ribbon synapses formed between photoreceptors and the horizontal and bipolar cells of the retina.
Higashide
T
T
Bascom Palmer Eye Institute, University of Miami School of Medicine, Florida 33136, USA.
McLaren
M J
MJ
Inana
G
G
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D029742:Caenorhabditis elegans Proteins; D016384:Consensus Sequence; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D019084:Fluorescent Antibody Technique, Indirect; D015801:Helminth Proteins / Q000032:analysis* / Q000235:genetics; D006801:Humans; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000235:genetics; D010786:Photoreceptor Cells / Q000737:chemistry*; D017729:Presynaptic Terminals / Q000737:chemistry*; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins; D017386:Sequence Homology, Amino Acid
D048868:Adaptor Proteins, Signal Transducing; D029742:Caenorhabditis elegans Proteins; D017931:DNA Primers; D005136:Eye Proteins; D015801:Helminth Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C098176:UNC-119 protein, C elegans; C098032:UNC119 protein, human