September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Identification of possible target binding site in retinal degeneration 3 (RD3) protein
Author Affiliations & Notes
  • Igor V Peshenko
    Research, Salus University, Elkins Park, Pennsylvania, United States
  • Elena V Olshevskaya
    Research, Salus University, Elkins Park, Pennsylvania, United States
  • Alexander M Dizhoor
    Research, Salus University, Elkins Park, Pennsylvania, United States
  • Footnotes
    Commercial Relationships   Igor Peshenko, None; Elena Olshevskaya, None; Alexander Dizhoor, None
  • Footnotes
    Support  NIH grants EY11522; Pennsylvania Department of Health CURE Formula Grant
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 3173. doi:
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      Igor V Peshenko, Elena V Olshevskaya, Alexander M Dizhoor; Identification of possible target binding site in retinal degeneration 3 (RD3) protein. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3173.

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

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Abstract

Purpose : Regulation of retinal membrane guanylyl cyclase, RetGC1, plays important role in rod and cone physiology. Retinal degeneration 3 (RD3) protein associates with RetGC, inhibits catalytic activity of the enzyme and prevents the cyclase activation by GCAPs [1,2]. RD3 is also crucial for the proper expression of both RetGC isozymes in photoreceptors [3]. The purpose of this study was to identify the target -binding site in RD3 molecule.

Methods : Non-tagged or GFP-tagged human RD3 was expressed in E. coli or in HEK293 cells, and the recombinant RD3 was then tested in RetGC activity assay in vitro or probed for co-localization with RetGC1 in HEK293 cells using the in cyto methodology previously described in detail [4].

Results : We probed the primary amino acid sequence of a human RD3 by introducing 3 to 6 amino acid residues long amino acid substitutions in those regions of the RD3 primary structure that were most conserved among different mammalian species. The GFP-tagged RD3 mutants were co-expressed with mOrange-tagged RetGC1 in living HEK293 cells to evaluate the effect of the substitutions on the ability of RD3 to bind and co-localize with RetGC1 in the membranes. The mutants that showed impaired binding to membrane cyclase in HEK293 cells also failed to properly suppress activation of RetGC1 by GCAP1 in vitro. We have identified two regions in the RD3 primary structure where the amino acid substitutions produced the most prominent effect.

Conclusions : We identified the putative cyclase–binding interface portion of the RD3 primary structure that can directly interact with its target enzyme, RetGC1. References: [1] Azadi et al. (2010) Proc. Natl. Acad. Sci. USA 107, 21158-63; [2] Peshenko et al. (2011) Biochemistry 50, 9511-19; [3] Zulliger et al. (2015) J. Biol. Chem. 290, 3488-99; [4] Peshenko et al. (2015) J. Biol. Chem. 290, 19584-96

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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