Temperature-Sensitive Interactions Between RPE and Rod Outer Segment Surface Proteins

Abstract

Phagocytosis of rod outer segments by the retinal pigment epithelium is distinguished by the two distinct temperature-dependent steps of binding and ingestion. This study was designed to see if retinal pigment epithelial (RPE) plasma membrane proteins interact with ROS plasma membrane proteins at temperatures favoring either binding or ingestion. A modified blot overlay assay was used whereby Western blots of RPE plasma membrane proteins were overlaid with biotinylated ROS plasma membrane proteins. RPE/ROS interactions were detected by streptavidin-HRP and the ECL method at 25°C (ingestion), 15°C (binding), and 4°C (little or no binding or ingestion). Unlabeled ROS proteins served as the negative control. Competition with excess unlabeled ROS proteins were used to test the specificity of the protein interactions. Some protein interactions were somewhat temperature dependent. For example, two RPE plasma membrane proteins (200 kDa and 173 kDa) interacted with ROS plasma membrane proteins at both 25°C and 15°C, but not at 4°C. A strongly labeled protein at 50 kDA protein was present at 25°C but weakly labeled at 15°C and at 4°C. Other protein interaction were more clearly temperature dependent. For example, a 110 kDa RPE protein interacted with ROS proteins only at 25°C. Another RPE protein (55 kDa) interacted only at 15°C. These latter data provide correlations between binding events in the assay and previously described stages of phagocytosis.