(Received for publication, December 18, 1995; and in revised form, February 21, 1996)
From the
Phosducin has recently been identified as a cytosolic protein
that interacts with the -subunits of G proteins and thereby
may regulate transmembrane signaling. It is expressed predominantly in
the retina but also in many other tissues, which raises the question of
its potential specificity for retinal versus nonretinal
-subunits. We have therefore expressed and purified different
combinations of
- and
-subunits from Sf9 cells and have also
purified transducin-
from bovine retina and a mixture of
complexes from bovine brain. Their interactions with
phosducin were determined in a variety of assays for
function: support of ADP-ribosylation of
by pertussis
toxin, enhancement of the GTPase activity of
, and
enhancement of rhodopsin phosphorylation by the
-adrenergic
receptor kinase 1 (
ARK1). There were only moderate differences in
the effects of the various
complexes alone on
, but there were marked differences in their ability
to support
ARK1 catalyzed rhodopsin phosphorylation. Phosducin
inhibited all
-mediated effects and showed little specificity
toward specific defined
complexes with the exception of
transducin-
(
), which was
inhibited more efficiently than the other
combinations. In a
direct binding assay, there was no apparent selectivity of phosducin
for any
combination tested. Thus, in contrast to
ARK1,
phosducin does not appear to discriminate strongly between different G
protein
- and
-subunits.
Guanine nucleotide-binding proteins (G proteins) are transducers
between heptahelical receptors and various effectors. Traditionally, G
proteins have been classified according to their -subunits, but in
the last years a plethora of effects have been assigned to the
-subunits. Thus, they have been shown to regulate adenylyl
cyclases, phospholipases C-
and A
, PI3-kinase, the
ADP-ribosylation factor and several ion channels (for reviews, see (1) and (2) ). In addition,
-subunits provide
a membrane attachment site for the
-adrenergic receptor kinase
(
ARK) (
)which allows translocation of the kinase from
the cytosol to the cell membrane and thus in close proximity to its
substrate receptors(3, 4) . Phosphorylation of G
protein-coupled receptors by
ARK is thought to represent the first
step of homologous desensitization; it is followed by binding of
proteins of the arrestin family which results in uncoupling of
receptors and their G proteins(5, 6) .
Another
cytosolic protein which interacts with G protein -dimers is
phosducin, a phosphoprotein originally purified from bovine retina as a
complex with the
-subunits of G
(7) . By
binding to G protein
-subunits phosducin can inhibit G
protein-mediated signaling(8, 9) . Furthermore,
phosducin can compete with
ARK for the
-subunits and can
thereby impair
ARK-mediated phosphorylation of
receptors(10) . The interaction of phosducin with G proteins is
markedly reduced following its phosphorylation by protein kinase
A(8, 11) .
The possibility of a common motif for
the interaction of all these proteins with -subunits has
attracted much recent interest. Binding to distinct types of adenylyl
cyclases, phospholipase C-
3, atrial K
channels,
and
ARK has been found to include a defined stretch of amino
acids(12) . However, this sequence cannot be found in
phosducin. Binding to
ARK involves the kinase's C terminus,
which partially overlaps with the pleckstrin-homology domain (PH
domain) of
ARK(13, 14) . The binding site of
phosducin responsible for interaction with
-subunits has been
mapped to its N terminus (15, 16) which contains
neither a PH domain nor the sequence mentioned above. Thus, the
interaction of phosducin with G protein
-subunits must be
different from those with other
-binding proteins.
Molecular cloning techniques have revealed multiple isoforms of
- and
-subunits. The most recent data indicate the existence
of five distinct
- and 10
-subunits(17, 18) . Compared to the highly
conserved
-subunits, the
-subunits are more divergent,
suggesting them to be the specificity-determining factor. The
functional differences of defined
combinations are limited;
regulation of particular types of adenylyl cyclases or stimulation of
phospholipase C-
isoforms and K
channels as well
as interactions with G protein
-subunits did not reveal striking
differences except for
, the
complex found in retinal rods, which showed lower potencies
in its actions(19, 20, 21) . Receptor
phosphorylation by
ARK was stimulated more potently by
-containing dimers and a preparation of
-subunits from bovine brain than by complexes with
or by
(22) .
