Inhibition of monoclonal antibody binding and proteolysis by light-induced phosphorylation of rhodopsin

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Abstract
Light-induced phosphorylation of rhodopsin in bovine rod outer segment disk membranes inhibits the binding of 3 carboxyl-terminal-specific anti-rhodopsin antibodies and the cleavage of the carboxyl-terminal region of rhodopsin by trypsin and Staphylococcus aureus V-9 protease. Two monoclonal antibodies, rho 3A6 and rho 1C4, which previously were shown to preferentially bind to the 8''-12'' and the 9''-18'' carboxyl-terminal segments of rhodopsin, respectively, are both highly sensitive to phosphorylation. When an average of one phosphate is incorporated per rhodopsin, the binding reactivity of rhodopsin for these antibodies decreases to 30% that of nonphosphorylated rodopsin as measured in radioimmune competition assays. Reactivity of the rho 1D4 antibody whose primary binding site is localized in the 1''-8'' C-terminal segment of rhodopsin is unaffected at this level of phosphorylation but decreases to 30% when 3 phosphates on average are incorporated per rhodopsin. Direct binding studies using 125I-labeled antibodies indicate that phosphorylation of rhodopsin decreases the maximum extent of rho 3A6 and rho 1C5 binding to rhodopsin. For rho 1D4, the maximum extent of binding is unaffected by phosphorylation, but the dissociation constant is increased by 10-fold. Phosphorylation of rhodopsin also inhibits cleavage of the 1''-9'' and 1''-7'' carboxyl-terminal peptides by trypsin and S. aureus V-8 protease, respectively. When an average of 1 phosphate/rhodopsin is incorporated, cleavage decreases to 40% that of nonphosphorylated rhodopsin as measured by high-performance liquid chromatography. Phosphorylation of rhodopsin had no effect on S. aureus cleavage of rhodopsin into the F1 (MW 25,000) and F2 (MW 12,000) fragments. The 9''-Thr or possibly the 11''-Ser which is in the primary binding domain of rho 3A6 and rho 1C5 antibodies and is in close proximity to the cleavage sites for trypsin and S. aureus protease is preferentially phosphorylated at low levels of phosphate incorporation. In related experiments, the binding of these carboxyl-terminal specific antibodies to rhodopsin in rod outer segment membranes is shown to inhibit light-induced phosphorylation of rhodopsin. These carboxyl-terminal antibodies and proteolysis may be useful probes for distinguishing phosphorylated from nonphosphorylated rhodopsin for in vitro and in vivo analysis.