(Received for publication, September 20, 1996, and in revised form, November 19, 1996)
From the University Stuttgart-Hohenheim, Institute of
Zoophysiology, 70599 Stuttgart, Federal Republic of Germany,
§ Duke University Medical Center, Howard Hughes Medical
Institute, Durham, North Carolina 27710, and ¶ University
of Würzburg, Institute of Pharmacology,
97078 Würzburg, Federal Republic of Germany
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
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
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
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 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 odor-induced 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 that is initiated by activating the second messenger-dependent kinase. The details of how these kinases interact, however, remain elusive. Several mechanisms seem plausible; second messenger-dependent kinase might directly phosphorylate and thus increase the enzymatic activity of receptor-specific kinases, similar to kinase-mediated activation of enzymes in regulating metabolic pathways (9). However, there is no evidence for this mechanism; in fact, the sequence of GRK3 does not contain any concensus site for PKA phosphorylation. Alternatively, the receptor-specific kinase may be under tonic control of an as yet unknown inhibitor, the phosphorylation of which by PKA may relieve this inhibitory constraint (1, 10).
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, 11-14).
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).
Phosducin, a major soluble phosphoprotein first discovered in mammalian
retinal tissue (16, 17) also binds -subunits of heterotrimeric
G-proteins (18) probably via structural domains within the
amino-terminal region which are homologous to the G
-binding domain of GRKs (19). The notion that both proteins may compete for
binding sites of the G
complex has recently been confirmed (10).
Moreover, phosducin is readily phosphorylated at serine residue 73 by
protein kinase A, and this modification significantly reduces its
binding affinity for G
-subunits (19-21). 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.
Materials
Sprague-Dawley rats were purchased from Charles River, Sulzfeld;
fresh bovine olfactory epithelium was obtained from a local slaughterhouse and stored at 70 °C.
The odorants citralva (3,7-dimethyl-2,6-octadiennitrile), hedione
(3-oxo-2-pentylcyclopentaneacetic acid methyl ester), and eugenol
[2-methoxy-4-(2-propenyl)phenol] were provided by Drom, Baierbrunn, Germany. Protein kinase A inhibitor (Walsh inhibitor) as
well as
N6,2-O-dibutyryladenosine
3
:5
-cyclic monophosphate (dibutyryl cAMP) were obtained from
Sigma. Radioligand assay kits for cAMP were provided
by Amersham Corp. Subtype specific polyclonal antibodies against
recombinant GRK3 were prepared as described previously (15); antibodies
against phosducin were raised in rabbits and goats using recombinant
phosducin as antigen and purified as described (22); horseradish
peroxidase-conjugated goat anti-rabbit IgG was obtained from Bio-Rad.
All other chemicals were obtained from Sigma. The
purity grade of all chemicals used was >99%.
Methods
Preparation of Phosducin MutantsA human phosducin plasmid
(pBS-33K) was kindly provided by Dr. T. Shinohara (National Institutes
of Health). An MluI site (ACGCGT) was introduced into
pBS-33K by a single base mutation at position 252 (A C) employing a
mutagenetic reverse primer containing a BsmI site. The
forward primer contained a sequence complementary to the 5
-end of
phosducin (including the start codon and 5
-nucleotides) and contained
a 5
-KpnI site. The polymerase chain reaction fragment
generated from these primers was subcloned into pBS-33K and digested
with KpnI/BsmI. Polymerase chain
reaction-cassette mutagenesis of Ser-73 to Ala and Asp was accomplished
using this new construct and the flanking
MluI/HindIII sites. The introduction of mutations
was verified by sequencing with Sequenase. The mutant phosducin
cDNAs were ligated into the pQE30 vector (Qiagen) as a fusion
protein with 10 amino acids containing a hexahistidine sequence. Fusion
protein constructs were introduced into the Escherichia coli
strain BL21 and induced as described previously (23, 24).
The induced cells were
lysed in 50 mM HEPES, 1% Tween 20, protease inhibitor
mixture (Boehringer Mannheim), pH 7.5, by freeze-thawing. The lysed
cells were centrifuged at 15,000 rpm (SS-34 rotor, Sorvall instruments)
for 20 min at 4 °C, and the pellet was resuspended in buffer A (50 mM HEPES, 300 mM NaCl, pH 7.5) containing 8 M urea. After 4 h incubation with rotation, the
solution was centrifuged at 15,000 rpm for 20 min. Ni-resin (proBond;
Invitrogen) was then added to the supernatant and incubated at 4 °C
for 1 h with rotation. The same volume of buffer A containing 4 M urea was added, and the beads were collected in a column.