The proteins of the
retinal signal transduction cascade are homologous but distinct from
those of receptor-mediated signaling(23) . They are restricted
in their expression to the retina and the developmentally related
pineal gland, and they show specificity toward each other when compared
with the proteins of other receptor systems. This applies also to the G
protein -subunits, with
being a retina-specific combination. Phosducin, on the other
hand, is highly expressed in the retina and pineal gland, but also in
other tissues(8) . This suggests that it may show less
specificity for the other components of the retinal signaling system
than has been found for other members of this system. In fact,
phosducin has been shown to inhibit the GTPase-activity not only of
G
but also of G
, G
, and G
preparations(8, 9) . Since the
-subunits appear to be the primary interaction partners for
phosducin, the aim of the present investigation was to examine the
specificity of interactions between defined G protein
combinations and phosducin.
G protein -subunits and
from bovine brain and transducin-
-subunits
from bovine retina were purified according to established
procedures(25, 26) . In order to reduce the
contamination of
with
-subunits to a
minimum, the usual purification process was extended by a further
chromatography step on a C7 heptylamine-Sepharose column.
Phosducin was expressed in Escherichia coli and purified following the procedure of Bauer et al.(8) .
In order to investigate effects of -subunits of
defined composition, the proteins were produced in Sf9 cells by
co-infection with recombinant baculoviruses directing the expression of
defined
- and
-subunits. The resultant
complexes
were then purified to >95% purity by chromatography on C7
heptylamine-Sepharose and DEAE-Sephacel as described
previously(22) . The
complexes used in this study
were
,
,
,
and
. Mock preparations of cells
infected with wild-type baculoviruses were prepared in the same manner
for control purposes. Transducin-
(
) and a bovine brain
preparation containing multiple
- as well as
-subunits were
prepared by established procedures to serve as controls.
All
preparations were able to support pertussis toxin-catalyzed
ADP-ribosylation of
(Fig. 1A). There
were no major differences between the different recombinant
complexes. The two native preparations, transducin-
and the
mixed preparation of
-subunits from bovine brain, were more
effective at lower concentrations than the recombinant preparations; at
higher concentrations this difference disappeared for
transducin-
and became small for the bovine brain
preparation.
Figure 1:
Pertussis toxin-mediated
ADP-ribosylation of . Stimulation by
-dimers (panel A) and inhibition by phosducin (panel B). A,
(2 pmol, 40 nM) purified from
bovine brain was ADP-ribosylated in the presence of pertussis toxin
with increasing amounts of different
combinations
(2-60 nM).
stands for
transducin-
,
for a mixture of
-subunits from bovine brain, CON for a mock
preparation from uninfected Sf9 cells. Nonlinear curve fitting to the
Hill equation gave the following E
and EC
values:
, 0.52 ± 0.04
mol/mol, 20.9 ± 3.8 nM;
0.65 ± 0.09 mol/mol, 25.4 ± 7.7 nM;
, 0.48 ± 0.04 mol/mol, 19.3
± 3.8 nM;
, 0.58
± 0.08 mol/mol, 33. 0 ± 9.3 nM;
, 0.68 ± 0.04 mol/mol, 13.7 ± 2.2
nM;
, 0.41 ± 0.02 mol/mol, 5.8
± 1.1 nM. Data are means ± S.E. of three
experiments. B, 0.8 pmol (16 nM)
was incubated with 0.8 pmol (16 nM)
-subunits
in the presence of various concentrations of phosducin (8 to 240
nM). The effects evoked by the individual
-dimers
alone were set to 100%. Nonlinear curve fitting to the Hill equation
gave the following I
and IC
values:
, 46 ± 9%, 32 ± 21
nM;
, 49 ± 11%, 49
± 41 nM;
, 51
± 6%, 40 ± 15 nM;
, 58 ± 11%, 26 ± 21
nM;
, 45 ± 5.5%, 34 ± 13
nM;
, 79 ± 13%, 34 ± 19
nM. Data are means ± S.E. of three
experiments.
