May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
Regulation of Arrestin Binding by the Number of Rhodopsin-Attached Phosphates
Author Affiliations & Notes
  • S. A. Vishnivetskiy
    Pharmacology, Vanderbilt University, Nashville, Tennessee
  • D. Raman
    Pharmacology, Vanderbilt University, Nashville, Tennessee
  • J. Wei
    University of Washington, Seattle, Washington
  • M. J. Kennedy
    University of Washington, Seattle, Washington
  • J. B. Hurley
    University of Washington, Seattle, Washington
  • V. V. Gurevich
    Pharmacology, Vanderbilt University, Nashville, Tennessee
  • Footnotes
    Commercial Relationships S.A. Vishnivetskiy, None; D. Raman, None; J. Wei, None; M.J. Kennedy, None; J.B. Hurley, None; V.V. Gurevich, None.
  • Footnotes
    Support NIH Grants EY11500, EY06641, EY13572
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 4661. doi:
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    • Get Citation

      S. A. Vishnivetskiy, D. Raman, J. Wei, M. J. Kennedy, J. B. Hurley, V. V. Gurevich; Regulation of Arrestin Binding by the Number of Rhodopsin-Attached Phosphates. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4661.

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

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Abstract

Purpose:: To determine the number of receptor-attached phosphates sufficient to enhance arrestin binding to light-activated (P-Rh*) and dark rhodopsin (P-Rh) and opsin (P-Ops).

Methods:: Rhodopsin was phosphorylated in purified ROS by endogenous rhodopsin kinase and regenerated. Rhodopsin fractions with different levels of phosphorylation were separated by chromatofocusing and reconstituted into liposomes. Rhodopsin orientation was determined with N-terminal antibody and the phosphorhodopsin species in each fraction were quantified by mass-spectrometry. Eighteen fractions with phosphorylation levels from 0 to 7 phosphates per rhodopsin were used in direct binding assay with P-Rh*, dark P-Rh, and P-Ops.

Results:: A single rhodopsin-attached phosphate does not enhance arrestin binding to any of the functional forms tested; two are necessary to induce high-affinity interaction, and three phosphates fully activate arrestin in case of P-Rh*. Higher levels of phosphorylation do not enhance arrestin binding or the stability of arrestin-P-Rh* complex. However, arrestin complexes with hyper-phosphorylated P-Rh* are less sensitive to high salt and release retinal faster, suggesting that arrestin can form functionally different complexes with P-Rh*. The presence of four or more phosphates significantly enhances arrestin binding to dark P-Rh and P-Ops. Arrestin complexes even with highly phosphorylated P-Rh and P-Ops demonstrate the same stability and salt sensitivity as with P-Rh and P-Ops carrying three phosphates, indicating that these forms of rhodopsin cannot fully activate arrestin regardless of the phosphorylation level.

Conclusions:: Arrestin quenches rhodopsin signaling after the second or third phosphate is added by rhodopsin kinase. These data favor the models that explain the reproducibility of a single photon response with no more than four steps of rhodopsin inactivation (three phosphorylation events followed by arrestin binding). Dark P-Rh and/or P-Ops with multiple phosphates can "attract" arrestin to the outer segment or retain it in this compartment. This phenomenon may contribute to the light adaptation in rods. Arrestin complex with heavily phosphorylated rhodopsin formed with certain disease-associated rhodopsin mutants has distinct characteristics that may contribute to the phenotype of these visual disorders.

Keywords: photoreceptors • phosphorylation • signal transduction 
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