The beads were washed with buffer A containing 4, 3, 2, and 1 M urea stepwise, and the washed beads were incubated with
buffer A containing 1 M urea overnight at 4 °C. The
refolded His-6-fusion proteins were eluted with buffer A containing 500 mM imidazole. The eluate was then dialyzed against 20 mM HEPES, 350 mM NaCl, 1 mM EDTA, 1 mM -mercaptoethanol, pH 7.5, for 2 h at 4 °C.
The protein concentration was determined by Bradford method
(Bio-Rad).
Enriched preparations of
olfactory cilia from rat and bovine olfactory epithelium were isolated
using the calcium-shock method described by Anholt et al.
(25) and Chen et al. (26). All isolation steps were
performed at 4 °C. The olfactory epithelium was washed in Ringer
solution (120 mM NaCl, 5 mM KCl, 1.6 mM K2HPO4, 25 mM
NaHCO3, 7.5 mM glucose, pH 7.4) and
subsequently transferred to Ringer solution containing 10 mM calcium. Detached cilia were separated by centrifugation
for 5 min at 7,700 × g. The supernatant was collected,
and the resulting pellet was resuspended again in Ringer solution with
10 mM CaCl2 and centrifuged as described above.
The supernatants were combined and centrifuged for 15 min at
27,000 × g. The pellet containing the detached cilia
was resuspended in TME buffer (10 mM Tris/HCl, 3 mM MgCl2, 2 mM EGTA, pH 7.4) and
stored at 70 °C.
Phosducin was immunoprecipitated from bovine isolated olfactory cilia preparations as described recently (27). Briefly, cytosolic fractions were prepared by homogenizing 8 mg of isolated cilia under liquid nitrogen and suspending them in 20 mM Tris, 5 mM EDTA, 5 mM EGTA, 20 mg/liter benzamidine, 20 µM phenylmethylsulfonyl fluoride, pH 7.4. After turraxing, the samples were centrifuged for 30 min at 50,000 × g, and the resulting supernatant was incubated for 1 h at 4 °C with pre-washed Protein A-Sepharose (Pharmacia Biotech Inc.). After an additional centrifugation step, 5 µg of affinity purified rabbit anti-phosducin antibody was added to the supernatant and incubated for 2 h before the immune complexes were bound by adding another aliquot of Protein A-Sepharose and pelleted by centrifugation. After washing the pellets with Brij buffer (1% Brij 96, 50 mM NaCl, 50 mM Tris/HCl, 10 mM EDTA, 10 mM EGTA, 20 mg/liter benzamidine, 20 µM phenylmethylsulfonyl fluoride, pH 7.4) the samples were resuspended in SDS-loading buffer containing 6 M urea and heated to 95 °C for 5 min to release the protein. As a control, the same procedure was done with cytosol that has been heated to 95 °C for 5 min followed by pelleting of the denatured protein. The samples were separated on SDS-polyacrylamide gels, and phosducin was detected using the IgG fraction of a second phosducin antiserum raised in goats (22) and the enhanced chemiluminescence system (Amersham Corp.).
Determination of Odor-induced Second Messenger ResponsesA rapid kinetic system was used to determine odorant-induced changes of second messenger concentrations in the subsecond time range. Stimulation experiments were performed at 37 °C as described previously (4). Syringe I contained the stimulation buffer (200 mM NaCl, 10 mM EGTA, 50 mM MOPS, 2.5 mM MgCl2, 1 mM dithiothreitol, 0.05% sodium cholate, 1 mM ATP, and 2 µM GTP, pH 7.4) and 12 nM free calcium calculated and adjusted as described by Pershadsingh and McDonald (28). Syringe II contained the olfactory cilia, and syringe III was filled with stop solution (7% perchloric acid). If not indicated otherwise, 205 µl of stimulation buffer was mixed with 20 µl of cilia; at the appropriate time (ms) the reaction was stopped by injection of perchloric acid. Quenched samples were stored on ice for 20 min and then analyzed for second messenger concentrations as described previously (29).
