December 2002
Volume 43, Issue 13
ARVO Annual Meeting Abstract  |   December 2002
The Role Of Amphipathic Alpha-helix I in Arrestin Membrane Translocation
Author Affiliations & Notes
  • VV Gurevich
    Pharmacology Vanderbilt University Nashville TN
  • SA Vishnivetskiy
    Pharmacology Vanderbilt University Nashville TN
  • M-G Velez
    Pharmacology Vanderbilt University Nashville TN
  • Footnotes
    Commercial Relationships   V.V. Gurevich, None; S.A. Vishnivetskiy, None; M. Velez, None. Grant Identification: Support: NIH grants EY11500 and GM63097
Investigative Ophthalmology & Visual Science December 2002, Vol.43, 1394. doi:
  • Views
  • Share
  • Tools
    • Alerts
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      VV Gurevich, SA Vishnivetskiy, M-G Velez; The Role Of Amphipathic Alpha-helix I in Arrestin Membrane Translocation . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1394.

      Download citation file:

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

  • Supplements

Abstract: : Purpose: Arrestin rapidly binds both in vivo and in vitro to phosphorylated light-activated rhodopsin (P-Rh*), thereby quenching signaling in rods. However, in vivo arrestin apparently stays in the complex with P-Rh* for a long time, at least until P-Rh* decays into phospho-opsin. To identify the mechanism that slows down arrestin dissociation, we studied it in direct binding assay. Methods: Tritiated arrestin was pre-bound to P-Rh* for 5 min at 37oC, then diluted 50-fold and allowed to dissociate on ice. Aliquots were taken every 15 min. and bound arrestin was measured using gel-filtration-based assay. Results: The half-life of the complex was found to be 123 + 7 min, i.e., extremely long. Our recent mutagenesis data suggest that in the process of arrestin binding to P-Rh* the three-element interaction involving beta-strand I, alpha-helix one, and arrestin C-tail is disrupted. One of the released elements, alpha-helix I (residues 100-111), is a typical amphipathic helix closely resembling helices used by many proteins as reversible membrane anchors. To test the possible role of this helix in arrestin binding, we performed alanine scanning mutagenesis. Virtually every point mutation reduces arrestin binding to P-Rh* by 10-20%. The triple mutation eliminating the three leucine residues aligned on one side of the helix reduces it by 35%, suggesting that these residues are involved in binding, even though the helix is localized on the opposite side of the N-domain relative to the other residues implicated in arrestin-P-Rh* interaction. We hypothesized that after its release the helix swings out and imbeds itself into the membrane, stabilizing the arrestin-P-Rh* complex. If that is so, the two hydrophobic residues on the N-domain, Val44 and Leu46, that are completely shielded by the helix in the basal state of arrestin, would become exposed and face the membrane, possibly serving as auxiliary membrane anchors. Alanine substitutions of either Val44 or Lue46 reduce arrestin binding to P-Rh* by about 10%, while double alanine substitution reduces it by 25%, supporting this idea. Moreover, V44A,L46A mutation in the context of a phosphorylation-independent arrestin-3A mutant also reduces its binding to both P-Rh* and Rh*. Conclusion: In the process of arrestin binding alpha-helix I likely moves out of its original position and serves (along with the newly exposed Val44 and Leu46) as a membrane anchor contributing to the high stability of arrestin-P-Rh* complex. Support: NIH grants EY11500 and GM63097 (V.V.G.)

Keywords: 580 signal transduction • 527 protein structure/function • 517 photoreceptors 

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.