July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Mutations in Leber's Congenital Amaurosis Disrupt Retinal Guanylate Cyclase Interaction with Rhodopsin in Vitro; Involvement In Phototransduction
Author Affiliations & Notes
  • Vladimir A Bondarenko
    Basic Science, Touro University Nevada, College of Osteopathic Medicine, Henderson, Nevada, United States
  • Footnotes
    Commercial Relationships   Vladimir Bondarenko, None
  • Footnotes
    Support  Touro University Nevada Research Grant
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 2370. doi:
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      Vladimir A Bondarenko; Mutations in Leber's Congenital Amaurosis Disrupt Retinal Guanylate Cyclase Interaction with Rhodopsin in Vitro; Involvement In Phototransduction. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2370.

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

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Abstract

Purpose : The purpose of this study is to demonstrate the direct communication between mammalian rhodopsin (Rho) and retinal guanylate cyclase (retGC) and to understand the role of their communication in phototransduction.

Methods : We expressed bovine wild type intradiscal domain of retGC or its mutants N129K and R313C in HEK293 cells and used soluble fractions in pull down assay to demonstrate interaction between these proteins and Rho.

Results : It has been shown that retGC activity in light-exposed ROS membranes was much higher than the activity of the same membranes isolated the under dark conditions suggesting that communication of retGC with activated rhodopsin is required for its full activation. To test this hypothesis we used pull down assay and a co-expression of fluorescent protein-tagged Rho and retGC in mammalian cells. Fluorescent protein-tagged bovine wild type or mutant intradiscal domains of retGC were transiently expressed in HEK293 cells. Soluble fractions containing intracellular domain of retGC were prepared from HEK293 cells transfected with plasmid vector expressing wild type or mutant proteins. We used photobleached and hypotonically washed rod outer segment (ROS) membranes as a source of photoactivated Rho. ROS membrane and soluble fractions were mixed together and upon incubation, ROS membranes were separated from soluble fraction and analyzed by western blot. The analysis demonstrated that only wild type but not mutant intradiscal domain of retGC was detected in ROS membrane fraction suggesting that mutation causing LCA disrupt the interaction between retGC and Rho. Moreover, co-expression of Rho and wild type retGC intradiscal domain, attached to different fluorescent proteins, confirmed that they colocalize in mammalian cell. This is additional evidence suggesting their interaction in vivo.

Conclusions : Based on our observations, we propose that photoactivated Rho and retGC directly interact in the intradiscal space and this communication is necessary for sensitization of retGC leading to more efficient recovery of photoreceptors to the dark state. Mutations N129K and R313C found in Leber’s Congenital Amaurosis (LCA) disrupt interaction between Rho and intradiscal domain of retGC in vitro and may interfere with sensitization of the enzyme which can lead to less efficient photoreceptor recovery to the dark state.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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