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N. Mori, Y. Ishiba, S. Kubota, A. Kobayashi, M.J. McLaren, G. Inana; Preferential Interaction of Mutant HRG4(UNC 119) With its Target Protein, ARL-2 . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1536.
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Purpose: HRG4 is a novel photoreceptor protein we have isolated by a differential cloning approach (Higashide et al., J. Biol. Chem. 271:1797-1804,1996). Immunofluorescence microscopy and immunocytochemistry localized HRG4 in the rod and cone photoreceptor synapses, establishing it as the first synaptic protein enriched in the photoreceptors (Higashide et al., Invest Ophthalmol Vis Sci. 39:690-698,1998). The HRG4 gene has been mapped to chromosome 17q11.2 and shown to consist of five exons (Higashide et al, Genomics. 57:446-450, 1999). A premature termination codon mutation in HRG4 was uncovered in a patient with late-onset cone-rod dystrophy, and transgenic mice expressing the identical mutation demonstrated a late-onset reduction in b-wave by ERG and retinal degeneration with marked synaptic degeneration (A. Kobayashi et al., Invest Ophthalmol Vis Sci. 41:690-698,2000). We have identified and confirmed ARL-2 as the interacting protein for HRG4 by the yeast 2-hybrid strategy (Kobayashi et al., FEBS Letter, in press). In the present study, interaction of wild-type and mutant HRG4 with ARL-2 was analyzed in order to investigate the molecular basis of pathogenicity in the patient and transgenic model. Methods: Recombinantly expressed GST-HRG4 (full, proximal, distal) hybrid proteins were used to pull down ARL2 in the expressed form and from retinal extracts. The levels of ARL2 and synaptic proteins and the efficiency of SNARE complex formation were analyzed in normal and transgenic retinas of different ages. Results: More ARL2 was bound by the mutant (proximal) compared to the wild-type (full) HRG4. After a temporary increase, the level of ARL2 decreased in the transgenics with age, as did many of the synaptic proteins and the efficiency of SNARE complex formation. Conclusions: The mutant HRG4 may be sequestering the target protein, ARL2, ineffectually, eventually leading to exhaustion of the ARL2 supply and disturbance in the synaptic vesicle function, consistent with the dominant negative mechanism originally postulated for this mutation.
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