May 2005
Volume 46, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2005
Characterisation of the Interaction Between the X–Linked Retinitis Pigmentosa Protein RP2 and Arl Proteins
Author Affiliations & Notes
  • R.J. Evans
    Molecular Genetics,
    Institute of Ophthalmology, UCL, London, United Kingdom
  • A.J. Hardcastle
    Molecular Genetics,
    Institute of Ophthalmology, UCL, London, United Kingdom
  • M.E. Cheetham
    Pathology,
    Institute of Ophthalmology, UCL, London, United Kingdom
  • Footnotes
    Commercial Relationships  R.J. Evans, None; A.J. Hardcastle, None; M.E. Cheetham, None.
  • Footnotes
    Support  Medical Research Council, UK
Investigative Ophthalmology & Visual Science May 2005, Vol.46, 5170. doi:
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      R.J. Evans, A.J. Hardcastle, M.E. Cheetham; Characterisation of the Interaction Between the X–Linked Retinitis Pigmentosa Protein RP2 and Arl Proteins . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5170.

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

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Abstract

Abstract: : Purpose: Mutations in the retinitis pigmentosa 2 (RP2) gene have been shown to cause X–Linked Retinitis Pigmentosa (XLRP). RP2 is a ubiquitous protein and the mechanisms by which mutations in RP2 lead to photoreceptor cell death are unknown. RP2 has been reported to interact with the ADP ribosylation factor (Arf)–like protein Arl3. The aim of this study was to confirm that Arl3 is a bona fide interacting protein partner for RP2, compare the binding with other Arl proteins and define the region of RP2 that interacts with Arl3. Methods: The yeast two–hybrid ‘Cytotrap’ system was used to analyse the interaction between RP2 and Arl proteins. Full length RP2 cDNA and a range of RP2 deletion mutants were cloned as in frame fusion proteins with human SoS in the plasmid pSoS. Human Arl2, Arl3 and Arl6 were cloned in frame into the ‘prey’ plasmid pMyr. Yeast were co–transformed with these plasmids and growth compared at permissive and non–permissive temperatures to identify protein:protein interactions. Expression of the SoS–RP2 fusion proteins were analysed by Western blotting. Results: Growth was not observed at the restrictive temperature when SoS–RP2 fusion was expressed alone. Co–transformation of pSoS–RP2 with pMyr–Arl3 and pMyr Arl2 resulted in growth at the non–permissive temperature demonstrating that Arl3 and Arl2 interact with RP2. Using RP2 deletion constructs it was possible to define the Arl3 binding domain of RP2 to a central region of the protein. When pMyr–Arl2 was co–transformed with SoS–RP2 constructs with low expression levels the interaction was abolished, suggesting a lower affinity of interaction with RP2 for Arl2 compared to Arl3. Arl6 does not appear to interact with RP2 as no growth was observed at the non–permissive temperature. Conclusions: Arl3 appears to the major Arl protein partner for RP2. Our data also define the central region of RP2 as containing the binding domain for Arl3. This data suggests Arl3 could play an important role in the retina and may be involved in retinal dystrophies.

Keywords: retinal degenerations: cell biology • retinitis • degenerations/dystrophies 
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