From the Institut für Pharmakologie und Toxikologie der Universität Würzburg, Versbacher Strasse 9, 97078 Würzburg, Germany
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ABSTRACT |
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The high affinity interactions of phosducin with
G-proteins involve binding of phosducin to the G-protein
subunits. Here we have investigated whether phosducin interacts also
with G-protein
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
and the
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
than for
subunits, by
the stable GTP analog guanosine 5'-(3-O-thio)triphosphate.
Isolated
o 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
subunit
complexes as well as purified
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 (G
), 110 nM (
o), and 200 nM
(
t). In functional experiments, the steady state GTPase
activity of isolated
o was inhibited by phosducin by
60% with an IC50 value of
300 nM,
whereas the GTPase activity of trimeric Go was inhibited by
90% with an IC50 value of
10 nM.
Phosducin did not inhibit the GTP-hydrolytic activity of isolated
o as measured by single-turnover assays, but it
inhibited the release of GDP from
o; the rate constant
of GDP release was decreased
40% by 500 nM phosducin,
and the inhibition occurred with an IC50 value for
phosducin of
100 nM. These data suggest that phosducin binds with high affinity to G-protein
subunits and with lower affinity to G-protein
subunits. We propose that the
subunit-mediated effects of phosducin might increase both the extent
and the rapidity of its inhibitory effects compared with an action via
the
subunit complex alone.
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INTRODUCTION |
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Heterotrimeric GTP-binding proteins (G-proteins) comprise a family
of regulatory proteins that couple transmembrane receptors for a
variety of neurotransmitters, hormones, and other stimuli to their
intracellular effectors (for reviews see Refs. 1-4). G-proteins are
composed of ,
, and
subunits. Upon activation by agonist,
receptors couple to their G-proteins and promote the exchange of bound
GDP for GTP. The resultant conformational change induces dissociation
of the GTP-bound
subunit
(G
)1 from the
subunit complex (G
). In this dissociated state
G
activates effectors such as adenylyl cyclases, other enzymes, or ion channels. Additionally, the free
subunits also interact with and regulate effectors (reviewed in Refs. 2 and 5). The
intrinsic GTPase activity of G
then leads to hydrolysis of the bound GTP, and this enables reassociation of GDP-bound G
with G
. The resulting reformation of
the trimeric G-protein terminates the signal.
A similar GTPase switch function occurs in the small molecular weight
monomeric GTP-binding proteins. For these proteins a large array of
regulatory proteins have been identified that regulate various steps in
the GTPase cycle (6). More recently, regulatory proteins have also been
discovered for the heterotrimeric G-proteins. Among these are the
growth cone protein GAP-43 and the RGS (regulators of
G-protein signaling) family members that have
been shown to activate the GTPase activity of several G-protein subunits (7-10) and a number of proteins that contain the structural
motif of the pleckstrin homology domain and that bind to G-protein
subunits (11), most notably the
-adrenergic receptor kinases
(12-15).
Phosducin is another type of regulator of G-protein signaling that is
thought to act via the subunits of G-proteins (16, 17). It was
initially discovered as a major retinal phosphoprotein that could be
copurified with the
and
subunits of transducin, Gt
(18). Its expression had been thought to be restricted to the retina
and the developmentally related pineal gland (18-20), but more
recently phosducin has been shown to be ubiquitously expressed (16,
21).
The molecular mechanisms of the interaction of phosducin with
G-proteins are not clear. The co-purification of phosducin from the
retina with the subunit complex of Gt (18) indicated high affinity interactions between these proteins, and both the N and
the C terminus of phosducin appear to contain high affinity binding
sites for the
subunit complex (22-25). From these functional as
well as structural observations it has been concluded that phosducin
interacts exclusively with the
subunits of G-proteins. Indeed,
studies on the effects of phosducin and phosducin-like protein on
Gt suggest that phosducin acts by "trapping" free
subunits; the GDP-bound
subunits would then be unable to
find free
subunits for reassociation and would therefore stay
inactive (17, 26, 27).