Phosducin inhibited these enhancing effects of all
complexes on pertussis toxin-catalyzed ADP-ribosylation of
. Fig. 1B shows the phosducin-mediated
inhibition of ADP-ribosylation in the presence of a constant,
submaximally effective concentration (16 nM) of the various
complexes. In this assay, only transducin-
showed
a distinct inhibition curve; its effects were antagonized to a greater
degree than those of other
complexes. This occurred even
though the enhancing effects of 16 nM transducin-
on
-ADP-ribosylation were not larger than those of the
recombinant
complexes and even smaller than those of the
bovine brain
preparation (Fig. 1, A and B). Among the recombinant
complexes there were only
very small differences in their inhibition by phosducin, with a slight
tendency toward better inhibition by
-containing
complexes versus
-containing complexes. These
minor differences were not statistically significant.
Interactions
between the complexes and
were also
investigated by measuring the enhancement of the intrinsic GTPase
activity of
which is exerted by
-subunits
in the presence of high concentrations of
Mg
(31) . All
preparations were
able to enhance the GTPase activity of
(Fig. 2A). Again, there were small differences
between the different recombinant
complexes; those
containing the
-subunit appeared
40% more
effective than those containing the
-subunit. Of the
native preparations, the brain preparation of
-subunit was
very effective, whereas transducin-
was the least effective
of all combinations. None of the combinations reached saturation within
the concentration range employed in these experiments; this was
particularly the case for the most potent combinations,
and the bovine brain
preparation.
Figure 2:
Stimulation of the GTPase activity of
by
-subunits (panel A) and its
inhibition by phosducin (panel B). A, the GTPase of
(1 nM) was stimulated in the presence of
high Mg
(10 mM) with increasing
concentrations of different
-dimers (1 to 25 nM).
Nonlinear curve fitting to the Hill equation gave the following
E
and EC
values:
, 614 ± 44%, 29.9 ± 3.4
nM;
, 347 ± 107%,
24.6 ± 12.8 nM,
, 338
± 18%, 15.8 ± 1.7 nM;
, 331 ± 27%, 24 ± 3.3
nM;
, 751 ± 163%, 47 ± 14
nM;
, 235 ± 52%, 16.5 ±
6.9 nM. Data are means ± S.E. of five experiments. B, the stimulatory effect of 24 nM of the different
complexes on 1 nM
(set to 100%)
was antagonized with various concentrations of phosducin (24 to 720
nM). I
and IC
values were
calculated using nonlinear curve fitting according to the Hill
equation:
, 65 ± 3.6%, 31
± 7.7 nM;
, 60
± 4.2%, 46 ± 13.1 nM;
, 58 ± 8.8%, 26 ± 19
nM;
, 71 ± 1.6%, 48
± 4.4 nM;
, 47 ± 4.8%, 165
± 49 nM;
, 87 ± 11.6%, 44
± 2.4 nM. Data are means ± S.E. of six
experiments.
Again, a submaximally effective concentration of
-subunits was chosen to examine the inhibitory effects of
phosducin (Fig. 2B). As was the case with the
ADP-ribosylation assays, phosducin was most efficacious in inhibiting
the effects of transducin-
. There was >40% inhibition at
the lowest concentration of phosducin, at which the concentrations of
phosducin and
-subunits were identical (24 nM), and
the inhibition was almost complete at higher concentrations of
phosducin. Transducin-
was followed by the various
recombinant
complexes, which were all inhibited in a very
similar manner by phosducin; maximal inhibition amounted to
60% in
all cases, and the IC
values were not significantly
different. The effects of the bovine brain
preparation, in
contrast, were not well antagonized by phosducin; there was almost no
effect at the lowest concentration of phosducin, and even at the
highest concentration of phosducin (720 nM) the inhibition was
only about 30%. These data suggest the possibility that the bovine
brain
preparation might contain some
complexes
which are not well recognized by phosducin.
