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Christina Locke, Nicole R Congrove, W Daniel Stamer, Brian S McKay; Myocilin is constitutively released with exosomes from RPE in situ. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2969.
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Myocilin is a ubiquitous protein that is found both soluble in the cytoplasm and associated with cytoplasmic vesicles. We have recently shown that myocilin enters the endocytic compartment of cells during receptor-mediated endocytosis and is later released from cells on the surface of exosomes. In this study, we tested the hypothesis that myocilin recruitment and release from RPE cells in situ is regulated by GPCR (GPR143) activation.
Using posterior segment prepared from fresh human donor eyes containing RPE with the retina removed, GPR143 (OA1) was stimulated with its endogenous ligand, L-DOPA and myocilin-associated exosome release was monitored over time. In a paired donor eyes, one was stimulated with 5 μM L-DOPA in DMEM while the contralateral eye was maintained in DMEM. The exosomes released into the media from the RPE were purified by differential ultracentrifugation. In parallel experiments, biotinylation was used to label surface membrane proteins prior to receptor stimulation to investigate whether myocilin was recruited to activated GPCRs in situ. We harvested total RPE protein, captured biotinylated proteins with bound cytoplasmic proteins using streptavidin conjugated beads. Samples were analyzed for myocilin content by western blot.
RPE in situ constitutively releases myocilin-associated exosomes. During a 25-minute period of media conditioning, 54% of total myocilin is released by the unstimulated RPE (n=4). Total is defined as myocilin in both the cellular and media compartments. Constitutive release of myocilin-associated exosomes was dramatically halted following activation of GPR143, with just 10% of t asotal of myocilin being released after stimulation. Similar to in vitro models, myocilin was recruited to the membrane compartment of RPE in situ after GPR143 activation (n=5).
Results show that myocilin-associated exosomes are continually produced by RPE cells in situ. GPR143 activation prompted two signal transduction-dependent outcomes in RPE; trafficking of myocilin to the endosomal compartment, and inhibition of myocilin-associated exosome release.
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