title: Rhodopsin/transducin interactions. II. Influence of the transducin-beta gamma subunit complex on the coupling of the transducin-alpha subunit to rhodopsin.
abstract: In these studies we have investigated the role of the beta gamma T subunit complex in promoting the rhodopsin-stimulated guanine nucleotide exchange reaction (i.e. the activation event) of the alpha T subunit. The results of these studies demonstrate that although the beta gamma T subunit complex increases the association of the alpha T subunit with lipid vesicles that lack the photoreceptor, the beta gamma T complex is not necessary for the binding of alpha T to lipid vesicles containing rhodopsin, provided sufficient amounts of rhodopsin are present. The rhodopsin-promoted GDP/guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) exchange reaction, within the rhodopsin-alpha T complex, then results in the dissociation of the alpha TGTP gamma S species from the rhodopsin-containing phospholipid vesicles. A second line of evidence for the occurrence of rhodopsin/alpha T interactions, in the absence of beta gamma T, comes from phosphorylation studies using the beta 1 isoform of protein kinase C. The phosphorylation of the alpha T subunit by protein kinase C is inhibited by beta gamma T, both in the absence and in the presence of rhodopsin, but is enhanced by rhodopsin in the absence of beta gamma T. These rhodopsin-alpha T complexes also appear to be capable of undergoing a rhodopsin-stimulated guanine nucleotide exchange event. When the guanine nucleotide exchange is allowed to occur prior to the addition of protein kinase C, the phosphorylation of the alpha T subunit is inhibited. Although beta gamma T is not absolutely required for the rhodopsin/alpha T interaction, it appears to increase the apparent affinity of the alpha T subunit for rhodopsin, both when rhodopsin was inserted into phosphatidylcholine vesicles and when soluble lipid-free preparations of rhodopsin were used. This results in a significant kinetic advantage for the rhodopsin-stimulated guanine nucleotide exchange event, such that the addition of beta gamma T causes a 10-fold promotion of the rhodopsin-stimulation [35S]GTP gamma S binding to alpha T after 1 min but provides less than a 20% promotion of the rhodopsin-stimulated binding after 1 h. The ability of beta gamma T to increase the association of alpha T with the lipid vesicle surface does not appear to contribute significantly to the ability of rhodopsin to couple functionally to alpha T subunits, and there appears to be no requirement for beta gamma T in the alpha T activation event, once the rhodopsin-alpha T complex has formed.
authors.affiliation: Department of Pharmacology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853.
authors.forename: W J
authors.initials: WJ
authors.lastname: Phillips
authors.forename: S C
authors.initials: SC
authors.lastname: Wong
authors.forename: R A
authors.initials: RA
authors.lastname: Cerione
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008081:Liposomes; D046911:Macromolecular Substances; D008954:Models, Biological; D010713:Phosphatidylcholines / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D012243:Rhodopsin / Q000302:isolation & purification / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D015293:Transducin / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D008081:Liposomes; D046911:Macromolecular Substances; D010713:Phosphatidylcholines; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D012243:Rhodopsin; D011493:Protein Kinase C; D015293:Transducin