April 2009
Volume 50, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2009
Improving Phosphorylation-Independent Arrestin1: Redesign of the Rhodopsin-Binding Surface
Author Affiliations & Notes
  • S. A. Vishnivetskiy
    Pharmacology, Vanderbilt University, Nashville, Tennessee
  • X. Song
    Pharmacology, Vanderbilt University, Nashville, Tennessee
  • H. E. Boyd
    Pharmacology, Vanderbilt University, Nashville, Tennessee
  • V. V. Gurevich
    Pharmacology, Vanderbilt University, Nashville, Tennessee
  • Footnotes
    Commercial Relationships  S.A. Vishnivetskiy, None; X. Song, None; H.E. Boyd, None; V.V. Gurevich, None.
  • Footnotes
    Support  NIH Grant EY11500
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 4792. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      S. A. Vishnivetskiy, X. Song, H. E. Boyd, V. V. Gurevich; Improving Phosphorylation-Independent Arrestin1: Redesign of the Rhodopsin-Binding Surface. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4792.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose: : To construct phosphorylation-independent arrestin1 mutant that can fully compensate for the defects in rhodopsin phosphorylation in vivo.

Methods: : Site-directed mutagenesis and direct binding assay were used to create arrestin1 that binds unphosphorylated light-activated Rh* with the same affinity that wild type (WT) arrestin shows for phosphorylated P-Rh*.

Results: : Previously we found that triple alanine substitution that detaches the C-tail from the body of the molecule (3A) enhances arrestin binding to Rh* in vitro. Transgenic expression of arrestin1-3A in mice lacking rhodopsin kinase or expressing mutant rhodopsin that does not have phosphorylation sites improves photoreceptor survival and facilitates photoresponse recovery (measured by double-flash ERG) up to three-fold, as compared to animals expressing WT arrestin on the same genetic backgrounds. However, photoresponse recovery in "rescued" animals was slower than in normal WT mice. We found that mutant arrestin-Rh* complex was less stable than WT arrestin-P-Rh* complex. Since receptor-binding surface of WT arrestin fits multi-phosphorylated rhodopsin best, we hypothesized that it must be redesigned for optimal fit to unphosphorylated Rh*. We identified mutations on the rhodopsin-binding concave sides of both arrestin domains that greatly increase the affinity of 3A mutant for Rh*. The levels of binding of several mutants to Rh* in vitro approaches that of WT arrestin to P-Rh*. Combinations of individual mutations improving arrestin affinity for Rh* further increases the binding.

Conclusions: : Targeted redesign of rhodopsin-binding surface of the phosphorylation-independent arrestin1 mutant is necessary to ensure better fit for the cytoplasmic tip of unphosphorylated Rh*. Higher affinity of new phosphorylation-independent versions of arrestin1 for Rh* will increase their ability to compensate for the defects in rhodopsin phosphorylation in vivo, enhancing their therapeutic potential in human disorders associated with excessive signaling by mutant forms of rhodopsin.

Keywords: protein structure/function • photoreceptors • signal transduction 
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×