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Trent J Bowen, Nicole R Congrove, W Daniel Stamer, Brian S McKay; Myocilin Mutations Alter GPCR Endocytosis. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2906.
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© ARVO (1962-2015); The Authors (2016-present)
Different mutations in myocilin cause glaucoma with distinct disease severity and age of onset. We previously showed that myocilin participates in G-protein coupled receptor (GPCR) endocytosis in a ligand-dependent manner. In this study, we tested the hypothesis that mutations in myocilin alter its function in endocytosis of GPR143 after ligand stimulation.
COS cells were transfected to co-express an isoform of myocilin (WT, G364V or P370L) and GPR143 (as a GFP fusion protein). GPR143 receptors were stimulated with their endogenous ligand (L-DOPA, 5 µM) for 0, 2, 5, 20, 40, and 60 minutes. The cells were washed with cold PBS and fixed using 4% paraformaldehyde. Fixed cells were permeabilized using TBST and stained using affinity-purified antibodies directed against human myocilin. Myocilin specific staining was visualized using a secondary antibody conjugated to TRITC. Confocal microscopy with a Zeiss LSM 510Meta-NLO multiphoton microscope was performed at 40x magnification at 488 nm and 543 nm excitation wavelengths. GFP and myocilin image stacks were produced separately and overlaid using ImageJ.
At time 0 (before ligand stimulation), WT myocilin displayed a diffuse cytoplasmic distribution, whereas GPR143 was both on the plasma membrane and in the Golgi apparatus. As early as 2 minutes after stimulation, the distribution of both myocilin and GPR143 changed, and both proteins localized to early endosomes. The proportion of both proteins localized to the early endosome continued to increase as the time course proceeded, and at 20 minutes both proteins were localized to a large late endosome. By 40 minutes, myocilin and GPR143 no longer co-localized, returning completely to time 0 distribution by 60 minutes. The P370L myocilin mutant did not co-localize with GPR143 after treatment until 40 minutes when both appeared in late endosomes. P370L returned to conditions similar to initial time 0 conditions at 60 minutes. G364V behavior following treatment was indistinguishable from WT.
Our results indicate that the P370L mutation alters myocilin’s function in receptor-mediated endocytosis. The G364V mutation does not alter its function in endocytosis, yet both P370L and G364V mutations cause glaucoma. These observations suggest that mutations in myocilin differentially affect receptor-mediated endocytosis which may account for the distinct disease severity and age of onset seen with glaucoma involving mutations in myocilin.
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