In all cases cilia were preincubated 10 min on ice with the recombinant phosducin mutants or the other modulators; control samples were incubated with TME buffer, which was used to dilute the protein. The concentrations of the different modulators indicated are the concentrations during pretreating the cilia.
Enzyme-linked Immunosorbent AssayOdor-induced
translocation of GRK3 from the cytosol to the membrane was performed
using the enzyme-linked immunosorbent assay technique as described
previously (15). Briefly, isolated olfactory cilia preparations in
hypotonic TME buffer were pretreated for 15 min on ice with solutions
of recombinant phosducin or the various modulators; control samples
were incubated only with TME medium, which was used to dilute the
protein. Prior to odor stimulation, cilia preparations (100 µl) and
the stimulation buffer containing an odorant dilution of citralva,
hedione, eugenol were adapted to 37 °C. After adding the stimulation
buffer (200 µl) the samples were incubated for 10 s at 37 °C,
and the reaction was stopped by freezing the samples in liquid
nitrogen. To separate the cytosolic and membrane fraction, samples were
directly centrifuged for 1 h at 48,000 × g;
during this period the samples became totally thawed without disturbing
the balance of membrane and cytosolic distribution of GRK3. After
resuspending the pellet in TME buffer, the protein concentration was
determined according to Bradford (30) using bovine serum albumin as a
standard. Microwell plates were coated with protein (200 ng to 1 µg
of protein/100 µl 50 mM NaHCO3, pH 9.5) at
4 °C overnight in a wet chamber. Unbound material was washed out
with TBS (10 mM Tris/HCl, pH 7.6, 150 mM NaCl)
and 0.1% gelatin. After preincubation with TBS gelatin containing 1%
bovine serum albumin for 30 min at room temperature in a wet chamber,
GRK3 antibody dilutions (1:1000 to 1:10,000) in TBS gelatin, 1% bovine
serum albumin were added and incubated for 2 h at 37 °C,
followed by horseradish peroxidase-conjugated goat anti-rabbit IgG
(1:15,000 in TBS gelatin, 1% bovine serum albumin). Each step was
followed by three TBS gelatin washes. Antibody binding was visualized
using a substrate solution containing 0.005%
3,5,3,5
-tetramethylbenzidine and 0.003% H2O2
in 100 mM sodium acetate/citrate buffer, pH 6.0; the enzyme
reaction was stopped after 20-30 min by adding 1 N
H2SO4; extinction was measured at 450 nm with a
Dynatech MR 700 microplate reader.
In order to verify if phosducin is indeed present in olfactory
cilia preparations, a combined immunoprecipitation/Western blot
analysis was performed using specific antibodies as described previously (27). As shown in Fig. 1, a strong
immunoreactive band of 33 kDa (lane 3) which comigrates with
recombinant phosducin (lane 1) is visualized in the
olfactory cilia preparation. The specificity of the reaction is
supported by the negative reaction in lane 2 (control) which
represents a boiled sample of cilia. Based on the labeling intensity,
the concentration of phosducin in the cilia preparation seems to be in
the same range as in other tissues (27).
To approach the question of whether phosducin may affect the kinetics
of odor-induced second messenger signaling, the effect of exogenous
phosducin on odor-induced cAMP responses in olfactory cilia was
analyzed. Cilia preparations from the rat olfactory epithelium were
preincubated with recombinant phosducin and subsequently stimulated
with a mixture of odorants (citralva, hedione, and eugenol, 1 µM each); the odorant-induced cAMP response was monitored in the subsecond time range. Stimulation of control samples elicited a
rapid and transient increase in the concentration of cAMP
(Fig. 2) as described previously (4). In samples
pretreated with recombinant phosducin the "onset kinetics" of the
odorant-induced cAMP signal were virtually unaffected; however, the
"off kinetics" were significantly changed; the elevated cAMP level
decayed with a much slower rate. These results indicate that exogenous
phosducin inhibited the rapid termination of the odor-induced second
messenger signal; thus phosducin seems to interfere with the
desensitization process.
