May 2004
Volume 45, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2004
Interactions of CRALBP and EBP50/NHERF–1
Author Affiliations & Notes
  • J.C. Saari
    Ophthalmology,
    Biochemistry,
    University of Washington, Seattle, WA
  • M. Nawrot
    Ophthalmology,
    University of Washington, Seattle, WA
  • T. Liu
    Biochemistry,
    University of Washington, Seattle, WA
  • D.C. Teller
    Biochemistry,
    University of Washington, Seattle, WA
  • J.W. Crabb
    Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, OH
  • Footnotes
    Commercial Relationships  J.C. Saari, None; M. Nawrot, None; T. Liu, None; D.C. Teller, None; J.W. Crabb, None.
  • Footnotes
    Support  NIH Grant EY02317, EY01730, EY06603, GM63020
Investigative Ophthalmology & Visual Science May 2004, Vol.45, 1262. doi:
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      J.C. Saari, M. Nawrot, T. Liu, D.C. Teller, J.W. Crabb; Interactions of CRALBP and EBP50/NHERF–1 . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1262.

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

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Abstract

Abstract: : Purpose: : To define ligand dependency and target sequence specificity associated with interactions between cellular retinaldehyde–binding protein (CRALBP) and PDZ–domains of ERM–binding phosphoprotein 50 (EBP50), also known as sodium hydrogen exchanger regulatory factor type 1 (NHERF–1). Both proteins are present in RPE and Müller cell microvilli and could interact in vivo. Methods: Ligand dependency was analyzed with serial dilutions of EBP50/NHERF–1 applied to nitrocellulose and treated with identical concentrations of apo–rCRALBP, rCRALBP·11–cis–retinal, rCRALBP·11–cis–retinol, or mutant apo–rCRALBPs (M225K and R233W). Interactions were detected with anti–CRALBP. Target sequence specificity was evaluated with the molecular modeling program MOE using published molecular coordinates of PDZ–1 in EBP50/NHERF–1 and C–terminal sequences of human, bovine, and mouse CRALBP. Results: Binding of CRALBP to recombinant EBP50 was independent of the occupancy of the CRALBP retinoid–binding pocket. CRALBP mutants associated with human retinitis punctata albescens and Bothnia dystrophy (M225K, R233W) bind to EBP50/NHERF–1 with comparable affinities. CRALBP C–terminal peptides NTAL (mouse) and NTAF (bovine, human) are predicted to form similar networks of H–bonds with the ß2–strand and α2–helix of PDZ1 of EBP50/NHERF–1. Both leucine and phenylalanine fit into the hydrophobic cavity of the PDZ1 peptide–binding groove. Conclusions: PDZ–domain proteins serve as scaffolds for the assembly of multi–protein complexes in which interaction through the C–terminal 3–4 amino acids of the target protein minimally perturbs its structure. The lack of retinoid dependency for interaction of CRALBP and EBP50/NHERF–1 is consistent with this function. Interaction of CRALBP, a visual cycle component, and EBP50/NHERF–1 within RPE microvilli may be a mechanism for assembly of a complex of visual cycle components.

Keywords: retinoids/retinoid binding proteins • retinal pigment epithelium • protein structure/function 
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