Phosducin, which tightly binds betagamma-subunits of heterotrimeric
G-proteins, has been conjectured to play a role in regulating second
messenger signaling cascades, but to date its specific function has
not been elucidated. Here we demonstrate a potential role for phosducin
in regulating olfactory signal transduction. In isolated olfactory
cilia certain odorants elicit a rapid and transient cAMP response,
terminated by a concerted process which requires the action of two
protein kinases, protein kinase A (PKA) and a receptor-specific kinase
(GRK3) (Schleicher, S., Boekhoff, I. Arriza, J., Lefkowitz, R. J.,
and Breer, H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1420-1424).
The mechanism of action of GRK3 involves a Gbetagamma-mediated translocation
of the kinase to the plasma membrane bound receptors (Pitcher, J.
A., Inglese, J., Higgins, J. B. , Arriza, J. L., Casey, P. J., Kim,
C., Benovic, J. L., Kwatra, M. M. , Caron, M. G., and Lefkowitz,
R. J. (1992) Science 257, 1264-1267). A protein with a molecular
mass of 33 kDa that comigrates on SDS gels with recombinant phosducin
and which is immunoreactive with phosducin antibodies is present
in olfactory cilia. Recombinant phosducin added to permeabilized
olfactory cilia preparations strongly inhibits termination of odorant-induced
cAMP response and odorant-induced membrane translocation of GRK3.
In addition, the cAMP analogue dibutyryl cAMP stimulates membrane
targeting of the receptor kinase. This effect is presumably due to
PKA-mediated phosphorylation of phosducin, which diminishes its affinity
for binding to the Gbetagamma-subunit, thereby making Gbetagamma
available to function as a membrane anchor for GRK3. A specific PKA
inhibitor blocks the odorant-induced translocation of the receptor
kinase. Consistent with this formulation, a non-phosphorylatable
mutant of phosducin (phosducin Ser-73 --> Ala) is an even more effective
inhibitor of desensitization and membrane targeting of GRK3 than
the wild-type protein. A phosducin mutant that mimics phosphorylated
phosducin (phosducin Ser-73 --> Asp) lacks this property and in fact
recruits GRK3 to the membrane and potentiates desensitization. These
results suggest that phosducin may act as a phosphorylation-dependent
switch in second messenger signaling cascades, regulating the kinetics
of desensitization processes by controlling the activity of Gbetagamma-dependent
GRKs.