Since membrane targeting of GRK3 is a prerequisite for terminating the
olfactory signaling cascade (15), we investigated if exogenous
phosducin may affect the odor-induced translocation of GRK3. Isolated
olfactory cilia were preincubated with exogenous phosducin and
stimulated with an odor mixture; subsequently membrane and cytosolic
fractions were separated and analyzed for receptor-specific kinase
immunoreactivity as described previously (15). In untreated cilia prior
to odorant stimulation, only 21.2 ± 3.6% of the GRK immunoreactivity was found in the membrane fraction and about 78.8 ± 3.6% was in the cytosol (Fig. 3). Stimulation with
odorants induced a significantly different distribution pattern of the GRK immunoreactivity, cytosol (54.6 ± 4.6) and membrane fraction (45.3 ± 4.6). In contrast, when cilia preincubated with phosducin were stimulated with odorants, the distribution pattern of kinase immunoreactivity did not change; most of the GRK remained in the cytosolic fraction. These results imply that exogenous phosducin prevents the translocation of GRK3 to the membrane-anchored
G and that this may be the critical step by which phosducin
interferes with the rapid desensitization (Fig. 2).
If phosducin is tonically inhibiting the desensitization process by
preventing GRK3 from docking onto -subunits, then its phosphorylation by PKA may release this constraint, since previous studies have shown that phosphorylation of phosducin by PKA diminishes its potential to interfere with the interaction of GRK with G
(10, 19). To assess the relevance of this hypothesis, experiments were
performed measuring membrane targeting of GRK3 in the presence of a
cAMP analogue which should activate protein kinase A, leading to
phosphorylation of endogenous phosducin.
The data indicate that in the presence of elevated cAMP levels, a
significantly higher portion of GRK3 was associated with the membrane
(Fig. 4). These results support the notion that protein kinase A may play a key role in controlling the membrane targeting of
GRK3 in olfactory cilia induced by "cAMP odorants."
This view was further scrutinized in experiments using a specific
inhibitor of protein kinase A. As shown in Fig. 5, the
translocation of GRK3 elicited by odorant stimulation is attenuated by
the Walsh inhibitor in a dose-dependent manner. These
results further substantiate the essential role of PKA-mediated
phosphorylation reactions in regulating the translocation of GRK.
To further approach the functional role of phosphorylated and
non-phosphorylated phosducin, distinct mutants were prepared. In the
phosducin-Ser-73 Ala mutant, serine 73 was changed to alanine, thus
preventing its PKA-mediated phosphorylation; the phosducin-Ser-73
Asp mutant, in which serine 73 was substituted by aspartic acid, should
mimic the phosphorylated form of phosducin due to its negatively
charged side chain. Analyzing each mutant for its capability to become
phosphorylated by PKA revealed that only wild-type phosducin undergoes
phosphorylation whereas both mutants (Ala, Asp) are not phosphorylated
by PKA (Fig. 6A). These results confirm
previous observations indicating that serine 73 is the target site for
phosphorylation of phosducin by PKA (21).
To characterize the functional properties of the different phosducin
isoforms, their binding capacity for -subunits of trimeric G-proteins was assessed. As shown in Fig. 6B, in control
samples (without fusion protein) there is no
binding detectable;
the wild-type phosducin as well as the phosducin-Ala mutant interacted with
-subunits to a similar extent. The pseudophosphorylated phosducin-Asp mutant displayed a significantly reduced binding to
-subunits, probably due to its reduced affinity.
Subsequently the mutated forms of phosducin were assessed for their
potency to affect termination of odor-induced second messenger signaling. The phosducin-Ala mutant, which cannot be modified by PKA
phosphorylation (Fig. 6A) and which efficiently interacts with G-subunits (Fig. 6B), prevented the rapid decay
of the odor-induced second messenger response (Fig.
7A); interestingly, this non-modifiable phosducin form was
even more effective than the native form (Fig. 2) suggesting that
exogenously applied native phosducin may be partially inactivated by
PKA phosphorylation during preincubation.
In cilia preparations preincubated with the pseudo-phosphorylated
mutant phosducin-Asp, the rapid termination of the odor-induced cAMP
signal was not blocked, but rather the cAMP signal decayed even more
rapidly (Fig. 7B); in addition, the intensity of the response was reduced. These results indicate that phosducin-Asp does
not attenuate but rather seems to facilitate the desensitization process. This could mean that an excess of pseudo-phosphorylated phosducin may expose more -subunits for GRKs targeting, thus causing a recruitment of GRKs. Therefore, it is conceivable that an
excess of phosphorylated phosducin may allow a membrane docking of GRK
even before receptor stimulation.
