June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Charge reversal of rhodopsin’s Glu134Arg135, reduced transducin activation without affecting rhodopsin phosphorylation
Author Affiliations & Notes
  • Kasey Rose
    Zilkha Neurogenetic Institute, University of Southern California, Los Angeles, California, United States
  • Sowmya Lokappa
    Zilkha Neurogenetic Institute, University of Southern California, Los Angeles, California, United States
  • Jeannie Chen
    Zilkha Neurogenetic Institute, University of Southern California, Los Angeles, California, United States
  • Footnotes
    Commercial Relationships   Kasey Rose None; Sowmya Lokappa None; Jeannie Chen None
  • Footnotes
    Support  This work was funded by EY12155.
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 3225. doi:
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      Kasey Rose, Sowmya Lokappa, Jeannie Chen; Charge reversal of rhodopsin’s Glu134Arg135, reduced transducin activation without affecting rhodopsin phosphorylation. Invest. Ophthalmol. Vis. Sci. 2023;64(8):3225.

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

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Abstract

Purpose : The motif (Glu134Arg135 or Asp134Arg135) is conserved in all known G protein-coupled receptors. Charge reversal of ERY to REY (Arg135Glu134) uncouples the light activated receptor (R*) from transducin activation. How this mutation affects R*’s coupling to rhodopsin kinase (GRK1) and arrestin (ARR1) is less understood. Here, using a transgenic mouse line expressing REY rhodopsin, we monitor how this mutation affects R* signaling, R* phosphorylation, and light-activated ARR1 translocation.

Methods : 2-month-old WT (C57BL/6J) and RhoRey/Rey (REY) transgenic mice were dark-adapted overnight before performing in vivo electroretinograms (ERG), or retinal isolation for isoelectric focusing (IEF). ERG recordings were obtained for a series of flashes ranging from 0.03mcd to 25cd. For IEF, dark-adapted retinae were directly isolated under dim red light after 100mcd light exposure for 5 mins.

Results : In vivo scotopic ERGs verified a substantial sensitivity reduction in REY mice (n=4) compared to wildtype (n=3). Light responses were not fully eliminated in REY retinae and were detected at flash intensities starting at 100mcd compared to 1mcd for WT. IEF showed all six differentially phosphorylated species of rhodopsin for REY (n=8) and wildtype (n=8) mice after 5min of 100mcd light exposures. Moreover, the rhodopsin populations from REY and wildtype retinae were similarly phosphorylated, suggesting unperturbed GRK1 activity.

Conclusions : Mutation of rhodopsin’s highly conserved ERY motif to REY strongly diminishes rod sensitivity to light without impacting phosphorylation of R* by GRK1, or signaling to neighboring bipolar cells. Because REY retinae do not degenerate, these mice provide a unique opportunity to investigate the functionality and interaction of the ERY motif with other phototransduction proteins.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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