(Received for publication, September 14, 1995; and in revised form, October 30, 1995)
From the
The -adrenergic receptor kinase (
ARK) modulates
-adrenergic and other G protein-coupled receptors by rapidly
phosphorylating agonist-occupied receptors at the plasma membrane. We
have recently shown that
ARK also associates with intracellular
microsomal membranes both ``in vitro'' and
``in situ''
(García-Higuera, I., Penela, P., Murga,
C., Egea, G., Bonay, P., Benovic, J. L., and Mayor, F., Jr. (1994) J. Biol. Chem. 269, 1348-1355), thus suggesting a
complex modulation of the subcellular distribution of
ARK. In this
report, we used recombinant
[
S]methionine-labeled
ARK to show that this
kinase interacts rapidly with a high affinity binding site (K
of 20 ± 1 nM) present
in salt-stripped rat liver microsomal membranes. Although
ARK
binding is not modulated by membrane preincubation with G protein
activators, the activity of bound
ARK toward rhodopsin or a
synthetic peptide substrate was markedly enhanced upon stimulation of
the endogenous heterotrimeric G proteins present in the microsomal
membranes by AlF
or mastoparan/guanosine
5`-(3-O-thio)triphosphate, thus strongly suggesting a
functional link between these proteins and membrane-associated
ARK. Interestingly,
ARK association with microsomal membranes
is not significantly affected by a fusion protein derived from the
carboxyl terminus of
ARK1 (the proposed location of the
subunit binding site), whereas it is markedly inhibited by fusion
proteins corresponding to the amino-terminal region of the kinase. The
main determinants of binding appear to be localized to an
60-amino
acid residue stretch (residues 88 to 145). Our results further indicate
a functional relationship between
ARK and heterotrimeric G
proteins in different intracellular organelles, and suggest that
additional proteins may be involved in modulating the cellular
localization of the kinase through a new targeting domain of
ARK.
A general feature of G protein-coupled receptors is that
exposure to agonists often leads to a rapid loss of receptor
responsiveness, a process termed desensitization or tolerance. Such
regulatory mechanisms are triggered every time a receptor is activated
and play a key role in signal integration and plasticity at the
cellular level. The -adrenergic receptor
(
AR)(
)-coupled adenylyl cyclase system has provided an
important model for the study of the molecular mechanisms of
desensitization (Benovic et al., 1988; Dohlman et
al., 1991; Kobilka, 1992; Lohse, 1993). Work from several
laboratories has shown that rapid agonist-specific desensitization of
the
AR is due to functional uncoupling from the transducing G
protein, which is initiated by phosphorylation of the
AR by
-adrenergic receptor kinase (
ARK), a serine-threonine kinase
that specifically phosphorylates the agonist-occupied form of the
receptor (Palczewski and Benovic, 1991; Lefkowitz, 1993; Haga et
al., 1994a).
ARK-mediated phosphorylation allows the
interaction with the
AR of an additional regulatory protein,
-arrestin, which precludes receptor interaction with G proteins
and blocks signal transduction (Lohse et al., 1990; Kobilka,
1992). The uncoupling process is followed by transient receptor
internalization away from the plasma membrane. Interestingly, emerging
evidence indicates that
ARK may be able to modulate a variety of G
protein-coupled receptors (Benovic et al., 1987; Kim et
al., 1993b; Kwatra et al., 1993; Richardson et
al., 1993). This, together with the recent characterization of
different
ARK-related kinases, which constitute the G
protein-coupled receptor kinase (GRK) multigene family, strongly
suggests that very similar mechanisms of regulation operate for all G
protein-coupled receptors (Inglese et al., 1993; Haga et
al., 1994a; Loudon and Benovic, 1994;
García-Higuera et al., 1994b).
However, there is little understanding of the protein-protein
interactions of this complex regulatory network, and of the changes in
the subcellular distribution of these proteins that take place upon
receptor activation. ARK has been described as a soluble,
cytosolic enzyme that transiently translocates to the plasma membrane
when the receptor is occupied by an agonist (Strasser et al.,
1986; Mayor et al., 1987;
García-Higuera and Mayor, 1992; Chuang et al., 1992). Recent data indicate that
subunits
of heterotrimeric G proteins are able to enhance
ARK activity
toward different G protein-coupled receptors (Haga and Haga, 1992;
Pitcher et al., 1992; Kameyama et al., 1993; Kim et al., 1993b), and both purified G proteins and isolated
subunits can interact with
ARK in vitro (Pitcher et al., 1992; Kim et al., 1993b).
