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S. J. Shelby, D. A. Thompson; Mertk Interactions With SH2-Domain Proteins in the Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2919.
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© ARVO (1962-2015); The Authors (2016-present)
Mertk is essential for phagocytosis of shed photoreceptor outer segment membranes by the retinal pigment epithelium (RPE). In immune cells, cytoskeletal reorganization needed for phagocytic uptake involves signaling by small G-proteins. The purpose of this study is to identify SH2-domain proteins activated downstream of Mertk and likely to regulate small G-proteins involved in cytoskeletal reorganization in the RPE.
A cDNA clone encoding the Mertk intracellular region (aa 571-999) was expressed as a 6x-His fusion protein that was purified using Ni2+-NTA agarose and gel filtration chromatography, then phosphorylated by addition of ATP. cDNA clones encoding the SH2-domains of Grb2, Vav1, Vav2, and Vav3 were expressed as GST-fusion proteins that were purified on glutathione agarose. Mertk interacting proteins in RPE extracts from RCS congenic and dystrophic rats were identified using Ni2+-NTA agarose pulldowns with recombinant Mertk, followed by western analysis. Interactions were confirmed in pulldowns with recombinant SH2-domains from Grb2 and Vav proteins. Expression in the RPE was assessed using RT-PCR, western, and immunohistochemical analysis.
Western analysis of Mertk pulldowns from rat RPE extracts obtained during peak phagocytic activity (1.5 hours post-light onset) showed interaction with endogenous Grb2, Vav1, PI3K, ShcA, and Annexin IV. Mertk pulldowns with cloned SH2-domains showed interactions with Grb2, Vav1, and Vav3, but not Vav2. Transcripts encoding Grb2, Vav, PI3K, and Shc were detected in mouse RPE total RNA. Expression of Grb2 and Vav1 was detected by western analysis of rat RPE protein homogenates, and by immunohistochemical analysis of albino mouse RPE cryosections.
In rodent RPE during peak phagocytosis, Mertk appears to interact with regulators of small G-protein signaling, as Grb2 acts upstream of the Ras signaling cascade and Vav1 is a guanine nucleotide exchange factor that activates the Rho family of small GTPases. Activation of Mertk in the RPE is thus likely to regulate at least two signaling pathways with the potential to regulate cytoskeletal reorganization involved in phagocytic uptake.
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