May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
Rhodopsin Binds to Elements in Both the N- and C-Domains of Arrestin
Author Affiliations & Notes
  • W.C. Smith
    Ophthalmology, University of Florida, Gainesville, FL, United States
  • A. Dinculescu
    Ophthalmology, University of Florida, Gainesville, FL, United States
  • J.H. McDowell
    Ophthalmology, University of Florida, Gainesville, FL, United States
  • P.A. Hargrave
    Ophthalmology, University of Florida, Gainesville, FL, United States
  • Footnotes
    Commercial Relationships  W.C. Smith, None; A. Dinculescu, None; J.H. McDowell, None; P.A. Hargrave, None.
  • Footnotes
    Support  NIH Grants EY006225 and EY006226, Research to Prevent Blindness Career Development Award
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 1514. doi:
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      W.C. Smith, A. Dinculescu, J.H. McDowell, P.A. Hargrave; Rhodopsin Binds to Elements in Both the N- and C-Domains of Arrestin . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1514.

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Abstract

Abstract: : Purpose: The crystal structure of arrestin shows that the protein has two domains of seven-stranded beta sandwiches. Several labs have shown that residues in the N-domain are involved in the binding interaction with photoactivated and phosphorylated rhodopsin (R*P). In this study, we show that rhodopsin-binding extends beyond the N-domain and includes residues in the C-domain. Methods: Scanning alanine mutagenesis was performed for amino acids 241-254 in bovine visual arrestin. Mutant arrestins were expressed in yeast, purified in milligram quantities, and binding to R*P assessed in a centrifugation assay using rhodopsin in rod outer segment membranes prepared from bovine retinas. Competitive inhibition using an anti-myc antibody was also performed with arrestins containing a myc epitope (EQKLISEEDL) inserted in loop structures of the protein. Cysteine mutants of arrestin were labeled with Alexa Fluor594, an environmentally sensitive fluorophore. Results: Scanning alanine mutagenesis of residues 241-254 showed that alanine substitution of Tyr-250, Ser-251, and Ser-252 had the most significant impact on binding to R*P (20-25% reduction compared to native arrestin). Placement of the ten amino acid myc epitope between Tyr-250 and Ser-251 (250myc251) also reduced binding to R*P by approximately 50%, although the tagged arrestin still retained good binding selectivity for R*P. The remaining selectivity for R*P in 250myc251 could be competitively inhibited using an anti-myc monoclonal antibody (IC50= 2 uM). Labeling of S251C with AlexaFluor594 produced an arrestin with strong fluorescence emission at 600 nm when excited with UV light. This emission was enhanced when arrestin was bound to R*P. Labeling of S156C and S199C with the same fluorophore also produced a strongly fluorescent arrestin, but with no change in fluorescence emission when bound to R*P. Conclusions: The results from alanine scanning mutagenesis and competition with the anti-myc antibody demonstrate that residues in the C-domain of arrestin, particularly 250-252, are involved in the binding interaction with R*P. The increase in fluorescence of the fluorophore-labeled S251C when bound to R*P provides independent confirmation that rhodopsin must be closely apposed to this region of the protein.

Keywords: protein structure/function • molecular biology 
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