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Wei Zhang, Nageswara Rao Kollu, Linjing Li, Manisha Anand, Hemant Khanna; RPGR, a retinal ciliopathy protein, localizes to cilia in a prenylation dependent manner. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3193. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
RPGR (retinitis pigmentosa GTPase regulator), a major cause of retinitis pigmentosa (RP) worldwide, is a ciliary protein involved in regulating protein trafficking and composition of cilia. We undertook this study to understand the mechanisms of localization of RPGR to the cilium.
N-terminal GFP or SBP-tagged full-length and mutant human RPGR constructs were transiently transfected into hTERT-RPE1 cells grown on glass cover slips. RPGR cellular localization was detected by anti-GFP antibody; cilia were stained with anti-ARL13B antibody. RPGR-interacting proteins were identified using Tandem Affinity Purification of SBP-RPGR complexes from HEK293 cells followed by mass spectrometry analysis. Co-immunoprecipitation was performed to ascertain the interaction of RPGR and identified proteins.
The C-terminal isoprenylation motif (-812CTIL815) of RPGR is essential for its localization to cilia. We found that this motif interacts with PDE6D (delta subunit of Phosphodiesterase), a prenyl-binding protein. Disruption of the -CTIL motif abrogated RPGR’s interaction with PDE6D. Selected human disease-causing mutations in RPGR perturb its ciliary localization and interaction with PDE6D.
Our results suggest that prenylation modulates the trafficking of a regulator of ciliary composition and indicate the involvement of mistrafficking of RPGR as a potential mechanism of associated retinal ciliopathy.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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