In contrast to the many studies on phosducin-G
interactions, potential interactions with G-protein
subunits have not been investigated. Such direct effects on
subunits may be proposed from the observation that the extent of inhibition of Go GTPase is often larger than would be expected from
antagonism of the function of
subunits alone. A possible
solution to this problem would be a direct interaction of phosducin
with
subunits. Therefore, the present study was undertaken to
define the nature of interactions of phosducin with the subunits of
G-proteins, using mostly Go as a model system.
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EXPERIMENTAL PROCEDURES |
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Protein Purification--
Recombinant phosducin was expressed in
Escherichia coli as described earlier using the plasmid
pET-phosducin (16). Following lysis of the bacteria by freeze thawing
and precipitation of DNA with 2% streptomycin and centrifugation at
50,000 × g for 10 min, the supernatant was applied to
a MonoQ column (Amersham Pharmacia Biotech) and eluted with a 0-500
mM NaCl gradient in 10 mM Tris-HCl, pH 7.4. Peak fractions of phosducin eluting at 250 mM NaCl were concentrated to
1 ml and were then further purified by gel
filtration on Superdex 200 (Amersham Pharmacia Biotech). The purity of
the preparations was >95% as determined by SDS-polyacrylamide gel electrophoresis and staining with Coomassie Blue. Mock preparations from E. coli transformed with "empty" vector were used
as controls as described earlier (16).
Phosphorylation of Phosducin--
The catalytic subunit of
protein kinase A was purified to apparent homogeneity from bovine heart
by the method of Sugden et al. (31). 1 µg of purified
phosducin was incubated with 2 units of the PKA catalytic subunit in a
buffer containing 10 mM Tris-HCl, pH 6.5, 5 mM
MgCl2, 0.1 mM EDTA, 70 mM KCl, 0.5 mM dithiothreitol, 0.1 mM ATP, 5 mg/ml bovine
serum albumin, 0.1% Tween 80, 0.2 mM benzamidine, 0.2 mM phenylmethylsulfonyl fluoride, and 1 µg/ml leupeptin
for 30 min at 0 °C and 30 min at 30 °C. At the end of the
phosphorylation procedure, PKA inhibitor peptide was added to a final
concentration of 10 µM. In preliminary experiments we
verified that this was sufficient to completely suppress further PKA
activity. Control samples were treated identically, but the PKA
catalytic subunit was absent during the incubations. The extent of phosphorylation was determined by addition of a small amount of
[-32P]ATP and measurement of the incorporation of
32P into phosducin; phosphorylated phosducin used in the
present experiments contained 0.8-1.0 mol phosphate/mol phosducin.
Binding of G-proteins and Phosducin to Anti-phosducin Antibody Columns-- An antiserum against phosducin was generated by immunization of rabbits with purified recombinant phosducin, and phosducin-specific antibodies were isolated by affinity chromatography (21, 32). 2 ml of antiserum were incubated with 1 ml of wet protein A-Sepharose (Amersham Pharmacia Biotech) for 1 h at room temperature under gentle rocking. The resin was washed twice with 10 volumes of 0.2 M sodium borate, pH 9.0, and was suspended in 10 volumes of 0.2 M sodium borate, pH 9.0. The cross-linker bis(sulfosuccinimidyl)suberate (Pierce) was then added to a concentration of 1 mg/ml. After shaking for 30 min at room temperature, the reaction was stopped by washing the resin with several volumes of 0.2 M ethanolamine, pH 8.0, followed by incubation with 0.2 M ethanolamine, pH 8.0, for 2 h at room temperature. The resin was then washed with 5 volumes of 100 mM glycine-HCl, pH 2.5, and 40 volumes of phosphate buffered saline.