To assess this notion, the GRK3 distribution in cilia preparations was
determined in the presence of the two phosducin mutants. As can be seen
in Fig. 8A, the phosducin-Ala
completely blocked the odor-induced translocation of GRK3 from
the cytosol to the membrane (Fig. 8A). Thus, the
non-phosphorylated form of phosducin, which actively interferes with
the desensitization process (Fig. 7A) and efficiently
interacts with G-subunits (Fig. 6A), prevents odor-induced translocation of GRK3.
To approach the properties of the phosphorylated form of phosducin on GRK3 distribution, cilia were incubated with phosducin-Asp but not stimulated with odorants (Fig. 8B). The results indicate that after preincubation with phosducin-Asp 34.7 ± 9.8% of the GRK3 immunoreactivity was located in the membrane fraction compared with 21.2 ± 3.6 in untreated cilia. Upon stimulation with odorants under the same conditions, the proportion of GRK bound to the membrane (44.6 ± 8.2%) increased only slightly. These results indicate that an excess of pseudo-phosphorylated phosducin elicits a membrane targeting of GRK3.
Experiments using exogenous phosducin and its mutated forms are
consistent with the hypothesis that phosducin may control the kinetics
of second messenger signaling in olfactory cilia by governing the
stimulus-dependent translocation of a receptor-specific kinase. This observation is in line with previous in vitro
studies demonstrating that binding of GRK2 to -subunits is
inhibited by phosducin (10). In addition, experiments employing
phosducin mutants support the view that phosphorylation of phosducin
may change its functional properties. A phosducin mutant, which cannot be phosphorylated, prevented the rapid decay of an odor-induced second
messenger response even more efficiently than the native form.
Furthermore, phosducin mutants that mimic the phosphorylated form, due
to a negatively charged side chain, caused a constitutive translocation
of GRK3 to the membrane suggesting that the phosphorylation status of
phosducin may allow an anchoring of GRK3 to
-subunits. This could
be due to the reduced binding affinity of phosphorylated phosducin to
-subunits as previously observed in experiments using fusion
proteins with the amino terminus of phosducin (19, 31). Moreover,
reconstitution experiments have shown that the competition of phosducin
and GRK for a common binding site on the G
-complex is antagonized
following phosphorylation of phosducin by PKA (10).
Together with previous in vitro observations the findings of
this study support a model in which phosducin is a
phosphorylation-dependent regulator governing the membrane targeting of
GRK; in its non-phosphorylated state phosducin strongly interacts with
the -subunits of trimeric G-proteins thus preventing the
anchoring of GRK3 to these sites. Upon phosphorylation, the affinity of
phosducin for the
-subunits is reduced and the blockade of GRK
anchoring is relieved, so that the GRK can dock onto
-subunits
and phosphorylate receptor proteins, thus uncoupling the transduction
cascade.
Previous studies have shown that a sequential interplay of two types of kinases is involved in terminating the odor-induced second messenger signal, a second messenger controlled kinase and a receptor-specific kinase (GRK3) (1, 5-7); both types of kinases are required for an effective desensitization of the olfactory cascade (1). The sequential interplay of the two enzymes could be explained if phosducin is the previously proposed endogenous inhibitor of GRK3 action (15). An odorant-induced rise of cAMP levels would activate protein kinase A leading to phosphorylation of phosducin which triggers the membrane targeting of GRK3 and phosphorylation of the receptor protein.
Whether termination of the alternative olfactory pathway, odorant-induced generation of inositol 1,4,5-trisphosphate, is controlled by a similar cascade is unclear. Previous studies have indicated that in this case protein kinase C plays a critical role, analogous to kinase A in the cAMP pathway (5). The lack of obvious consensus sites for phosphorylation by protein kinase C makes it less likely that phosducin may also be involved in the phospholipase C pathway. However, recent studies have demonstrated that stimulation of protein kinase C elicits an activation of GRK due to a phosphorylation-mediated translocation to the plasma membrane (32).
We thank Caroline Reck for excellent technical assistance and Julie Pitcher for excellent suggestions during the preparation of the manuscript.