Fate of E-cadherin in early RPE cultures: transient accumulation of truncated peptides at nonjunctional sites.
OBJECTIVE
E-cadherin is known to accumulate variably and slowly at junctions of cultured human RPE cells. The intent of this investigation was to determine what limits E-cadherin protein accumulation in RPE cells by analyzing cultures at early postplating intervals when junctions of the dominant cadherin (N-cadherin) are first forming.


METHODS
RPE cell lines hTERT-RPE1 and ARPE-19 and RPE cultures established from human donors were analyzed within 48 hours after plating for E-cadherin gene and protein expression (by RT-PCR and Western blotting, respectively) and for protein distribution (by immunofluorescence and immuno-electron microscopy), including codistribution with markers for organelles. Cell surface localization was analyzed by biotinylation and trypsin cleavage of extracellular cadherin domains.


RESULTS
The E-cadherin gene was constitutively expressed by RPE cultures, but the protein did not accumulate substantially in early RPE cultures. Instead small amounts of newly synthesized E-cadherin were detectable only transiently, peaking within a few hours after plating, at which time the protein was in the form of peptides of variable size rather the predicted 120-kDa molecular mass. Immunoreactive E-cadherin peptides did not traffic to the cell surface and localize to junctions. Rather they codistributed with several organelles including the endoplasmic reticulum (ER; but not the Golgi), sites of protein degradation (proteasomes, lysosomes, and autophagosomes) and unusual compartments (centrosomes and apposed to subdomains of the mitochondrial network).


CONCLUSIONS
The results suggest that in RPE cells posttranscriptional mechanisms involving altered protein processing and rapid turnover exist to limit E-cadherin accumulation. The consequence may be to limit E-cadherin-specific inductive properties in the RPE, a cell type in which N-cadherin is the normal dominant cadherin.
Burke
Janice M
JM
Department of Ophthalmology, Medical College of Wisconsin, Madison, 53226, USA. jburke@mcw.edu
Hong
Jeehee
J
D001710:Biotin / Q000378:metabolism; D015153:Blotting, Western; D015820:Cadherins / Q000235:genetics / Q000378:metabolism*; D018929:Cell Culture Techniques; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D016253:Microscopy, Immunoelectron; D015388:Organelles / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism*; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D015820:Cadherins; D010446:Peptide Fragments; D001710:Biotin