April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
Network of RPGR and Its Interacting Proteins Regulates Primary Cilia Maintenance and Photoreceptor Intersegmental Transport
Author Affiliations & Notes
  • H. Khanna
    Ophthalmology-Kellogg Eye Ctr, University of Michigan, Ann Arbor, Michigan
  • C. A. Murga-Zamalloa
    Ophthalmology-Kellogg Eye Ctr, University of Michigan, Ann Arbor, Michigan
  • Footnotes
    Commercial Relationships  H. Khanna, None; C.A. Murga-Zamalloa, None.
  • Footnotes
    Support  EY007961, FFB, MEBTC
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 1106. doi:
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      H. Khanna, C. A. Murga-Zamalloa; Network of RPGR and Its Interacting Proteins Regulates Primary Cilia Maintenance and Photoreceptor Intersegmental Transport. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1106.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : Mutations in the cilia-centrosomal protein RPGR account for >70% of XLRP cases. RPGR associates with ciliary proteins mutated in Nephronophthisis (NPHP; cystic kidney disease), RP and cerebellar defects. These include NPHP5, NPHP6/CEP290 and NPHP8/RPGRIP1L. To investigate how dysfunction of RPGR results in photoreceptor degeneration, we sought to dissect the network of RPGR complexes with NPHP proteins in the retina.

Methods: : We utilized known-bait and known-prey yeast two-hybrid analysis to identify NPHP (prey) proteins that directly interact with RPGR (bait). Immunoprecipitation of RPGR from wild type (WT) and mutant mouse retinas was performed to evaluate the integrity of the RPGR-NPHP complexes. To dissect the RPGR-NPHP complexes, we performed serial immunodepletion (SID) of NPHP proteins followed by immunoprecipitation (IP) of RPGR from bovine retinal extracts. The precipitated proteins were tested for the presence of other NPHP proteins in RPGR complexes.

Results: : We show that the N-terminal domain of RPGR directly interacts with NPHP1, NPHP2, NPHP4, and NPHP5. Deletion in the NPHP-interacting domain of RPGR abrogates its interaction with NPHP proteins in the Rpgr-ko mouse retina. SID of NPHP proteins from bovine retinal extracts revealed that RPGR exists in at least two different complexes with the tested NPHP proteins. In one complex, RPGR associates with NPHP4, NPHP6 and NPHP8. In a second complex, RPGR associates with NPHP1, NPHP2, and NPHP5. Moreover, RPGR’s complex with NPHP4 and NPHP8 is disrupted in the retina of Nphp6/Cep290-mutant mouse. We also evaluated the role of RPGR in maintaining the integrity of the protein complexes formed by NPHP proteins. To this end, we found that NPHP2, NPHP4, NPHP5, NPHP6, and NPHP8 exist in multiprotein complexes in mouse retina. The integrity of the NPHP-complex is disrupted perturbed in the Rpgr-ko mouse retina.

Conclusions: : Our results indicate that RPGR plays a central role in orchestrating NPHP-complex formation in the retina and provide mechanistic insights into the pathogenesis of retinal degeneration in XLRP and associated ciliary diseases. We propose a model wherein different RPGR-NPHP complexes regulate vesicular trafficking and cargo docking at the photoreceptor cilium.

Keywords: photoreceptors • retinal degenerations: cell biology • retina 
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