In order to study this
hypothesis further, we investigated another effect of G protein
complexes, the enhancement of the phosphorylation of G
protein-coupled receptors by the
-adrenergic receptor kinase 1
(
ARK1). We had reported earlier that there are marked differences
between the ability of defined
complexes in enhancing
receptor phosphorylation by
ARK1(22) . Using rhodopsin
(
830 nM) as the substrate and a submaximally effective
concentration of
-subunits (50 nM), the combination
caused the largest enhancement,
followed by the modestly effective combination
and the bovine brain
preparation, while
and
as well as transducin-
had
only very small effects (Fig. 3A).
Figure 3:
Enhancement of ARK-mediated
phosphorylation of rhodopsin by different
-subunits (panel A) and inhibition by phosducin (panels B and C). A, rod outer segments (50 pmol) were
phosphorylated by 5 nM purified
ARK1 and 50 nM of different
-dimers in a reaction volume of 60 µl.
The concentration of 50 nM
-subunits was chosen
because it had a submaximal effect on rhodopsin phosphorylation. Data
are means ± S.E. of three experiments. B and C, inhibition of the stimulatory effect of
-subunits
on
ARK-mediated rhodopsin phosphorylation by phosducin. Rhodopsin
was phosphorylated with
ARK1 and 50 nM
-dimers
in the presence of increasing concentrations of purified phosducin
(0.025 to 1 µM). The data shown in panels B and C are identical, except that the values in panel C were normalized to the values in the absence of phosducin.
Nonlinear curve fitting to the Hill equation gave the following
IC
values:
, 123
± 9 nM;
, 104
± 47 nM;
780 ± 280
nM. Data are means ± S.E. of three
experiments.
The enhancing
effects of -subunits on
ARK1-mediated rhodopsin
phosphorylation were inhibited by phosducin (Fig. 3B).
The presence of high concentrations of phosducin (1000 nM, i.e. 20-fold above
) essentially abolished the
effects of all
combinations. The enhancing effects of
several
combinations (
,
, and transducin-
) were too
small to allow a reliable determination of the inhibitory effects of
phosducin. For the remaining combinations, it can be seen that
phosducin inhibited the effects of the two recombinant
combinations, i.e.
and
, more potently than those of the
bovine brain
preparation (Fig. 3C). The
EC
values were 105 and 123 nM for
and
, respectively, and 780 nM for the bovine brain
preparation. In contrast, the
maximal extent of inhibition appeared to be similar for the three
preparations.
Finally, we developed an enzyme-linked
immunosorbent assay procedure to directly determine the binding of
phosducin to the various complexes. The various
-dimers were immobilized in wells of microtiter plates and
phosducin was then incubated in the wells to allow binding to the
immobilized
-subunits. Phosducin bound to all
complexes in a saturable manner with similar characteristics (Fig. 4). Contrary to the data shown above, there were no
differences in the affinity of phosducin for the different
combinations. There were minor differences in the maximal binding, but
most notably transducin-
and the bovine brain
preparation had equal binding capacity for phosducin. Thus, phosducin
appeared to show no selectivity in the direct interaction with
-subunits, whereas in functional assays the effects of
transducin-
were antagonized more efficiently.
Figure 4:
Direct binding of phosducin to immobilized
-dimers. Different
combinations (300 ng/well) were
bound to 96-well microtiter plates. Various concentrations of purified
phosducin (0.1 to 20 µg) were added in reaction volumes of 100
µl and bound phosducin was determined with affinity-purified
anti-phosducin antibodies followed by a color reaction. E
and EC
values were determined with the help of
non-linear curve fitting to the Hill equation:
, 0.58 ± 0.05 A
, 0.61 ± 0.20 µM;
, 0.70 ± 0.08 A
, 0.73 ± 0.26 µM;
, 0.72 ± 0.06 A
, 0.55 ± 0.16 µM;
, 0.77 ± 0.10 A
, 1.05 ± 0.41 µM;
, 0.82 ± 0.06 A
,
0.44 ± 0.11 µM;
, 0.82
± 0.08 A
, 0.51 ± 0.17
µM. Data are means ± S.E. of four
experiments.
