Stimulation of phospholipase A2 activity in bovine rod outer segments by the beta gamma subunits of transducin and its inhibition by the alpha subunit.

Abstract

In the rod outer segments (ROS) of bovine retina, light activation of phospholipase A2 has been shown to occur by a transducin-dependent mechanism. In this report, the transducin-mediated stimulation of phospholipase A2 is shown to require dissociation of the apr heterotrimer. Addition of transducin to dark-adapted transducin-poor ROS stimulated phospholipase A2 activity only with coincident exposure to white light or, in the dark, with addition of the hydrolysis-resistant GTP analog, guanosine 5'-[y-thio]triphosphate (GTP[v-S]). Both light and GTP[y-S] induced dissociation of the transducin subunits and led to severalfold increases in the phospholipase A2 activity of transducin-rich, but not transducin-poor, ROS. In contrast, pertussis toxin treatment of transducin, which stabilizes the associated state of this G protein, prevented the stimulation of phospholipase A2 by exogenous transducin in the presence of light. Addition of purified transducin subunits to dark-adapted transducin-poor ROS revealed that phospholipase A2 stimulation occurred by action of the Py subunits. This is in contrast to the transducinmediated increase in cGMP phosphodiesterase activity, where activation occurs by action of the a subunit. The a subunit, which itself slightly stimulated phospholipase A2 activity, inhibited the P8y-induced stimulation of phospholipase A2. This inhibition appears to be the result of subunit reassociation since addition of GTP[y-S] abolished the inhibitory effect of the a subunit on the Pr-induced increase in phospholipase A2, while pertussis toxin treatment of the subunits further inhibited phospholipase A2 activity. Modulation of phospholipase A2 activity by the transducin subunits is, therefore, a mode of action for these subunits in signal transduction.Guanine nucleotide-binding regulatory proteins, G (or N) proteins, play a major role in receptor-mediated signal transduction (1-3). These G proteins are heterotrimers, which, upon ligand-induced stimulation, dissociate into a and P8y subunits (1, 3). The a subunits of these G proteins serve as substrates for specific bacterial toxins (4-7), have high affinity GTP binding sites (1, 2), and an intrinsic GTPase activity that plays a role in cycling the G proteins through their active (dissociated) and inactive (associated) states (1-3). The dissociated GTP-bound forms of the G protein a subunits are the active effectors of intracellular regulatory proteins such as adenylate cyclase and cGMP phosphodiesterase (1-3). The 8y subunits, in contrast, function primarily to facilitate the reassociation of the heterotrimeric complex, thereby deactivating the dissociated a subunits of those G proteins that share common Py subunits (1, 8). The f3y complex also appears to be required for or to enhance the interaction of specific G proteins with activated receptors (9, 10).In the rod outer segments (ROS) of the retina, the major G protein is transducin (2). The dissociated a subunit of this G protein couples the light activation of rhodopsin to stimulation of cGM...

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