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J.M. Burke, Y. Youn, J. Hong; E-cadherin in RPE Cells. Why Isn't It Always at Junctions? . Invest. Ophthalmol. Vis. Sci. 2003;44(13):374.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: E-cadherin expression by RPE cells is of interest because when it localizes to junctions it can induce a basal polarity of Na/K ATPase, a phenotype that is atypical for RPE. E-cadherin can be found in cultured RPE cell junctions, but only in late confluency after the junction of the dominant RPE cadherin (N-cadherin) is already formed. Here we analyzed the expression and distribution of E-cadherin in RPE cells shortly after plating to attempt to determine what prevents E-cadherin from accumulating in forming junctions. Methods: Human RPE cells from adult donors or RPE cell lines hTERT-RPE1 and ARPE-19 were studied 6-72 hrs after plating at near confluent densities. E-cadherin expression was analyzed by RT-PCR and by western blotting, using antibodies directed against cytoplasmic (clone 36) or extracellular (H-108, HECD-1) E-cadherin domains. E-cadherin distribution was analyzed by cell surface biotinylation, by controlled trypsinization of surface proteins, and by immunostaining. Immunoprecipitation (IP) was used to determine complexing with catenins. N-cadherin in RPE and E-cadherin in MDCK cells, which express E-cadherin as their dominant cadherin, were similarly analyzed. Results: In cultured RPE cells, both from donors and cell lines, E-cadherin mRNA is constitutively present, but E-cadherin protein does not co-localize with N-cadherin at forming junctions. Rather E-cadherin protein localizes transiently to cytoplasmic compartments that stain with markers for ER, Golgi, lysosomes and possibly proteasomes. Clone 36 and H-108 (but not HECD-1) immunoreactive E-cadherin protein also shows a stable (resistant to detergent extraction) distribution to the centrosome. These E-cadherin staining patterns were not seen in MDCK cells. Unlike N-cadherin, E-cadherin in RPE was not found in a complex with beta-catenin and was not detected on the cell surface. Blotting revealed no full length (120 kD) E-cadherin protein in RPE. Rather peptides were detected that may represent unprocessed protein (135kD) or truncated forms (dominant peptides of 30, 40, 48, 60, 80-84, or 100kD). Conclusions: E-cadherin is constitutively expressed by RPE cells but the protein is processed, presumably by proteolysis, to yield forms that fail to bind beta-catenin and to traffic to the cell surface. The peptides accumulate in biosynthetic organelles, degradative compartments, or unusual locations (centrosomes). The results suggest that a proteolytic mechanism(s) exists in RPE cells to prevent full length E-cadherin from localizing to forming junctions at a critical interval when it could exert an undesirable morphoregulatory effect.
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