Polarity and developmental regulation of two PDZ proteins in the retinal pigment epithelium

Invest Ophthalmol Vis Sci. 2001 Dec;42(13):3274-82.


Purpose: Identification of binding partners for ezrin, an actin-binding protein crucial for morphogenesis of apical microvilli and basolateral infoldings in RPE cells.

Methods: Rat eyes, rat primary RPE, the rat RPE-J cell line, and a clonal line of RPE-J cells transfected with human ezrin cDNA were analyzed by immunofluorescence microscopy and immunoblot. Immunofluorescence localization of two ezrin-binding proteins was performed in cryosections of rat eyes of various ages and in monolayers extracted with the detergent Triton X-100 and fixed in paraformaldehyde. The interaction of both proteins with ezrin and gluthathione-S-transferase (GST)-ezrin fusion proteins was analyzed by SDS-PAGE and immunoblot.

Results: Immunofluorescence microscopy of adult rat eyes detected a polarized distribution of ERM (ezrin, radixin, and moesin)-binding phosphoprotein of 50 kDa (EBP50) at the apical microvilli and synaptic-associated protein of 97 kDa (SAP97) at the basolateral surface of RPE cells, which overlapped with ezrin. These two PDZ (postsynaptic density protein [PSD-95]/disc large [DLG]-A/ZO-1) domain proteins had a similar polarized distribution and high resistance to detergent extractability, indicative of cytoskeletal association, both in primary cultures of rat RPE and in a clonal RPE-J cell line expressing high levels of transfected ezrin. RPE cell lysates from rat retinas of various postnatal ages revealed increasing levels of EBP50 and SAP97 compared with alphav integrin, a protein expressed at constant adult levels from birth. GST pull-down and immunoprecipitation experiments demonstrated a direct interaction between EBP50 and SAP97 and ezrin.

Conclusions: The data indicate that EBP50 localizes at the apical microvilli, whereas SAP97 localizes at the basolateral surface of RPE cells, probably through a direct interaction with ezrin.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Aging / metabolism*
  • Animals
  • Carrier Proteins / metabolism*
  • Cell Line
  • Cell Membrane / metabolism
  • Cell Polarity / physiology
  • Cytoskeletal Proteins
  • Discs Large Homolog 1 Protein
  • Humans
  • Intracellular Membranes / metabolism
  • Membrane Proteins
  • Nerve Tissue Proteins / metabolism*
  • Phosphoproteins / metabolism*
  • Pigment Epithelium of Eye / cytology
  • Pigment Epithelium of Eye / growth & development
  • Pigment Epithelium of Eye / metabolism*
  • Pigment Epithelium of Eye / physiology
  • Rats
  • Sodium-Hydrogen Exchangers*
  • Tissue Distribution


  • Adaptor Proteins, Signal Transducing
  • Carrier Proteins
  • Cytoskeletal Proteins
  • DLG1 protein, human
  • Discs Large Homolog 1 Protein
  • Dlg1 protein, rat
  • Membrane Proteins
  • Nerve Tissue Proteins
  • Phosphoproteins
  • Sodium-Hydrogen Exchangers
  • ezrin
  • sodium-hydrogen exchanger regulatory factor