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.