Abstract
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.
Highlights
The olfactory system responds precisely to iterative stimulation; this characteristic feature is due to the phasic responses of receptor cells [3], based on a rapid termination of the odorinduced primary reaction [4]
These results indicate that exogenous phosducin inhibited the rapid termination of the odor-induced second messenger signal; phosducin seems to interfere with the desensitization process
This observation is in line with previous in vitro studies demonstrating that binding of GRK2 to ␥-subunits is inhibited by phosducin [10]
Summary
The olfactory system responds precisely to iterative stimulation; this characteristic feature is due to the phasic responses of receptor cells [3], based on a rapid termination of the odorinduced primary reaction [4]. Recent studies have indicated that olfactory signaling is terminated by uncoupling the transduction cascade; the second messenger signal elicited by odors is turned off by a negative feedback reaction controlled by phosphorylation of odorant receptors mediated by two types of enzymes, a second messenger controlled kinase and a receptor specific kinase (GRK)1 [1, 5,6,7,8]. It has recently been found that interaction of GRKs with ␥-subunits of heterotrimeric G-proteins (G␥-subunits) is required for the cytosolic GRKs to be translocated to the membrane and subsequently phosphorylate the agonist-occupied receptors [2, 11,12,13,14] These findings were extended by the observation that membrane targeting of GRK by docking onto ␥-subunits is an essential prerequisite for turning off the second messenger cascade [15]. In this study we set out to explore the hypothesis that phosducin might be capable of serving as a PKA controlled inhibitor of GRK in olfactory cilia
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