To study binding of G-proteins or their subunits to phosducin, equal molar amounts of phosducin and Go (orCo-precipitation of Phosducin-His6 and G-protein
Subunits with Ni-NTA-Agarose--
As an alternative to antibody
immobilization of phosducin in the phosducin/G-protein binding assays
we used immobilization of hexahistidine-tagged phosducin on
Ni-NTA-agarose. Purified phosducin-His6 (20 nM)
was incubated with various concentrations of G or
o purified from bovine brain, or
t
purified from bovine retina, in phosphate-buffered saline containing
0.05% cholate (140 mM NaCl, 30 mM KCl, 6.5 mM Na2HPO4, pH 7.3) for 30 min at
4 °C. The mixture was centrifuged at 14,000 rpm for 10 min, and 40 µl of Ni-NTA resin (Qiagen) were added to the supernatant to bind
phosducin-His6 (plus associated proteins). After 10 min at
4 °C, the resin was pelleted by centrifugation and washed twice in
the same buffer with intervening short centrifugations. G-protein subunits retained together with phosducin-His6 were
detected by taking up the beads in SDS-sample buffer followed by
SDS-polyacrylamide gel electrophoresis and Western blotting as
above.
Determination of GTPase Activity--
The steady state GTPase
activity of Go or o was measured as
described earlier by determining the formation of
32Pi from [
-32P]GTP (33). 0.1 pmol (2 nM) of Go or
o was
assayed in buffer containing 0.1% Lubrol and 25 mM
MgSO4. Phosducin was present at various concentations. The
incubations lasted for 30 min at 30 °C and were terminated by
addition of 500 µl of 1% charcoal in 2 mM
NaH2PO4, pH 2.
Determination of [-32P]GDP
Release--
o (0.5 pmol/time point) was incubated with
500 nM (or the indicated concentrations) phosducin and 1 µM [
-32P]GTP (105 cpm/time
point) in a buffer containing 50 mM Hepes, pH 7.6, 0.1% Lubrol, 1 mg/ml bovine serum albumin, and either 100 µM
or 25 mM free Mg2+, added as MgSO4,
for 30 min at 20 °C, to allow binding of GTP and hydrolysis to GDP.
The [
-32P]GDP release reaction was started by addition
of GppNHp to a concentration of 100 µM. Aliquots of 50 µl were withdrawn at the indicated intervals, and the reaction was
stopped by addition to ice-cold stop solution (10 mM Hepes,
pH 7.6, 50 mM NaCl, 5 mM MgSO4, 5 mM NaF, 10 mM AlCl3). Samples were
applied to BA-85 nitrocellulose filters (Schleicher & Schuell, 0.45 µm), the tubes were rinsed twice with 2 ml of stop solution, and each
rinse was applied to the filter. Filters were then washed two times
with 20 ml of stop solution and counted by liquid scintillation
counting. Phosducin was present in these assays either in varying
concentrations as indicated or, for kinetic studies, in 50-fold excess
over
o.
Data Analysis-- Kinetic data were fitted assuming exponential functions as described earlier (34). Concentration response curves were fitted to the Hill equation, and binding data were analyzed with a nonlinear binding analysis program as described (34, 35).
The concentration response curve of the GTPase inhibition of Go by phosducin (see Fig. 6) was also analyzed assuming an interaction of phosducin with both the
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(Eq. 1) |
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RESULTS |
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To demonstrate directly the binding of phosducin to the subunits
of Go, we incubated phosducin with Go or
o in solution and then immobilized phosducin to detect
which subunits were bound to phosducin. This was done by covalently
coupling phosducin-specific antibodies raised in rabbits (32) to
protein A-Sepharose, which allowed specific retention of phosducin (and
proteins bound to phosducin) on small columns. In preliminary
experiments we verified that the antibodies showed no cross-reactions
with any of the subunits of Go, so that the Go
subunits should only be retained on the columns if they were bound to
phosducin. Phosducin (plus associated proteins) was then eluted from
the columns with 100 mM glycine-HCl, pH 2.5, and the
eluates were analyzed for the presence of
and
subunits of
Go in Western blots. (We did not analyze the presence of
subunits due to a lack of high affinity antibodies and also because
the
subunit complex does not appear to dissociate under
physiological conditions.) Fig.
