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Abstract
Rhodopsin, the prototypical G-protein-coupled receptor, which is densely packed in the disc membranes of rod outer segments, was proposed to function as a monomer. However, a growing body of evidence indicates dimerization and oligomerization of numerous G-protein-coupled receptors, and atomic force microscopy images revealed rows of rhodopsin dimers in murine disc membranes. In this work we demonstrate by electron microscopy of negatively stained samples, blue native- and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, chemical crosslinking, and by proteolysis that native bovine rhodopsin exists mainly as dimers and higher oligomers. These results corroborate the recent findings from atomic force microscopy and molecular modeling on the supramolecular structure and packing arrangement of murine rhodopsin dimers.
2-ME, 2-mercaptoethanol; AFM, atomic force microscopy/microscope; BN-PAGE, blue native-polyacrylamide gel electrophoresis; CCB, Coomassie brilliant blue G-250; DM, n-dodecyl-β-D-maltoside; DSP, dithiobis(succinimidyl propionate); DTT, dithiothreitol; EDTA, ethylene diamine tetra-acetic acid; GPCR, G protein-coupled receptor; HEPES, N-(2-hydroxyethyl) piperazine-N′-2-ethanesulfonic acid; LC-SPDP, succinimidyl 6-[3-(2-pyridyldithio)-propionamido]hexanoate; ROS, rod outer segment(s); SB, sample buffer; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; TEM, transmission electron microscopy
2-ME, 2-mercaptoethanol; AFM, atomic force microscopy/microscope; BN-PAGE, blue native-polyacrylamide gel electrophoresis; CCB, Coomassie brilliant blue G-250; DM, n-dodecyl-β-D-maltoside; DSP, dithiobis(succinimidyl propionate); DTT, dithiothreitol; EDTA, ethylene diamine tetra-acetic acid; GPCR, G protein-coupled receptor; HEPES, N-(2-hydroxyethyl) piperazine-N′-2-ethanesulfonic acid; LC-SPDP, succinimidyl 6-[3-(2-pyridyldithio)-propionamido]hexanoate; ROS, rod outer segment(s); SB, sample buffer; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; TEM, transmission electron microscopy