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Solène Roux, Isabelle S Audo, Ana Fakin, Andrew Webster, Christina Zeitz, Emeline F Nandrot; Characterization of the effect of MERTK patient mutations on RPE cells in vitro. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4030.
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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 segment tips (POS) following a circadian rhythm. MerTK defects lead to rod-cone dystrophies in human patients and to retinitis pigmentosa in rodents. Some patients display fundus autofluorescence, suggesting that phagocytosis is not completely absent. MERTK mutations span the whole gene sequence and are thus very useful to identify amino acids critical for protein function.
We used site-directed mutagenesis to reproduce one deletion and 18 missense/nonsense mutations on the mouse MerTK cDNA, including new mutations identified in our French cohort. Constructs were expressed in the rat RPE-J cell line and in MerTK-deficient RPE primary cultures from RCS rats. Presence of each mutant protein at the cell surface was checked by immunofluorescence labelings. Effect of mutations on MerTK activation and RPE phagocytosis was assessed by challenging cells with FITC-labeled purified photoreceptor outer segment (POS) fragments.
Some mutant proteins were sequestered inside the cell, such as human A740V and R844C, partially retained in the biosynthetic pathway or appeared to form aggregates at the cell surface. For Ig-Like domain 2 C262F and tyrosine kinase domain F703V mutants, dominant negative effects triggered the impairment of all steps of RPE phagocytosis. In other cases, changes such as G654A, H721Q, R727Q and R732X only increased numbers of POS tethered by RPE cells. Interestingly, another amino acid 727 mutant, R727G, decreased POS uptake as did N329S. In contrast, the c.2070-2074del mutant located shortly upstream of amino acid 727 increased POS internalization. In addition, observed effects sometimes varied depending on the type of RPE cell used: total phagocytosis increase by R775X in RPE-J cells expressing endogenous MerTK was translated into increased internalization by MerTK-deficient RPE primary cultures.
While sequestration of MerTK inside the cytoplasm totally hinders its function, the different effects of mutations located in close-by areas suggest that several factors can lead to the phenotype. Indeed, MerTK acts as a dimer and also needs to be able to bind ligands (Ig-Like domains) and signaling molecules (tyrosine kinase domain). Thus, its 3-D conformation that can be modified by amino acid changes is crucial.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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