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Generation of TYRO3 Receptor Tyrosine Kinase Clones to Study Interactions with SH2 Domain Proteins in the Retinal Pigment Epithelium
Author(s) -
Harris Lauren,
Shelby Shameka
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.760.21
Subject(s) - mertk , grb2 , retinal pigment epithelium , receptor tyrosine kinase , microbiology and biotechnology , biology , proto oncogene tyrosine protein kinase src , visual phototransduction , tyrosine kinase , autophosphorylation , sh2 domain , retina , biochemistry , retinal , kinase , chemistry , signal transduction , protein kinase a , neuroscience
The retina is comprised of cone and rod photoreceptors that must continually be maintained in order to preserve visual acuity. Daily light exposure to the outer portions of the photoreceptors, termed outer segments (OS), leads to photo‐oxidative stress. To combat potential retinal damage caused by light exposure, the Retinal Pigment Epithelium (RPE) phagocytizes spent outer segments. Disruption of OS phagocytosis leads to the accumulation of debris that blocks the flow of oxygen and nutrients to the retina. This will eventually lead to atrophy of the retina and, ultimately, blindness. Previous studies have demonstrated the requirement of Mer Receptor Tyrosine Kinase (MERTK) in the process of OS phagocytosis. Protein interactions between MERTK and SH2 domain proteins Grb2, P85α, Src, and Vav3 have also been shown to be necessary for OS phagoctytosis. Recent studies suggest that TYRO3, a familial receptor tyrosine kinase to MERTK, can compensate in the absence of MERTK. As such, we hypothesized that TYRO3 may bind to SH2 domain proteins known to bind to MERTK. To test the similarities of interactions between MERTK, TYRO3, and associated SH2 domain proteins, various clones of TYRO3 were generated, and GST‐tagged SH2 domain proteins were overexpressed and purified. GST‐SH2 domain proteins Grb2, P85α, Src, Vav1, and Vav3 were overexpressed and purified via affinity chromatography, followed by gel‐filtration chromatography. Two truncated TYRO3 proteins that included the kinase domain and cytoplasmic tail (residues 470 – 809 and 498 – 809) were successfully cloned into a pRSET vector and recombinantly overexpressed. Additionally, full length TYRO3 was successfully cloned into pcDNA3.1 His vector for overexpression in mammalian cultured cells. Further experiments will adequately purify the truncated TYRO3 protein and identify interactions between TYRO3 and SH2 domain proteins recombinantly via Ni‐NTA pull‐down assays. Confirmation of interactions identified in‐vitro will be conducted in mammalian cells transfected with TYRO3. This study has generated the tools necessary to further identify components of the RPE phagocytic mechanism. Elucidation of this mechanism will be instrumental in identifying future retinal disease genes and understanding the impact on proteins that may be involved in Age‐related Macular Degeneration. Support or Funding Information Florida Southern College Faculty‐Student Summer Research Collaboration Grant