Effect of monoclonal antibody binding on alpha-beta gamma subunit interactions in the rod outer segment G protein, Gt.
The guanyl nucleotide binding regulatory protein of retinal rod outer segments, called Gt, that couples the photon receptor rhodopsin with the light-activated cGMP phosphodiesterase, can be resolved into two functional components, alpha t and beta gamma t. The effect of monoclonal antibody binding to the alpha t subunit of Gt on subunit association has been investigated in the present study. It was previously shown that this monoclonal antibody, mAb 4A, blocks interactions with rhodopsin and its epitope was located within the region Arg310-Phe350 at the COOH terminus of the alpha t subunit. In this paper, we show that mAb 4A disrupts the Gt complex. Gt migrates in 5-20% linear sucrose density gradients as a monomer, with a sedimentation coefficient of 4.1 +/- 0.07 S, while in the presence of mAb 4A, the alpha t and beta gamma t subunits show sedimentation coefficients of 7.7 +/- 0.2 and 3.7 +/- 0.1 S, respectively. The beta gamma t subunit migrates with the same sedimentation rate as pure beta gamma t. Nonimmune rabbit IgG does not modify the sedimentation behavior of Gt. The Fab fragment of mAb 4A also dissociates the Gt complex, as suggested by the change of the sedimentation rate of alpha t. This effect of mAb 4A on Gt subunit association was also confirmed by immunoprecipitation studies in the presence of detergent. In the presence of detergent, subunit association is not affected, but the formation of Gt oligomers and, therefore, the nonspecific precipitation of beta gamma t subunit are reduced.(ABSTRACT TRUNCATED AT 250 WORDS)
Mazzoni
M R
MR
Department of Physiology and Biophysics, University of Illinois, College of Medicine, Chicago 60680.
Hamm
H E
HE
D000244:Adenosine Diphosphate; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001120:Arginine; D002417:Cattle; D002499:Centrifugation, Density Gradient; D000939:Epitopes; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007140:Immunoglobulin Fab Fragments; D010649:Phenylalanine; D010786:Photoreceptor Cells / Q000378:metabolism*; D011233:Precipitin Tests; D011487:Protein Conformation; D012374:Rod Cell Outer Segment / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D010649:Phenylalanine; D000244:Adenosine Diphosphate; D001120:Arginine; D019204:GTP-Binding Proteins