ISSN:
1432-1912
Keywords:
Key words G-protein
;
GTPase-cycle
;
Mastoparan
;
Phosducin
;
Protein kinase A
Source:
Springer Online Journal Archives 1860-2000
Topics:
Medicine
Notes:
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 βγ 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 (25mM), and enhanced GDP release at low Mg2+ concentrations (100μM). Likewise, phosducin inhibited basal GTPase activity at 25mM Mg2+ and stimulated at 100μM 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 βγ subunits on αo. Titration of the effects of phosducin on the GDP release and GTPase activity of Go and on the βγ subunit-dependent ADP-ribosylation of αo by pertussis toxin indicated an apparent affinity of ≈20nM. We conclude that via high-affinity interactions with G-protein βγ 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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/PL00005181
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