1A shows that using trimeric
Go the
as well as
subunits were retained on the
column in the presence of phosducin and that there was virtually no
retention of either
or
subunits when mock preparations were
used instead of phosducin. The retention of the
as well as the
subunits was sensitive to phosphorylation of phosducin by PKA (Fig.
1B). For both subunits, PKA-mediated phosphorylation decreased the amount of retention by more than 80% as determined by
densitometric analysis. This corresponds roughly to the 80-100% stoichiometry of phosducin phosphorylation that was achieved with PKA
in these assays (see "Experimental Procedures") and suggests that
phosphorylated phosducin binds the subunits of Go weakly if
at all.
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Preincubation of Go with GTPS to dissociate
o from the
subunits markedly reduced the amount
of retained
o but caused only a minor reduction of
retained
subunits (Fig. 1C). This result is compatible
with the notion that phosducin binds to the
subunit complex with
high affinity, whereas binding to
o is of low affinity.
However, this experiment does not indicate whether the residual binding
of
o is due to incomplete dissociation of Go
by GTP
S or whether it does indeed represent direct low affinity binding of
o to phosducin.
Direct low affinity binding of o to phosducin was then
demonstrated with isolated
o. The preparations of
o used in these experiments were devoid of residual
subunits by the criterion of Western blots (data not shown).
Incubation of such
o with phosducin led to the retention
of
o on the column, but, as expected for a low
affinity interaction, the amounts of
o retained were small (Fig. 2). Again, this binding was
essentially abolished when PKA-phosphorylated phosducin was used
(Fig. 2).
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To confirm direct binding of subunits to phosducin, a second
strategy involving another method to purify and to immobilize phosducin
was used. To this end, a hexahistidine tag was added to the C terminus
of phosducin, and phosducin-His6 was purified to apparent
homogeneity via binding of this hexahistidine tag to Ni-NTA-agarose.
When 50 nM phosducin-His6 were co-incubated with various concentrations of purified G
and then
pelleted with Ni-NTA-agarose, G
were co-precipitated
as detected by Western blots (Fig.
3A). Much less
G
was precipitated in the absence of phosducin,
indicating a relatively low nonspecific binding of G
to the resin. A semiquantitative analysis of this experiment by
densitometry (Fig. 3B) revealed that the binding of
G
to phosducin-His6 was saturable with an apparent affinity of 25 nM. Analysis of four similar
experiments gave an average apparent affinity of 55 ± 22 nM.
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Analogous experiments were then done with purified o
(Fig. 4). Again, there was a
phosducin-His6-dependent precipitation of
o with Ni-NTA-agarose. A semiquantitative analysis
showed saturable binding of
o to
phosducin-His6 with an apparent affinity of 85 nM (Fig. 4), and seven similar experiments gave an average value of 110 ± 12 nM.
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Because Gt and its subunits can be purified better than
Go (and also because phosducin was initially discovered in
the visual system) such experiments were also carried out with
t, which had been purified to apparent homogeneity from
bovine retina (Fig. 5). These experiments
gave essentially similar results as those obtained with
o, but higher concentrations of
t were
required. The apparent affinity was 160 nM in the
experiment shown in Fig. 5 and 200 ± 53 nM in a total
of four separate experiments.
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These data provide direct evidence for binding of phosducin not only to
the subunit complex but also to G-protein
subunits. Functional assays were then used to determine the effects of such binding to
subunits, again using
o because of its
substantial intrinsic GTPase activity. Fig.
6 shows the effects of phosducin on the
steady state GTPase activity of
o and trimeric
Go. Phosducin inhibited the steady state GTPase activity of
o in a concentration-dependent manner;
maximal inhibition was
60%, and the IC50 value was
somewhat higher (
300 nM) than expected from the direct
binding assays. As already shown earlier for trimeric Go
(16) no such inhibition was seen with PKA-phosphorylated phosducin
(data not shown). These results demonstrate a direct inhibitory effect
of phosducin on the GTPase activity of
o in agreement
with its direct binding.
