Transducin inhibition of light-dependent rhodopsin phosphorylation: evidence for beta gamma subunit interaction with rhodopsin
Department of Biochemistry and Molecular Pharmacology
Animals; Antigens; Arrestin; Blotting, Western; Cattle; Eye Proteins; G-Protein-Coupled Receptor Kinase 1; Immunologic Techniques; Light; Macromolecular Substances; Phosphorylation; Protein Kinases; Retinal Pigments; Rhodopsin; Rod Cell Outer Segment; Transducin
Biochemistry | Biochemistry, Biophysics, and Structural Biology | Molecular Biology
Rhodopsin kinase was purified from bovine retina rod outer segments as a 62-64-kDa protein that phosphorylated purified rhodopsin reconstituted into egg phosphatidylcholine/phosphatidylethanolamine liposomes. A competition binding assay in which transducin competes with rhodopsin kinase for binding sites on rhodopsin was used to assess the interaction of purified transducin subunits with rhodopsin. Preincubation of purified holotransducin with rhodopsin, in the absence of guanosine triphosphate, blocked the ability of the kinase to phosphorylate rhodopsin. Transducin-dependent inhibition of phosphorylation was relieved when guanosine 5'-(3-O-thio)triphosphate was present during the preincubation. Resolved alpha and beta gamma transducin subunits, in the absence of guanosine triphosphate, were each capable of specifically blocking phosphorylation of rhodopsin. A maximally effective concentration of T alpha or T beta gamma (1 microM) subunits inhibited phosphorylation of rhodopsin (0.23 microM) 45-65%. A similar concentration of reconstituted transductin (T alpha and T beta gamma) or native holotransducin (T alpha beta gamma) inhibited phosphorylation greater than 98%. The results indicate that rhodopsin must have a binding site for T beta gamma as well as a binding site for T alpha, and each subunit influences the recognition of bleached rhodopsin by rhodopsin kinase.
Mol Pharmacol. 1988 Oct;34(4):452-60.
Kelleher DJ, Johnson GL. (1988). Transducin inhibition of light-dependent rhodopsin phosphorylation: evidence for beta gamma subunit interaction with rhodopsin. Biochemistry and Molecular Pharmacology Publications. Retrieved from https://escholarship.umassmed.edu/bmp_pp/191