G protein-mediated signaling systems comprise a series of
proteins, including the receptors, the G protein subunits, the
effectors and various partially cytosolic proteins. The steadily
increasing number of isoforms for all of these proteins that are being
discovered raises the question of protein-protein specificity. A fairly
consistent pattern is the specificity of the proteins that are involved
in phototransduction: rhodopsin, G, cGMP-phosphodiesterase,
rhodopsin kinase, and arrestin are all expressed only in the retina
(and in the developmentally related pineal gland). Furthermore, in in vitro assays they show marked specificity toward each
other. For example, rhodopsin kinase is much better in phosphorylating
rhodopsin than are
-adrenergic receptors, whereas the reverse is
true for
-adrenergic receptor kinase(32) . Similarly,
arrestin binds much better to rhodopsin than to
-adrenergic
receptors, while
-arrestin prefers
-adrenergic
receptors(28) . At the level of the signaling chain itself
(receptor/G protein/effector) the issue of specificity is less
clear-cut. While a series of experiments with antisense
oligonucleotides against individual G protein
-,
- and
-subunits have suggested a remarkable specificity for certain
receptor/ion channel coupling (33, 34, 35, 36) , reconstitution
experiments have shown only moderate specificity of receptor/G protein
coupling and various degrees of specificity for regulation of effectors
by G protein
subunits(19, 20, 22, 37, 38) .
Phosducin differs from the other proteins of such signaling chains
in its expression pattern: while it is very abundant in the retina and
in the pineal gland, it is also expressed in many other
tissues(8) . Since phosducin interacts primarily with the G
protein -subunits, and since the retinal
-dimer
is not found in other tissues, the
presence of phosducin in nonretinal tissues would make sense only if it
exhibited little or no selectivity toward specific
-subunits.
This question was addressed in the present study by investigating
the interaction of phosducin with a number of defined G protein
complexes in several functional assays as well as in a
direct binding assay. For this purpose, defined
complexes
were produced in Sf9 cells with the help of recombinant baculoviruses.
The baculovirus expression system has been used by several groups for
producing functional G protein subunits in high
quantities(19, 22, 39, 40) . For the
synthesis of functional
-subunits it is necessary to express
both subunits simultaneously. Their functionality can be ascertained by
their ability to support the ADP-ribosylation of G protein
-subunits by pertussis toxin. In our hands, all the combinations
tested so far were able to enhance the ADP-ribosylation of
equally well. We interpret this finding as evidence for equal
functional activity of the different
preparations. Similar
observations have been made by others regarding the ADP-ribosylation of
native
and
, whereas for
produced in E. coli (which was not
myristoylated) transducin-
was less efficient and
potent(19, 20, 37) .
Another functional
test for complexes which likewise involves interaction with
the
-subunits is their ability to alter the intrinsic GTPase
activity of the
-subunit. In the case of
,
-subunits activate the GTPase in the presence of high
concentrations of Mg
whereas in the presence of low
concentrations of Mg
(below 1 mM) they exert
an inhibitory effect(31) . In our studies, we measured modest
differences in the activity of the different
-dimers on
, which were about 2-fold for the best
(
) versus the worst
(transducin-
) combination. The pattern of selectivity was
somewhat different from the one found in the ADP-ribosylation assays,
suggesting that there are different requirements for the
-subunits in the two different types of experiments.
Investigations by Ueda et al.(20) on the inhibition of
-subunit GTPase activity by
-subunits have also failed to reveal significant selectivity
with the exception of
, which was
less potent in all assays.
A third, quite different, test for the
functionality of the complexes is their ability to serve as
membrane anchors for
ARK to enhance agonist-dependent receptor
phosphorylation(3, 4) . In an earlier study we had
found differences between defined
complexes in their ability
to enhance
ARK-mediated rhodopsin phosphorylation(22) .
Similar differences were found here, with
being most efficient and transducin-
least.
Phosducin had inhibitory effects on all of these -mediated
effects. Among the different
combinations, the stimulatory
effects of transducin-
both on ADP-ribosylation and GTPase
activity were more efficiently antagonized, while we did not detect any
real differences between the recombinant dimers consisting of
,
,
, and
. Phosducin also inhibited the
-mediated
enhancement of rhodopsin phosphorylation. However, the divergent
enhancing potential of the
complexes allowed a
characterization of this inhibition only in the case of
,
,
and
.
