Binding sites of photoreceptor-specific antibodies COS-1, OS-2 and AO

The chicken red-sensitive cone visual pigment (iodopsin) and several synthetic peptides of cone and rod visual pigments were used to find the binding sites of our photoreceptor-specific antibodies with immunocytochemistry. The ability of iodopsin to block immunolabeling with monoclonal antibodies COS-1 and OS-2 furnished direct evidence that both antibodies are specific to visual pigments. Immunocytochemistry on whole-mount retinas with and without detergent, as well as electron microscopic labeling of cone photoreceptor membranes revealed the binding sites of COS-1 and OS-2 to be on the cytoplasmic side of the membrane. By testing several synthetic peptides, mainly from the C-terminal region of the cone visual pigments, we found that the domain consisting of the last 6 amino acids of the human red/green-, and the chicken red-sensitive cone pigments completely blocked immunolabeling with COS-1, while the sequence consisting of the last 12 amino acids of the human blue cone pigment was effective to block the binding of OS-2. Both monoclonals can be regarded therefore C-terminal specific antibodies. OS-2 was found to bind to the dark-adapted photopigment more strongly than to the light-adapted one. The binding of the polyclonal rhodopsin antibody AO was almost entirely inhibited by the N-terminal synthetic peptide of bovine rhodopsin indicating that this antibody binds primarily to the N-terminal domain of rhodopsin in a tissue environment.