Inhibition of monoclonal antibody binding and proteolysis by light-induced phosphorylation of rhodopsin.
Light-induced phosphorylation of rhodopsin in bovine rod outer segment disk membranes inhibits the binding of three carboxyl-terminal-specific anti-rhodopsin antibodies and the cleavage of the carboxyl-terminal region of rhodopsin by trypsin and Staphylococcus aureus V-8 protease. Two monoclonal antibodies, rho 3A6 and rho 1C5, which previously have been 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 rhodopsin 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 three 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 one phosphate per 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 (Mr 25 000) and F2 (Mr 12 000) fragments.(ABSTRACT TRUNCATED AT 250 WORDS)
Molday
R S
RS
MacKenzie
D
D
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002417:Cattle; D010450:Endopeptidases; D000939:Epitopes / Q000032:analysis*; D007700:Kinetics; D008027:Light; D010446:Peptide Fragments / Q000032:analysis; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D010786:Photoreceptor Cells / Q000378:metabolism*; D011863:Radioimmunoassay; D012168:Retinal Pigments / Q000378:metabolism*; D012243:Rhodopsin / Q000276:immunology / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D012697:Serine Endopeptidases
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes; D010446:Peptide Fragments; D010761:Phosphorus Radioisotopes; D012168:Retinal Pigments; D012243:Rhodopsin; D010450:Endopeptidases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase