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.
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