Unraveling the Molecular Mystery of Retinal Pigment Epithelium Phagocytosis
Retinal pigment epithelium (RPE) phagocytosis is essential for clearance of shed photoreceptor outer segments (POS) to prevent retinal degeneration. Despite the importance, our understanding of the molecular mechanisms of RPE phagocytosis is relatively limited. Phagocytosis ligands, receptors, and signaling cascades are traditionally identified on case-by-case basis with challenges. It is even more daunting to identify a new signaling pathway in the absence of any molecular probe. We developed a phagocytosis-based functional cloning strategy with open reading frame (ORF) phage display and identified tubby-like protein 1 (Tulp1) as a new phagocytosis ligand in an unbiased manner. We used Tulp1 as a molecular probe and characterized the well-known MerTK as the receptor of Tulp1. Tulp1 as a genuine MerTK ligand was independently validated by co-immunoprecipitation, MerTK autophosphorylation and MerTK-dependent intracellular signaling. Moreover, Tulp1 was characterized as a bridging molecule with a C-terminal phagocytosis prey-binding domain. These results suggest that Tulp1 is a genuine RPE phagocytosis ligand and that ORF phage display is a valid technology for unbiased identification of phagocytosis ligands, which will further lead to their receptors and signaling cascades. Thus, this new strategy will facilitate the unbiased mapping of molecular mechanisms of RPE and other phagocytes.
KeywordsRetinal pigment epithelium Phagocytosis Phagocytosis ligand Tubby-like protein 1 Tulp1 MerTK ORF phage display
We thank Dr. Douglas Graham for technical help with MerTK phosphorylation. This study was supported by NIH R01EY016211, R01EY016211-05S1, P30-EY014801, and an institutional grant from Research to Prevent Blindness.
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