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The GTPase activity of trimeric Go under the same
conditions (i.e. basal activity in 0.1% Lubrol), was
inhibited by phosducin up to 90% with an IC50 value of
10 nM (Fig. 6). Assuming that this inhibition is caused
both by a direct effect of phosducin on
o and the well
known effect via G
, we also analyzed this inhibition
curve, assuming that it has a
-dependent and an
-dependent component as described under "Data
Analysis." This analysis gives a fit indicating that the
-dependent component causes 73% inhibition with an
IC50 value of
7 nM, whereas the
-dependent component causes an additional 19%
inhibition with an IC50 value of
120 nM. The
latter affinity is in the range of the IC50 values
determined from the direct effects of phosducin on
o;
likewise, the extent of inhibition (
19/(100-73), i.e.
70%) is similar to the
60% inhibition seen in the GTPase assays with
o alone. This two-component fit is statistically
significantly better (p < 0.01 by F-test) than the
simple one-component fit, but the differences between the two curves
are very small.
The GTPase cycle contains two main steps: hydrolysis of GTP to GDP and
release of GDP (followed by very rapid binding of GTP). The effects of
phosducin on the first step in o were assayed in
single-turnover experiments. In these assays,
[
-32P]GTP is bound to Go in the absence of
Mg2+, conditions that suppress the enzymatic activity of
G-proteins. The addition of Mg2+ plus a large excess of a
stable GTP analog then allows the monitoring of the hydrolysis of the
bound [
-32P]GTP. Fig. 7
shows that even a high concentration (2 µM) of phosducin had no appreciable effect on this catalytic step. GTP hydrolysis occurred with a rate constant of 4.8 ± 0.4 min
1 in
the absence and 3.9 ± 0.4 min
1 in the presence of
phosducin. The amount of [
-32P]GTP hydrolyzed was
almost identical under the two conditions (0.51 ± 0.01 pmol/pmol
Go under control conditions versus 0.56 ± 0.02 in the presence of phosducin). These data indicate that the
GTP-hydrolytic step of Go is not significantly affected by phosducin.
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However, phosducin did impair the GDP release step of isolated
o. The presence of 0.5 µM phosducin
reduced the rate constant of GDP release from isolated
o
from 0.23 ± 0.01 to 0.14 ± 0.01 min
1 (Fig.
8). Concentration response curves for
this inhibitory effect were done by monitoring the amount of GDP
remaining bound to
o 5 min after initiation of the
release reaction (Fig. 9). Phosducin caused a concentration-dependent increase in the amount of
bound GDP, and this effect was half-maximal at
100 nM.
This concentration is in agreement with the apparent affinity of
phosducin for
o determined in the direct binding assays
(Fig. 4B).
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DISCUSSION |
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Phosducin is a widely distributed inhibitor of the GTPase activity
of trimeric G-proteins (16, 17, 21, 22, 26). In addition to inhibition
of the enzymatic activity of G-proteins themselves, it has also been
shown to inhibit G-protein-mediated activation of adenylyl cyclase in
the -adrenergic receptor system (16) and of cGMP phosphodiesterase
in the rhodopsin system (17). In intact cells its overexpression
impairs cAMP production induced by stimulation of
-adrenergic
receptors (32). In the present study we have addressed the role of the
G-protein subunits in this process and in particular whether in
addition to the well demonstrated interactions with G-protein
subunits phosducin might also have effects on the
subunits.
Our data indicate that phosducin can indeed interact directly with
G-protein subunits. This was shown in direct binding experiments as
well as in functional assays. In the direct binding experiments two
different factors were used to immobilize phosducin: specific
affinity-purified antibodies and a C-terminal hexahistidine tag. These
two strategies first permitted the use of two different initial
purification procedures for phosducin and second should result in very
different kinds of nonspecific binding of the G-protein subunits. With
the antibody method no detectable amounts of G-protein subunits were
retained with the use of mock preparations from E. coli
instead of phosducin (Fig. 1A), indicating first that the
antibodies showed no significant reactivity toward any of the G-protein
subunits and second that the retention of G-protein subunits was not
due to a contaminating protein. We conclude that these assays did
indeed measure the interaction of phosducin with G-protein subunits.
