Phosducin, Potential Role in Modulation of Olfactory Signaling

Abstract

Phosducin, which tightly binds ␤␥-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 G␤␥-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 odorantinduced 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 G␤␥-subunit, thereby making G␤␥ 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 3 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 3 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 G␤␥-dependent GRKs.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, 5-8). These observations raise the possibility that the two kinases act sequentially in a reaction cascade tha...

Mutational Analysis of Gβγ and Phospholipid Interaction with G Protein-coupled Receptor Kinase 2

“…Previous in vitro experiments demonstrated that a 28-mer synthetic peptide representing Trp-643 through Ser-670 of GRK2 prevented association between G␤␥ and GRK2 (9). When permeabilized olfactory cilia were treated with the same peptide, both membrane translocation of GRK3 and desensitization of odorant receptors were inhibited (55). These studies suggest, in agreement with our observations, that in a physiologically relevant setting disruption of G␤␥ binding is sufficient to block efficient GRK3-mediated receptor phosphorylation (55).…”

“…When permeabilized olfactory cilia were treated with the same peptide, both membrane translocation of GRK3 and desensitization of odorant receptors were inhibited (55). These studies suggest, in agreement with our observations, that in a physiologically relevant setting disruption of G␤␥ binding is sufficient to block efficient GRK3-mediated receptor phosphorylation (55). Thus, it is interesting to consider what purpose is served by this combined regulation of GRK2/3.…”

Internalization of the Human Nicotinic Acid Receptor GPR109A Is Regulated by Gi, GRK2, and Arrestin3

“…sibility of linking G i to the phosphorylation of activated GPR109A via protein kinase A, but our observation that the siRNA knockdown of GRK2 completely inhibited GPR109A internalization indirectly excludes a role for protein kinase A in the regulation of GPR109A internalization. Many studies suggest that GRK2 activity also requires the binding of the G␤␥ subunit (28,59), and the plasma membrane translocation of these kinases is regulated by their association with the G␤␥ subunit (29,60). It is then more likely that PTX pretreatment abolishes GPR109A internalization by inhibiting the association of G␤␥ subunit with GRK2.…”

Function of phosducin-like proteins in G protein signaling and chaperone-assisted protein folding

“…Fourth, evidence was accumulating for a similar role for Pdc in other G protein signaling systems. It was found that addition of Pdc in vitro or over-expression of Pdc in cultured cells resulted in inhibition of Gβγ-mediated GRK2 [41], GRK3 [42], PLCβ [43] and AC2 [44] activity. These results suggested that Pdc might be a general G-protein regulator through its ability to sequester Gβγ.…”