May 2005
Volume 46, Issue 13
ARVO Annual Meeting Abstract  |   May 2005
The MAP Activities of the Full–Length Retinitis Pigmentosa 1 (RP1) Protein
Author Affiliations & Notes
  • N.R. Benitah
    Ophthalmology, University of Pennsylvania, Philadelphia, PA
  • Q. Liu
    Ophthalmology, University of Pennsylvania, Philadelphia, PA
  • E.A. Pierce
    Ophthalmology, University of Pennsylvania, Philadelphia, PA
  • Footnotes
    Commercial Relationships  N.R. Benitah, None; Q. Liu, None; E.A. Pierce, None.
  • Footnotes
    Support  NIH Grant EY12910, RPB, FFB, Rosanne Silbermann Foundation
Investigative Ophthalmology & Visual Science May 2005, Vol.46, 1704. doi:
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      N.R. Benitah, Q. Liu, E.A. Pierce; The MAP Activities of the Full–Length Retinitis Pigmentosa 1 (RP1) Protein . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1704.

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

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Abstract: : Purpose: Mutations in the RP1 gene are a common cause of dominant retinitis pigmentosa (RP). All of the mutations in RP1 identified to date are predicted to produce mutant proteins which contain the N–terminal 1/3 to 1/2 of RP1. The mechanism by which these truncated N–RP1 proteins cause photoreceptor cell death remains to be determined. The RP1 protein has previously been shown to be a photoreceptor–specific microtubule associated protein (MAP). Both the full–length and N–Rp1 proteins stabilize the photoreceptor axoneme in vivo. However, in vitro tests of RP1 MAP activities have been limited to the N–RP1 protein due to difficulties with expression of the full–length protein in heterologous systems. We have used adenoviral vectors to express full–length RP1 in cultured cells, and tested the MAP activities of the full–length protein. Methods: Three recombinant adenoviral vectors were created containing the coding sequences for full–length human RP1 (FL), a truncated N–RP1 form with previously demonstrated MAP activity (amino acids 1–683), and a form from which exons 2 and 3, which encode the microtubule binding domain, were deleted (designated Δ2–3). All three viruses contain a C–terminal V5 epitope tag. COS–7 cells were infected with the viruses at an MOI of 104 PFU/cell for 24 hours, followed by treatment with varying concentrations of the microtubule–destabilizing drug nocodazole. The cells were then fixed and stained with antibodies to V5 and α–tubulin. Results: 20% of cells infected with the N–RP1 virus expressed the protein, compared to 3% of cells infected with the FL virus and <0.1% of cells infected with the Δ2–3 virus. As demonstrated previously, the truncated N–RP1 protein associated closely with microtubules and stabilized them against the depolymerizing effects of nocodazole. The FL–RP1 protein was also associated with microtubules, but did not appear to prevent nocodazole–induced depolymerization to the same degree as N–RP1. Too few cells expressed the Δ2–3 protein to evaluate its effects on microtubule stability. Conclusions: Adenovirus is capable of generating expression of the full–length RP1 protein, although with less efficiency than the truncated N–RP1 protein. The full length RP1 protein does not appear to stabilize microtubules in heterologous cells as well as the N–RP1 protein. This suggests that domains in the C–terminal portion of RP1 could modulate the MAP activity of the N–terminal microtubule–binding domain. This may help explain how mutations in RP1 cause retinal degeneration. Additional experiments are in progress to define the regions in RP1 which modulate its MAP activity.

Keywords: photoreceptors • retinal degenerations: cell biology • proteins encoded by disease genes 

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