March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Synergistic interaction of Tubby and Tubby-like Protein 1 (Tulp1)
Author Affiliations & Notes
  • Gabriela S. Alvarado
    Ophthalmology, Bascom Palmer Eye Inst, Univ of Miami, Miami, Florida
  • Nora B. Caberoy
    Ophthalmology, Bascom Palmer Eye Inst, Univ of Miami, Miami, Florida
  • Yixiong Zhou
    Ophthalmology, Bascom Palmer Eye Inst, Univ of Miami, Miami, Florida
  • Wei Li
    Ophthalmology, Bascom Palmer Eye Inst, Univ of Miami, Miami, Florida
  • Footnotes
    Commercial Relationships  Gabriela S. Alvarado, None; Nora B. Caberoy, None; Yixiong Zhou, None; Wei Li, None
  • Footnotes
    Support  This work is partially supported by NIH R01GM094449-01A1, R01EY016211-05S1, 1K99EY020865-01A1, and an institutional grant from Research to Prevent Blindness
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 6568. doi:
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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)

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Abstract

Purpose: : 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.

Methods: : 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.

Results: : 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.

Conclusions: : 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.

Keywords: retinal degenerations: cell biology • phagocytosis and killing • protein purification and characterization 
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