Generation of Tyro3 Receptor Tyrosine Kinase Clones to Study Interactions with SH2 Domain Proteins in the Retinal Pigment Epithelium
Florida Southern College
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 phagocytosis. Recent studies suggest that TYRO3, a familial receptor tyrosine kinase to MERTK, can compensate in the absence of MERTK. As such, I 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 (Grb2, P85α, Src, Vav1, Vav2, and Vav3), various clones of TYRO3 were generated. Two truncated TYRO3 proteins that included the kinase domain and cytoplasmic tail (residues 470 – 890 and 498 – 890) were successfully cloned into a pRSET vector and recombinantly overexpressed. These clones were then purified and potential interactions between the purified Tyro3 and the SH2 domain proteins, which were generously provided by Dr. Shameka Shelby, were assessed using Ni- NTA pulldown assays; however, future pulldowns will need to be conducted to obtain conclusive results. Additionally, full length TYRO3 was successfully cloned into pcDNA3.1 His vector for overexpression in mammalian cultured cells. Further experiments will confirm the identified interactions in-vitro and 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.
Tyro3 receptor, Tyrosine Kinase Clones, Retinal Pigment