May 2006
Volume 47, Issue 13
ARVO Annual Meeting Abstract  |   May 2006
Watching Arrestin Bind: Differential Engagement of Arrestin Residues by Different Functional Forms of Rhodopsin
Author Affiliations & Notes
  • V.V. Gurevich
    Pharmacology, Vanderbilt, Nashville, TN
  • S.M. Hanson
    Pharmacology, Vanderbilt, Nashville, TN
  • D.J. Francis
    Biophysics, Medical College of Wisconsin, Milwaukee, WI
  • S.A. Vishnivetskiy
    Pharmacology, Vanderbilt, Nashville, TN
  • W.L. Hubbell
    JSEI, UCLA, Los Angeles, CA
  • C.S. Klug
    Biophysics, Medical College of Wisconsin, Milwaukee, WI
  • Footnotes
    Commercial Relationships  V.V. Gurevich, None; S.M. Hanson, None; D.J. Francis, None; S.A. Vishnivetskiy, None; W.L. Hubbell, None; C.S. Klug, None.
  • Footnotes
    Support  NIH grants EY11500, GM63097, AI58024, GM70642, EY00331, EY05216, GM07628
Investigative Ophthalmology & Visual Science May 2006, Vol.47, 824. doi:
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      V.V. Gurevich, S.M. Hanson, D.J. Francis, S.A. Vishnivetskiy, W.L. Hubbell, C.S. Klug; Watching Arrestin Bind: Differential Engagement of Arrestin Residues by Different Functional Forms of Rhodopsin . Invest. Ophthalmol. Vis. Sci. 2006;47(13):824.

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

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Purpose: : To determine arrestin residues engaged by its preferred target, phosphorylated light–activated rhodopsin (P–Rh*), as well as dark P–Rh and Rh*.

Methods: : We used site–directed spin labeling/electron paramagnetic resonance (EPR) of free and receptor–bound arrestin, site–directed mutagenesis of exposed charged residues on the receptor–binding surface of arrestin and a direct binding assay. Purified arrestin mutants spin labeled in 29 different positions and 42 mutants with surface charges neutralized or reversed were tested.

Results: : As many as 22 charge mutants decrease arrestin binding to P–Rh* by 15–47%, and one mutation significantly increases it. The mutations that affect binding to dark P–Rh are predominantly localized in the N–domain, whereas the mutations specifically affecting Rh* interaction form a cluster in the C–domain of arrestin. Upon arrestin binding to dark P–Rh the mobility of spin label in seventeen different positions in arrestin decreases, suggesting that these elements are engaged in binding. Light activation of rhodopsin induces further reduction in label mobility in seven positions. Interestingly, in two positions the label immobilized by dark P–Rh interaction reverts to higher mobility in complex with P–Rh*. These data demonstrate that arrestin elements binding dark P–Rh and P–Rh* differ, and indicate that the conformation of arrestin bound to these forms of rhodopsin is different. The binding to phosphorhodopsin (active or inactive) releases the arrestin C–tail, as judged by the loss of spin–spin interaction in a doubly labeled protein. The most dramatic binding–induced changes occur in the N–domain, particularly in the flexible "finger" loop between b–strands V and VI and in and around the three–element interaction.

Conclusions: : Different partially overlapping groups of residues on the concave sides of both arrestin domains are engaged in its binding to P–Rh*, dark P–Rh and Rh*, supporting the sequential multi–site model of the arrestin–rhodopsin interaction. Arrestin binding to P–Rh* involves more than 30 residues, suggesting that an equally extensive surface of rhodopsin must be involved in arrestin binding.

Keywords: photoreceptors • protein structure/function • signal transduction 

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