Differential immunogold-dextran labeling of bovine and frog rod and cone cells using monoclonal antibodies against bovine rhodopsin

Abstract

Eleven monoclonal antibodies against different segments of bovine rhodopsin were used with immunogold-dextran markers to label Lowicryl thin sections of bovine and frog retinal photoreceptor cells for visualization by transmission electron microscopy. Antibodies against the C-terminus, F1-F2 loop and N-terminus of rhodopsin were all observed to label bovine rod outer segments (ROS) densely, but to label rod inner segments (RIS) only sparsely. Most antibodies bound 200-600 gold particles per micron2 in the ROS, 10-60 gold particles per micron2 in the RIS and 5-20 particles per micron2 on the Lowicryl resin. One antibody against the N-terminus and one antibody against the C-terminus resulted in the binding of over 1000 particles per micron2 in bovine ROS. Cone outer segments (COS) were labeled with only one antibody, rho 3D6, having a specificity for the 1'-4' C-terminus of bovine rhodopsin. Ninety per cent of the COS were observed to be labeled with this antibody. Immunogold-dextran labeling was also used to study the cross-reactivity of these antibodies to rhodopsin in red and green frog ROS and COS. Monoclonal antibodies directed against sites along the F1-F2 loop, and the N-terminus labeled red frog ROS densely, but did not label either green ROS or COS. Three C-terminal specific antibodies against binding sites along the 1'-8' segment labeled both green and red ROS, but a higher extent of labeling was observed on the green ROS. Antibody rho 3D6, which bound to bovine COS, also labeled frog COS. These results indicate that the F1-F2 loop and segments along to the N-terminus and the C-terminus of bovine rhodopsin show a high degree of homology with corresponding regions of frog rhodopsin from red ROS; the C-terminal 1'-8' segment of bovine rhodopsin is closely related to the corresponding segment of frog rhodopsin from green ROS; and the 1'-4' C-terminus of bovine rhodopsin is highly homologous to segments of opsin from most bovine and frog COS. The labeling of frog red ROS in relation to multiple forms of rhodopsin observed by SDS-gel electrophoresis is discussed.