May 2007
Volume 48, Issue 13
ARVO Annual Meeting Abstract  |   May 2007
Each Rhodopsin Molecule Binds Its Own Arrestin
Author Affiliations & Notes
  • V. V. Gurevich
    Pharmacology, Vanderbilt University, Nashville, Tennessee
  • S. M. Hanson
    Pharmacology, Vanderbilt University, Nashville, Tennessee
  • E. V. Gurevich
    Pharmacology, Vanderbilt University, Nashville, Tennessee
  • S. A. Vishnivetskiy
    Pharmacology, Vanderbilt University, Nashville, Tennessee
  • M. R. Ahmed
    Pharmacology, Vanderbilt University, Nashville, Tennessee
  • X. Song
    Pharmacology, Vanderbilt University, Nashville, Tennessee
  • Footnotes
    Commercial Relationships V.V. Gurevich, None; S.M. Hanson, None; E.V. Gurevich, None; S.A. Vishnivetskiy, None; M.R. Ahmed, None; X. Song, None.
  • Footnotes
    Support NIH Grant EY11500
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 1110. doi:
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      V. V. Gurevich, S. M. Hanson, E. V. Gurevich, S. A. Vishnivetskiy, M. R. Ahmed, X. Song; Each Rhodopsin Molecule Binds Its Own Arrestin. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1110.

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

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Purpose:: Arrestins are ubiquitous regulators of the most numerous family of signaling proteins, G protein-coupled receptors. Two models of the arrestin-receptor interaction have been proposed: the binding of one arrestin to an individual receptor or to two receptors in a dimer. These models have different biological implications. To resolve this controversy, we determined the binding stoichiometry in vivo and in vitro.

Methods:: In vivo we used rod photoreceptors where rhodopsin and arrestin are expressed at comparably high levels. We genetically manipulated their expression using arrestin and rhodopsin homo- and hemizygous mice (A+/+Rh+/+, A+/-Rh+/+, A+/+Rh+/-, and A+/-Rh+/-), as well as transgenic mice expressing high levels of arrestin to generate animals with arrestin-rhodopsin ratio ranging from 0.4 to 2.7. We took advantage of light-dependent arrestin translocation where arrestin localization in the light is determined by its binding to activated rhodopsin in the outer segment. We also used arrestin-rhodopsin ratios from 0.5 to 3.3 in vitro with purified proteins carefully quantified by amino acid analysis to measure direct binding between the two proteins that cannot be complicated by the participation of any other cellular components.

Results:: In vivo we found that the maximum amount of arrestin that moves to the rhodopsin-containing compartment exceeds 80%, but not 100%, of the molar amount of rhodopsin present in the outer segment. We only observed incomplete translocation in mice where photoreceptors expressed arrestin in excess of rhodopsin. In vitro experiments confirmed that when only these two proteins are present, arrestin "saturates" rhodopsin at a one-to-one ratio. We also found that arrestin dimers and tetramers completely dissociate in the process of its binding, demonstrating that only monomer can bind rhodopsin, so that the observed binding stoichiometry cannot be explained by arrestin oligomer interaction with rhodopsin oligomer.

Conclusions:: Both the extent of arrestin translocation in mice and the level of binding of purified arrestin to pure phosphorhodopsin in isolated disc membranes can only be rationalized in the context of one-to-one binding model. Thus, a single rhodopsin molecule is necessary and sufficient to bind arrestin in vivo and in vitro. Remarkable structural conservation among receptors and arrestins strongly suggests that all arrestin subtypes bind individual molecules of their cognate G protein-coupled receptors.

Keywords: signal transduction • protein structure/function • photoreceptors 

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