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Emeline F Nandrot, Dorine Neel, Ludovic Carduner, Eglantine Heude; Investigation of MerTK intracellular tyrosine kinase residues used for RPE phagocytosis. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2317.
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© ARVO (1962-2015); The Authors (2016-present)
Mer tyrosine kinase (MerTK) receptors expressed at the apical surface of retinal pigment epithelial (RPE) cells are required to internalize shed photoreceptor outer segments tips (POS) following a circadian rhythm. MerTK defects lead to rod-cone dystrophies in human patients and to retinal phenotypes resembling retinitis pigmentosa in rodents. Some patients display fluorescence in their eye fundus, suggesting that phagocytosis is not completely absent. MerTK receptors are activated via tyrosine phosphorylation and display 17 tyrosine residues in their cytoplasmic tail. Therefore, we set out to characterize which of these amino acids are crucial for daily MerTK function.
We cloned rat and mouse MerTK cDNAs. We used site-directed mutagenesis to produce point mutations in each tyrosine residue of MerTK cytoplasmic tail, either constitutively inactivating or activating them. We transfected either the RPE-J cell line or MerTK-defective RPE cells from RCS rats in primary culture. We then challenged these cells with FITC-labeled POS to analyze effect of these mutations on their phagocytic capability and on MerTK activation.
Constitutively inactivating some MerTK tyrosine residues decreases POS phagocytosis when compared to wild-type MerTK (e.g. Tyr544). In other cases, activating some MerTK tyrosines decreases POS phagocytosis while inactivating them increases phagocytosis (e.g. Tyr867). Alternatively, modifying the activity of some other tyrosines does not have any effect on POS phagocytosis (e.g. Tyr924).
The mutations tested had varioust effects on POS phagocytosis, either stimulating or inhibiting POS uptake. This suggests that MerTK tyrosines can carry different roles in daily POS elimination, that could be used at different times of day. In addition, it is possible that several tyrosine residues function simultaneously, as it has been shown for macrophages.
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