With the hexahistidine tag method there was detectable nonspecific
precipitation of both G
and G
(Figs.
4-6), presumably due to the fact that the washing conditions could not
be chosen as harsh as in the antibody method. However, this nonspecific
binding was relatively low and allowed the clear detection of saturable
specific binding of G
,
o, and
t to phosducin. The demonstration of direct binding to
t as well as
o is important for two
reasons. First,
t can be purified to apparent
homogeneity, indicating that no other proteins are required to effect
phosducin-G
interactions. Second, it indicates that
phosducin might affect G-protein
subunits also in the visual system.
The analysis of these binding assays by densitometry can be only
semiquantitative due to the nonlinearity of the detection method.
However, the apparent affinities correlate fairly well with those
determined in functional assays; the affinities of phosducin for
G reported by various groups using various methods is
in the range of 8-80 nM (16, 22, 23, 36-38). The value
found in our direct binding assay was 55 nM, which is in the upper range of the reported functional values. It is about 10-fold
better than the previously obtained values utilizing
G
directly immobilized on microtiterplates, which
presumably resulted in distortion of G
(37).
The apparent affinity of phosducin for o determined in
the direct binding assay was 110 nM, whereas in the
functional assays IC50 values of 100 nM (GDP
release) and 300 nM (GTPase) were obtained; the analysis of
the inhibition of Go-GTPase with a
- and an
-dependent component gave an IC50 value of
the
-component of 120 nM. These affinities agree within
the accuracy of the methods used.
The affinity of phosducin for o is lower than that for
the
subunit complex. However, an affinity in the range of
100-300 nM is still below the concentration of phosducin
in most tissues, which is about 1 µM (21). Thus, this
affinity is sufficient to allow phosducin-G
interactions
in vivo. Likewise, the high concentrations of phosducin in
the retina (18-21) are above the levels required for the low affinity
interaction with
t. Furthermore it appears that these
interactions do indeed contribute to the inhibition of G-protein
function by phosducin; the Go GTPase activity was inhibited
by phosducin by up to 90% (Fig. 6). Under the conditions of these
assays (in particular 0.1% Lubrol and high Mg2+),
G
causes a 2-4-fold activation of the GTPase
activity of
o (39). Thus, if phosducin acted only to
"trap" G
, it should inhibit the GTPase activity
of Go by at most 50-75%. This is clearly less than the
observed inhibition of 90% and suggests an additional mode of
inhibition. The analysis of this inhibition curve with two components
(
and
) did indeed result in a significantly better fit. Even
though the improvement is only small, this two-component fit is
entirely compatible in qualitative and in quantitative terms with the
other data obtained here; it gives a G
-mediated inhibition by 73% with an IC50 value of 7 nM,
compatible with the ranges given above, and a G
-mediated
inhibition of the remaining activity by 70% with an IC50
value of 120 nM, compatible with the affinities and the
extent of inhibition seen in assays with isolated
o.
The functional effects of phosducin on o consist in an
inhibition of GDP release but no effect on the catalytic step of the GTPase cycle. Thus, the effects of phosducin on Go appear
to be composed of a direct inhibition of GDP release from
o, and an antagonism of the effects of the
subunit complex on the function of
o. Because the
subunits have stimulatory effects on
o under activating
conditions, such as in the presence of high Mg2+
concentrations, mastoparan, or active receptors (39), the direct and
the G
-mediated effects of phosducin on
o are additive under the conditions of stimulated
Go activity.
The effects of phosducin on G should not only cause an
increased inhibition of G-protein function compared with
G
-mediated effects alone, but they should also
increase the rapidity of this inhibition. This is because
trapping of G
subsequent to G-protein activation
would leave GTP-bound G
free to interact with effectors
and disrupt the G-protein cycle only after the first round of GTP
hydrolysis. In contrast, a direct effect on G
might
already affect signaling in this first cycle of G-protein activation.
Taken together these data suggest that the interactions of phosducin
with
o are of functional relevance.
