We are investigating the mechanisms involved in the interaction between rhodopsin-containing membranes and the cells of the retinal pigment epithelium (RPE). Little is known of the processes involved in regulating this key phase of the phenomenon of rod renewal. Preliminary evidence has been obtained indicating the presence of receptors for rhodopsin on the RPE surface. The basic experimental design for these studies involves the use of RPE cells of the embryonic chick maintained in cell culture as the biological systems, and rhodopsin-liposomes, rod outer segments (ROS), and disc membranes prepared from the ROS as the target rhodopsin- containing membranes. After incubation under various conditions, the association between the cells and the membranes will be quantitated by means of a radioimmunoassay for rhodopsin. Interference in this process has been observed upon coating the membranes with Fab fragments obtained from polyclonal anti-rhodopsin IgG. Monoclonal antibodies raised against bovine rhodopsin will be characterized in terms of reactivity against specific amino acid sequences of rhodopsin, and Fab fragments prepared from them. The influence of these molecules on the binding may provide information concerning domains of rhodopsin recognized by the RPE. We have observed the binding of rhodopsin and rhodopsin- containing membranes by membranes from bovine RPE. We will explore the nature of this binding, its specificity, and attempt to characterize the components on the RPE that mediate this effect. All of these studies are directed toward investigating whether or not there are receptors for rhodopsin on the RPE. If receptors for rhodopsin are revealed, their isolation will be attempted using rhodopsin affinity chromatoraphy and antiidiotype-antibody technology. We will explore further the possibility that glycosylation of """"""""receptors"""""""" on the RPE are involved in the binding of rhodopsin- membranes. The effects of various inhibitors of glycosylation on the process will be examined, such as tunicamycin, swainosonine and castanospermine. Further studies will be carried out dealing with the influence of various carbohydrates on the binding process, using RPE and mouse macrophage cells, with special attention paid to the effect of neoglycoproteins.
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