While stimulation of rhodopsin
phosphorylation by or
were equally well inhibited by
phosducin, the effect on
was less pronounced and
appeared to require higher concentrations of phosducin. Similarly, we
also observed only a weak inhibition on the
-mediated stimulation of
-GTPase. We speculate that the mixture of
-subunits from bovine brain might contain
complexes which are only poorly recognized by phosducin.
Whereas the
functional assays suggested a preference of phosducin for
transducin-, we found no real differences in affinity or
maximal binding when we assayed phosducin binding to immobilized
-subunits directly. Thus, the apparent selectivity of
phosducin for transducin-
in the functional assays may be
caused by events which occur subsequent to the initial binding step. An
alternative explanation is that transducin-
has a better
solubility than the other
complexes and that the binding
assay was done with immobilized
-subunits while the
ADP-ribosylation and GTPase assays were carried out in solution. In
this context it is important to note that the affinities measured in
the direct binding assay with immobilized
-subunits were
10-fold lower than those determined in the functional assays in
solution. Likewise, the affinities of phosducin for
and
found in the ADP-ribosylation and GTPase assays were
3-fold
higher than those determined in the
ARK assay, where the
-subunits were integrated in the rod outer segment membranes.
Similar discrepancies have been noted earlier. For example, Heithier et al.(41) reported severalfold higher affinities of
complexes for
in detergent-containing
solution than in lipid vesicles.
Since transducin- differs
from
or
only in its
-subunit, the small functional specificity of
phosducin for transducin-
must be due to the presence of
.
contains a different
isoprenylation, farnesyl instead of geranylgeranyl in all other G
protein
-subunits. The functional importance of this farnesyl,
compared to other prenyl modifications for the interaction with
receptors, has very recently been elucidated by Kisselev et
al.(42) . Alternatively, the slightly different effects of
may be due to its rather different amino acid
sequence: the amino acid identity of
with
or
is less than 40%, while the sequence
identity between
and
is about
80%(18) .
Overall, however, the interactions of phosducin
with the various complexes appear to be quite similar. This
relative lack of specificity goes in line with the nonretinal
expression of phosducin, since it enables an interaction of phosducin
with multiple G proteins and thus an interference with multiple
signaling pathways. It contrasts with the more pronounced selectivity
of
ARK for certain
complexes where
was the preferred partner and
transducin-
ineffective (22) (see also Fig. 3A). This agrees with our hypothesis mentioned in
the introduction that the
-binding domains of
ARK and
phosducin are dissimilar. While some homologies between the N terminus
of phosducin and the C terminus of
ARK have been
noted(14, 15) , phosducin contains neither a PH domain
nor the
-binding consensus sequence
Gln/Asn-X-X-Glu/Asp-Arg/Lys proposed by Chen et
al.(12) . Furthermore, phosducin-like protein (43) with its much shorter N terminus which shows no similarity
at all to the C terminus of
ARK, also interacts with G protein
-subunits(44) , supporting the differences in the
-binding mechanisms between phosducin and
ARK.
While
these data suggest that the binding sites on G protein
-subunits for
ARK and phosducin might be different, they
must clearly be overlapping, since phosducin and
ARK have been
shown to compete for
binding(10) . Furthermore, the
binding sites for phosducin and for
ARK appear to overlap also
with the binding site for the
-subunits, since both proteins
interfere with coupling between the
- and the
-subunits.
Thus the
-subunits appear to possess various distinct but
overlapping binding sites for different proteins.
Reconstitution
experiments employing defined G protein complexes have
largely failed to reveal much specificity. This contrasts with
antisense experiments which indicate that G proteins of very specific
-,
-, and
-composition are required for efficient
coupling of certain receptors to ion
channels(33, 34, 35, 36) . This
raises the possibility that selectivity may be encoded in the cellular
organization rather than in protein-protein interactions. Further
studies are required to determine whether similar rules might also
govern the interactions of phosducin with G protein
-subunits.