Direct interactions of phosducin with G-protein subunits were not
observed by Lee et al. (17) and Yoshida et al.
(26) in their investigations on transducin (Gt). These
authors found no comigration of phosducin and
t on gel
filtration columns and no effect of phosducin on the binding of
t to rod outer segment membranes. There are two possible
explanations for this discrepancy. First, we found that the affinity of
phosducin for
t is lower than that for
o.
Second, the assays used by these authors, which involve physical
separation of proteins by chromatography or by centrifugation and
washing, may be less sensitive to interactions of low affinity than the
assays used here. In fact, gel filtration chromatography of phosducin
has been shown to involve interactions with the matrix (26) that might
well result in disruption of low affinity phosducin-
subunit
interactions, and the affinity of phosducin for Gt reported
in binding assays with rod outer segment membranes (26) is
10-fold
lower than that found in our assays.
In our hands, phosphorylation of phosducin by PKA impaired binding to
G as well as to
o. This was seen both
in direct binding and in GTPase assays. The data about the effect of
phosphorylation on the phosducin/transducin-
interaction are
conflicting, depending on the assay used. Phosphorylated phosducin no
longer coeluted with transducin-
from gel filtration columns but
still inhibited transducin-
binding to rod outer segment membranes (26). It was concluded that phosphorylation might not alter
the affinity of phosducin for transducin-
but rather affect the
character of the interaction (26). However, Hawes et al.
(22) observed a loss of G
binding upon
phosphorylation of phosducin similar to our data. Because
phosphorylation of phosducin would be required to impair
G
binding by the C-terminal as well as the N-terminal
binding sites, it is plausible to assume that it involves a major
alteration of the structure of phosducin (24). Under these
circumstances it is not surprising that phosphorylation of phosducin
would also impair its interactions with G
.
The molecular mechanisms of the interaction between phosducin and
G remain to be elucidated. The crystal structure of phosducin complexed with the
subunits of Gt (24)
suggests that phosducin binds with its C-terminal domain, particularly with an essential stretch of a few essential amino acids close to the C
terminus (25), to the side of the
-propeller, whereas its less well
defined N terminus is stretched out on the face of the propeller
covering sites where G
interacts with G
. Detailed studies will be required to elucidate
G
-binding sites in phosducin. Furthermore, we do not
know whether the interactions of phosducin with Go involve
two separate binding events (one with G
and another
one with
o), two-step binding (first to
G
and then to the
trimer), or a single
composite reaction to form a tetrameric phosducin-
complex.
In summary, we believe that our data support interactions of phosducin
with G as well as G
. At low
concentrations, phosducin appears to act preferentially by binding to
G
and by neutralizing G
effects on
Go activation. At higher concentrations, a direct
inhibition of
o causes additional inhibition of
G-protein function. Both types of interactions occur with affinities in
the lower range of physiological phosducin concentrations in many
tissues. Direct effects of phosducin on G
are predicted to have two major effects compared with the previously presumed exclusive action via G
; they would increase the
extent as well as the rapidity of its inhibitory effects. Our
observations suggest that complex interactions of phosducin with
G-protein subunits play a role under physiological circumstances. These interactions provide an additional level to the many mechanisms (40)
that regulate transmembrane signaling via G-protein-coupled receptors.
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ACKNOWLEDGEMENTS |
---|
We thank Stefan Müller for
purified o, Werner Schnepp for purified
t,
and Cornelius Krasel for purified PKA. Some G
and
G
antibodies were kindly provided by G. Schultz and
R. Schulz. We thank M. Heck and K. P. Hofmann for helpful discussions regarding the curve fitting of the GTPase experiments.
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FOOTNOTES |
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* This work was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 49-931-2015400;
Fax: 49-931-2013539.
1
The abbreviations used are: G,
G-protein
subunit; G
, G-protein
subunit
complex; GTP
S, guanosine 5'-(3-O-thio)triphosphate; PKA,
protein kinase A; Ni-NTA, nickel-nitrilotriacetic acid.
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REFERENCES |
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