RHODOPSIN TRANSDUCIN INTERACTIONS .2. INFLUENCE OF THE TRANSDUCIN-BETA-GAMMA SUBUNIT COMPLEX ON THE COUPLING OF THE TRANSDUCIN-ALPHA SUBUNIT TO RHODOPSIN

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(T)GTP-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 [S-35]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.