April 2009
Volume 50, Issue 13
ARVO Annual Meeting Abstract  |   April 2009
Analysis of Mer Tyrosine Kinase Receptor Domains Regulating Retinal Phagocytosis
Author Affiliations & Notes
  • E. F. Nandrot
    Department of Genetics, UPMC, Inserm UMR_S968, Institut de la Vision, Paris, France
  • E. Clérin
    Department of Genetics, UPMC, Inserm UMR_S968, Institut de la Vision, Paris, France
  • S. S. Bhattacharya
    Department of Genetics, UPMC, Inserm UMR_S968, Institut de la Vision, Paris, France
    Department of Genetics, University College London, Institute of Ophthalmology, London, United Kingdom
  • Footnotes
    Commercial Relationships  E.F. Nandrot, None; E. Clérin, None; S.S. Bhattacharya, None.
  • Footnotes
    Support  Fondation Bettencourt Schueller, Fondation Voir et Entendre, Agence Nationale de la Recherche
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 1864. doi:
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      E. F. Nandrot, E. Clérin, S. S. Bhattacharya; Analysis of Mer Tyrosine Kinase Receptor Domains Regulating Retinal Phagocytosis. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1864.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : Mer tyrosine kinase (MerTK) receptors are expressed at the apical surface of retinal pigment epithelium (RPE) cells. These receptors are required to internalize photoreceptor outer segments tips (POS) that are shed once daily. Rodent strains defective for MerTK develop retinal phenotypes resembling human retinitis pigmentosa. In humans, mutations were observed in patients with cone-rod and rod-cone dystrophies. Some patients display fluorescence in the fundus, suggesting that phagocytosis was not completely absent. Despite its obvious importance, regulation of MerTK function during RPE phagocytosis remains poorly understood. Therefore, we set out to characterize amino acids crucial for MerTK to internalize POS.

Methods: : We cloned rat and mouse MerTK cDNAs. We used site-directed mutagenesis was used to produce several point mutations in different intracellular domains of MerTK. When targeting the tyrosine residues, we either constitutively inactivated or activated them. We then analyzed effect of these mutations on the phagocytic capability of transfected MerTK-deficient NRK-49F and RPE cells by challenging them with FITC-labeled POS and quantifying the corresponding signals. We assessed activity of the mutants by analyzing their phosphorylation patterns on immunoblots.

Results: : In NRK-49F fibroblasts, constitutively activating some MerTK tyrosines increased POS phagocytosis compared to wild-type MerTK (e.g. Tyr825). In contrast, mutation of the kinase domain activity decreased POS phagocytic activity. In RPE-J cells, POS binding increased with normal MerTK and with inactivation of some tyrosines (e.g. Tyr825 or Tyr924). Conversely, constitutive activation of some of the same tyrosines reduced POS binding (e.g. Tyr825). POS internalization did not change significantly with most of the mutations tested compared to the control plasmid. Additionally, mutation of the kinase domain did not have any drastic effect.

Conclusions: : The mutations tested had different effects on POS phagocytosis, suggesting that each MerTK domain has a specific role in POS removal. Moreover, NRK-49F fibroblasts and RPE-J cells reacted differently to the mutations. This might be explained by the presence of the endogenous MerTK already present in RPE-J cells whereas NRK-49F are devoid of any MerTK protein. Understanding how MerTK receptor's function is regulated to perform this crucial phagocytic task will give us insights into normal and pathologic retinal function, and could help us design therapies for specific forms of retinal dystrophies.

Keywords: retinal pigment epithelium • phagocytosis and killing • receptors: pharmacology/physiology 

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