Interactions of Phosducin with the Subunits of G-Proteins

• Published in: The Journal of biological chemistry 1998-04, Vol.273 (16), p.9465-9471
• Main Authors: Bauer, Petra H., Blüml, Klaus, Schröder, Stefan, Hegler, Jutta, Dees, Christian, Lohse, Martin J.
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.273.16.9465

The high affinity interactions of phosducin with G-proteins involve binding of phosducin to the G-protein βγ subunits. Here we have investigated whether phosducin interacts also with G-protein α subunits. Interactions of phosducin with the individual subunits of G o were measured by retaining phosducin-G-protein subunit complexes on columns containing immobilized anti-phosducin antibodies. Both the α and the β subunits of trimeric G o were specifically retained by the antibodies in the presence of phosducin. This binding was almost completely abolished for both subunits following protein kinase A-mediated phosphorylation of phosducin and was reduced, more for α than for β subunits, by the stable GTP analog guanosine 5′-(3- O -thio)triphosphate. Isolated α o was also retained on the columns in the presence of phosducin but not in the presence of protein kinase A-phosphorylated phosducin. Likewise, purified G-protein βγ subunit complexes as well as purified α subunits of G o and G t were precipitated together with His 6 -tagged phosducin with nickel-agarose; this co-precipitation occurred concentration-dependently, with apparent affinities for phosducin of 55 n m (G βγ ), 110 n m (α o ), and 200 n m (α t ). In functional experiments, the steady state GTPase activity of isolated α o was inhibited by phosducin by ≈60% with an IC 50 value of ≈300 n m , whereas the GTPase activity of trimeric G o was inhibited by ≈90% with an IC 50 value of ≈10 n m . Phosducin did not inhibit the GTP-hydrolytic activity of isolated α o as measured by single-turnover assays, but it inhibited the release of GDP from α o ; the rate constant of GDP release was decreased ≈40% by 500 n m phosducin, and the inhibition occurred with an IC 50 value for phosducin of ≈100 n m . These data suggest that phosducin binds with high affinity to G-protein βγ subunits and with lower affinity to G-protein α subunits. We propose that the α subunit-mediated effects of phosducin might increase both the extent and the rapidity of its inhibitory effects compared with an action via the βγ subunit complex alone.