Abstract
The high affinity interactions of phosducin with G-proteins involve
binding of phosducin to the G-protein betagamma subunits. Here we
have investigated whether phosducin interacts also with G-protein
alpha subunits. Interactions of phosducin with the individual subunits
of Go were measured by retaining phosducin-G-protein subunit complexes
on columns containing immobilized anti-phosducin antibodies. Both
the alpha and the beta subunits of trimeric Go 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 alpha than for beta subunits, by the stable GTP analog guanosine
5'-(3-O-thio)triphosphate. Isolated alphao 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 betagamma
subunit complexes as well as purified alpha subunits of Go and Gt
were precipitated together with His6-tagged phosducin with nickel-agarose;
this co-precipitation occurred concentration-dependently, with apparent
affinities for phosducin of 55 nM (Gbetagamma), 110 nM (alphao),
and 200 nM (alphat). In functional experiments, the steady state
GTPase activity of isolated alphao was inhibited by phosducin by
approximately 60% with an IC50 value of approximately 300 nM, whereas
the GTPase activity of trimeric Go was inhibited by approximately
90% with an IC50 value of approximately 10 nM. Phosducin did not
inhibit the GTP-hydrolytic activity of isolated alphao as measured
by single-turnover assays, but it inhibited the release of GDP from
alphao; the rate constant of GDP release was decreased approximately
40% by 500 nM phosducin, and the inhibition occurred with an IC50
value for phosducin of approximately 100 nM. These data suggest that
phosducin binds with high affinity to G-protein betagamma subunits
and with lower affinity to G-protein alpha subunits. We propose that
the alpha subunit-mediated effects of phosducin might increase both
the extent and the rapidity of its inhibitory effects compared with
an action via the betagamma subunit complex alone.
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