Polarity and developmental regulation of two PDZ proteins in the retinal pigment epithelium.
OBJECTIVE
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.
Bonilha
V L
VL
Department of Ophthalmology, Margaret Dyson Vision Research Institute, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA.
Rodriguez-Boulan
E
E
D048868:Adaptor Proteins, Signal Transducing; D000375:Aging / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D016764:Cell Polarity / Q000502:physiology; D003598:Cytoskeletal Proteins; D000074288:Discs Large Homolog 1 Protein; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000254:growth & development / Q000378:metabolism* / Q000502:physiology; D051381:Rats; D017923:Sodium-Hydrogen Exchangers; D014018:Tissue Distribution; D000099149:Ezrin
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D000074288:Discs Large Homolog 1 Protein; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D017923:Sodium-Hydrogen Exchangers; D000099149:Ezrin; C513314:DLG1 protein, human; C501119:Dlg1 protein, rat; C093376:sodium-hydrogen exchanger regulatory factor