Effects of phosducin on the GTPase cycle of Go

Abstract

The cytosolic phosphoprotein phosducin is an inhibitor of G-protein GTPase activity and G-protein-mediated signalling. Here we investigate the effects of phosducin on individual steps of the GTPase cycle of Go, and the role of the G-protein betagamma subunits in mediating these effects. Phosducin was expressed in E. coli and purified to apparent homogeneity. Phosducin inhibited the MAS-7-stimulated as well as basal steady-state GTPase activity of Go, but did not affect the GTP-hydrolytic step. It slowed the release of GDP from Go in the presence of high Mg2+ concentrations (25 mM), and enhanced GDP release at low Mg2+ concentrations (100 microM). Likewise, phosducin inhibited basal GTPase activity at 25 mM Mg2+ and stimulated at 100 microM Mg2+. All of these effects were lost following phosphorylation of phosducin by protein kinase A (PKA). These observations are compatible with the hypothesis that phosducin antagonizes the influence of betagamma subunits on alpha(o). Titration of the effects of phosducin on the GDP release and GTPase activity of Go and on the betagamma subunit-dependent ADP-ribosylation of alpha(o) by pertussis toxin indicated an apparent affinity of approximately 20 nM. We conclude that via high-affinity interactions with G-protein betagamma subunits phosducin decreases the proportion of active GTP-bound G-proteins by slowing GDP-release without affecting GTP-hydrolysis, and that thereby it inhibits G-protein-mediated signalling.