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Gabriela S. Alvarado, Nora B. Caberoy, Yixiong Zhou, Wei Li; Synergistic interaction of Tubby and Tubby-like Protein 1 (Tulp1). Invest. Ophthalmol. Vis. Sci. 2012;53(14):6568.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in either tubby or tubby-like protein 1 (Tulp1) cause retinal degeneration with undefined mechanisms. We recently identified both proteins with unconventional secretion as novel MerTK-specific phagocytosis ligands for retinal pigment epithelium (RPE) cells. The purpose of this study is to characterize the possible interaction between tubby and Tulp1 for synergistic MerTK activation and RPE phagocytosis.
Tubby-binding proteins were screened by open reading frame (ORF) phage display as a new technology for protein-protein interactions. The interaction of tubby and Tulp1 was verified by yeast two-hybrid assay and co-immunoprecipitation. Tubby and Tulp1 were analyzed for their capacity to stimulate RPE ingestion of photoreceptor outer segments (POS) by phagocytosis assay and to activate MerTK with receptor phosphorylation by Western blot.
ORF phage display screening with purified tubby N-terminal domain (Tubby-N) as bait identified Tulp1 as a tubby-binding protein. The interaction of tubby and Tulp1 was independently verified by yeast two-hybrid assay and co-immunoprecipation. Both proteins were characterized with MerTK-binding motif(s). Tubby or Tulp1 alone stimulated RPE phagocytosis of POS, which was blocked by excessive MerTK extracellular domain. The combination of tubby and Tulp1 synergistically stimulated RPE ingestion of POS vesicles. MerTK receptor dimerization is known to facilitate receptor activation and phosphorylation. Tubby or Tulp1 alone induced MerTK activation. The combination of tubby and Tulp1 synergistically activated MerTK with receptor phosphorylation.
These data suggest that tubby and Tulp1 form a heterodimer, thereby synergistically activating the phagocytosis receptor and stimulating RPE phagocytosis. It is possible that the formation of tubby and Tulp1 may also play important roles in their cytoplasmic and nuclear functions.
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