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Shameka Shelby, Kecia Feathers, Lin Jia, Frank Mei, Debra Thompson; MERTK regulation of the Rab GTPase effector GDI1 from the superfamily of GDI/CHM proteins. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2647.
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© ARVO (1962-2015); The Authors (2016-present)
We have previously shown that tyrosine phosphorylation of GDP dissociation inhibitor alpha (GDI1) occurs during peak phagocytic uptake in congenic RCS rats, but is not detected in dystrophic RCS rats. The purpose of the present study was to determine the mechanism of MERTK-mediated phosphorylation of GDI1 and its role in the phagocytic mechanism of the retinal pigment epithelium (RPE).
Protein expression in mouse retina/RPE/choroid cryosections, and in cultured RPE-J cells challenged with rod outer segments, was evaluated using immunohistochemical analysis. Protein-coding transcripts in total RNA from mouse retina and RPE/choroid were assayed using RT-PCR. Expression constructs for MERTK, GDI1, and mutant GDI1 were generated using cDNAs from human RPE/choroid. Protein-protein interactions were evaluated by western analysis of immunoprecipitations from transfected HEK-293T cells using antibodies for phosphotyrosine, GDI1, SRC, MERTK, incubated plus or minus SRC inhibitors (PP1 and PP2, and analog PP3).
Tyrosine phosphorylation of GDI1 was seen in HEK-293T cells co-transfected with wild-type MERTK, but not with a MERTK mutant lacking kinase activity. GDI1 mutants lacking key tyrosine residues exhibited markedly lower levels of phosphorylation. SRC activation was shown to occur upstream of GDI1 phosphorylation, and the use of inhibitors established that SRC activity was essential in this mechanism. The expression patterns of Mertk, Gdi1, and Src were seen to overlap in the apical region of mouse RPE, and all three proteins were detected on phagosomes in RPE-J cells challenged with outer segments. Transcripts encoding Rab5 isoforms involved in early endosome/phagosome formation were detected in mouse RPE/choroid. In transfected cells, interaction of RAB5B and GDI1 was seen in the absence of MERTK activity, but not in cells transfected with active MERTK.
The finding that MERTK regulates the interaction of GDI1 with Rab GTPases identifies a novel signaling mechanism in which MERTK appears likely to play a direct role regulating membrane trafficking. Such a role could potentially link the mechanism of phagocytic uptake with the processing of early phagosomes. The functional relationship between MERTK and GDI1, which bears significant homology with CHM/REP1 that is mutated in choroideremia, points to the exceptional importance of the GDI/CHM family of proteins in RPE function.
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