Abstract
Abstract: :
Purpose: CD81 is a small membrane protein associated with the regulation of cell–cycle of cultured astrocytes and retinal pigment epithelium (RPE). To begin to understand the role of CD81, this study identifies the interactions of CD81 with other membrane proteins and intracellular proteins in RPE cells. Methods: Immunoprecipitation studies used a human RPE cell line (D407) transfected with rat CD81 (D407–rCD81). Membrane proteins were surface labeled, the cells were lysed in mild detergent and CD81 was immunoprecipitated. Colocalization of proteins was defined by double–labeled immunofluorescence methods in sections of rat eyes, rat primary RPE cells and human RPE cell line. Results: When rat CD81 was immunoprecipitated from the D407–rCD81 cells several surface labeled proteins and unlabeled proteins were associated with the rat CD81. Cross–blotting experiments identified many of these associated proteins, including CD81, CD9, CD151, EWI–F, EWI–2, alpha 3 integrin, beta 1 integrin and beta 5 integrin. We have also observed several associated intracellular proteins including SAP97, EBP50 and Ezrin. Our immunohistochemical studies reveal at least two specific CD81 complexes, one in the microvilli containing EBP50 and a second at the basolateral surface containing Sap97. Colocalization of CD81 with EWI–F or EWI–2 was also shown in cultured human RPE cells. Conclusions: CD81 forms at least two molecular complexes within the RPE cell, each with unique proteins and intracellular links. At the present time we are defining the specific complex associate with the microvilli on the apical surface. We know that this complex includes EBP50/Ezrin and beta 5 integrin. One cannot help but speculate that the CD81 complex in the microvilli plays a role in the phagocytosis of shed rod outer segments. The second complex containing SAP97 on the lateral and basal surface of the cells is ideally positioned to aid the cell in stabilizing its connections with Bruch’s membrane and the adjacent RPE cells. Current efforts are designed to test the hypothesis that CD81 is associated with compartmentalization of these molecular complexes.
Keywords: cell adhesions/cell junctions • proliferation • retinal pigment epithelium