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C.–H. Sung, C. Vega, W. Jun, J.–Z. Chuang; Structural and functional impairment of endocytic pathways by retinitis pigmentosa mutant rhodopsin–arrestin complexes . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3609.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Nearly 100 mutations of the rhodopsin gene have been identified in patients with autosomal dominant RP (adRP). Several mutant bovine rhodopsins with amino acid changes at Arg135 have been shown to exhibit higher affinity to rhodopsin kinase and v–arrestin (v–arr) in vitro. There are three known human adRP–associated rhodopsin missense mutations at Arg135 (R135G, R135L, R135W). We examined the interaction between v–arr and Arg135 mutants in transfected HEK cells. The cellular defects caused by Arg135 and v–arr interaction were analyzed both in vitro and in vivo. Methods: Human wild type (wt) or mutant rhodopsins were singly transfected or co–transfected with GFP–v–arr into HEK cells. Transfected cells were examined by immunohistochemistry, biochemical, and endocytosis assays. Expression of mutant rhodopsins in rat retinas was achieved with an in vivo transfection procedure. Results: Although wt rhodopsin predominantly accumulated on the plasma membrane, Arg135 mutant rhodopsin appeared on both the cell surface and on intracellular vesicles of transfected cells. Whole cell phosphorylation experiments suggested that Arg135 mutant is constitutively highly phosphorylated. The Arg135 mutant, but not the wt rhodopsin, was found to be tightly associated with v–arr biochemically. Co–expression experiments suggested that mutant rhodopsin recruited the cytosolic v–arr to the plasma membrane, and that the rhodopsin–arrestin complex was internalized into the endocytic pathway. Furthermore, the rhodopsin–v–arr complexes altered the morphology of endosomal compartments and severely damaged receptor–mediated endocytic functions. Arg135 mutant expression in the transfected rat retinas was employed to corroborate the in vitro results. Conclusions: Arg135 mutations in rhodopsin cause a severe form of RP, consistent with the unique biochemical and cellular defects in these mutant proteins. Because the defects of Arg135 mutant rhodopsins are distinct from those previously described for class I and class II RP mutations, we propose that they be named class III. Impaired endocytic activity may underlie the pathogenesis of RP caused by class III rhodopsin mutations.
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