May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
Mutational Analysis of the Visual Arrestin Dimer Interface
Author Affiliations & Notes
  • S.M. Buckheister
    Pharmacology, Vanderbilt University, Nashville, TN, United States
  • M.E. Eisley
    Biophysics, Johns Hopkins University, Baltimore, MD, United States
  • S.A. Vishnivetskiy
    Biophysics, Johns Hopkins University, Baltimore, MD, United States
  • C. Hubbell
    Biochemistry, University of California, Los Angeles, CA, United States
  • C.S. Klug
    Biophysiscs, Medical College of Wisconsin, Milwaukee, WI, United States
  • W.L. Hubbell
    Biophysiscs, Medical College of Wisconsin, Milwaukee, WI, United States
  • K.G. Fleming
    Biophysiscs, Medical College of Wisconsin, Milwaukee, WI, United States
  • V.V. Gurevich
    Biophysiscs, Medical College of Wisconsin, Milwaukee, WI, United States
  • Footnotes
    Commercial Relationships  S.M. Buckheister, None; M.E. Eisley, None; S.A. Vishnivetskiy, None; C. Hubbell, None; C.S. Klug, None; W.L. Hubbell, None; K.G. Fleming, None; V.V. Gurevich, None.
  • Footnotes
    Support  NIH grants EY11500, GM63097 (VVG), GM57534 (KGF), EY05216, EY00331 (WLH),training grant GM07628(SMB)
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 1517. doi:
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      S.M. Buckheister, M.E. Eisley, S.A. Vishnivetskiy, C. Hubbell, C.S. Klug, W.L. Hubbell, K.G. Fleming, V.V. Gurevich; Mutational Analysis of the Visual Arrestin Dimer Interface . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1517.

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

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Abstract

Abstract: : Purpose: Intracellular signaling by light-activated rhodopsin is rapidly quenched by its phosphorylation and subsequent binding of visual arrestin. It has been shown previously that arrestin dimerizes at physiological concentrations. The crystal structure of visual arrestin reveals several possible dimer interfaces. We have identified key residues that form ion pairs at each interface. Our goal is to determine which residues are involved in dimerization and in the process identify the true dimer interface in solution. Methods: Point mutations disrupting putative salt bridges between monomers in a dimer and double mutations restoring these salt bridges were generated by PCR. The function of these mutants was tested in direct binding assay using tritiated arrestin and four functional forms of rhodopsin: dark phosphorylated, light-activated phosphorylated (P-Rh*), dark, and light-activated (Rh*). Analysis of dimerization was performed at 4oC, 22oC, and 37oC and pH 6.0, 7.2, and 8.5 by analytical centrifugation and light-scattering. Results: Binding data show that none of the point mutations (R18A, D138A, D138R, D162A, D162K, K166A, K166E, K235A, E346A, E346K or F197C) significantly alter binding to rhodopsin with the exception of R18E and K235E, which reduce binding to P-Rh* by one-half. Three double mutations (R18E/D138R, D162K/K235E, K166E/E346K) also do not significantly alter binding. The dimerization constant is affected by temperature and pH. For wild type and truncated arrestin (1-378) the constant decreases (i.e., dimerization increases) with increasing temperature. Truncated arrestin, which binds both P-Rh* and Rh* with high affinity, had a consistently lower propensity to dimerize than wild type. Mutations E346K and F197C also alter dimerization affinity. Conclusion: The deletion of the arrestin C-tail (which is not resolved in the crystal structure) reduces its tendency to dimerize, suggesting that it is involved in dimerization. This may be the result of the disruption of direct interactions involving the C-tail or the result of a conformational change that occurs in the molecule when these residues are absent. Further analysis of the point and double mutants is necessary to identify elements critical for dimer formation thereby defining the dimer interface in solution.

Keywords: photoreceptors • protein structure/function • signal transduction 
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