The developmental regulation of CD81 in the rat retina

PURPOSE: The tetraspanin CD81 is expressed in MUller glial cells and retinal pigment epithelium (RPE). CD81 and other members of the tetraspanin family link extracellular interactions of cells into intracellular cascades. This study examined the developmental expression of CD81 and protein-protein interactions linking CD81 to intracellular proteins. METHODS: We used synthetic peptides of the C-terminal intracellular domains of CD81 to probe fusion proteins of PDZ domains blotted to nitrocellulose membranes, then confirmed the relationships between the PDZ proteins using immunoprecipitation methods. Colocalization of the associated proteins was analyzed across development, using double-label immunohistochemical methods in the retina of Sprague-Dawley rats. RESULTS: The C-terminal intracellular sequences of CD81 bound to three putative PDZ domains that potentially represented domains on Sap97 and EBP50. In immunoprecipitation experiments using RPE cells, CD81 coprecipitated with both proteins, EBP50 and Sap97. Like CD81, EBP50 and Sap97 are expressed at low levels immediately after birth and upregulated during the first two postnatal weeks, reaching almost adult levels at postnatal day 20. In the RPE layer, synapse-associated protein 97 (Sap97) and CD81 were associated with the basolateral surface of the cells; ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) localizing with CD81 was found on microvilli at the inner surface of RPE cells. CONCLUSIONS: These results support the hypothesis that CD81 is associated with the final stages of RPE cell maturation, establishing key molecular interactions linking the cell membrane proteins into macromolecular complexes containing PDZ protein scaffolds.