Therefore, it has been proposed that the interaction of
subunits with
ARK would help to target the kinase to the periphery
of the plasma membrane and to increase its activity toward the
activated receptor. The
-binding domain of
ARK has been
localized to a 125-amino acid stretch in the COOH-terminal region of
the enzyme (Kameyama et al., 1993; Koch et al.,
1993); this region partially overlaps with a pleckstrin homology domain
present in
ARK (Touhara et al., 1994). Moreover, we have
recently reported that, in addition to being a soluble enzyme that
transiently translocates to the plasma membrane, a significant amount
of
ARK is associated with internal microsomal membranes in a
variety of tissues and cell lines
(García-Higuera and Mayor, 1994;
García-Higuera et al., 1994a),
thus raising new questions regarding the functional role of this
kinase. Subcellular fractionation studies and indirect
immunofluorescence and immunogold electron microscopy localization in
cultured cells confirmed the association of
ARK with microsomal
structures in situ (García-Higuera and Mayor, 1994;
García-Higuera et al., 1994a). In
our previous report, cell-free association experiments indicated that
ARK peripherally associates with a protein component of the
microsomal membranes by means of electrostatic interactions
(García-Higuera et al., 1994a),
in a way reminiscent of its transient interaction with the plasma
membrane.
In this paper, we have used a direct binding assay with
[S]methionine-labeled
ARK (Kim et
al., 1993b) to further characterize the kinase binding site in the
microsomes and investigated the functional consequences of such
interaction. Our data support a functional link between
ARK and
microsomal heterotrimeric G proteins and suggest the existence of a new
targeting domain of
ARK involved in the modulation of the complex
subcellular distribution of this important regulatory kinase.
Previous studies from our laboratory have shown that ARK
is associated with microsomal membranes in rat liver and many other
tissues and cell lines (García-Higuera et al., 1994a). The kinase can be completely extracted from
the membranes by mild salt treatment (200 mM NaCl), thus
suggesting a peripheral association based on electrostatic
interactions. Moreover, we reported (by using activity measurements and
immunoblot analysis), that recombinant purified
ARK1 was able to
interact with a protein component of salt-stripped rat liver microsomal
membranes in a cell-free system
(García-Higuera et al., 1994a).
In these experiments, membrane-associated and unbound
ARK were
separated by centrifugation at 250,000
g followed by
kinase quantitation in the pellet or the supernatant. In order to gain
further insight into the mechanisms of association of
ARK with
microsomal membranes, we have utilized a direct binding assay using
purified recombinant [
S]methionine-labeled
ARK1, which has recently been used for investigating the
interaction of
ARK with phospholipid vesicles containing purified
G protein subunits (Kim et al., 1993b). In the assays shown
below,
S-labeled
ARK was incubated under different
experimental conditions with stripped rat liver microsomal membranes,
and the amount of membrane-associated
ARK was then determined by
measuring the radioactivity in the high-speed pellets. Nonspecific
binding of
ARK was estimated by performing parallel experiments in
the presence of 200 mM NaCl, a condition known to completely
inhibit the kinase binding to the microsomal vesicles
(García-Higuera et al., 1994a).
This has enabled a quantitative and detailed characterization of the
interaction of
ARK with this physiological membrane preparation.
Fig. 1A shows that ARK binding to stripped
microsomal membranes is very rapid (half-maximal binding achieved in
less than 1 min) and results in the association of most (
75%) of
the kinase present in the assay. Scatchard analysis (Fig. 1B) indicates that the interaction process is
saturable and appears to involve one population of high affinity
binding sites (K
of 20 ± 1 nM).
Similar results are obtained using lower concentrations of membranes.
Interestingly, such kinetic and affinity data are similar to those
reported for the interaction of
ARK1 with purified heterotrimeric
G proteins or
subunits (58 ± 14 and 32 ± 5
nM, respectively) (Kim et al., 1993b). In order to
better understand
ARK association with microsomal membranes, we
tried to obtain further information on the type of proteins(s)
responsible for the interaction, on the effect of binding on
ARK
activity, and on possible modulators of
ARK association and
function.
Figure 1:
Characterization of S-labeled
ARK binding to stripped microsomal
membranes. A, time course of direct binding. Labeled
ARK
(20 nM, 500 cpm/pmol) was incubated in buffer A with stripped
microsomal membranes (0.56 µg of protein/µl) at 37 °C for
the times indicated and membrane-associated
ARK was determined
after high-speed centrifugation as detailed under ``Experimental
Procedures.'' Specific binding was the difference between counts
in the absence and presence of 200 mM NaCl. Data points are
the mean ± S.E. of two independent experiments done in
duplicate. B, representative Scatchard analysis of
ARK
binding. Labeled
ARK (5-80 nM, 350 cpm/pmol) was
incubated in buffer A with stripped microsomal membranes (0.4 µg of
protein/µl) for 15 min at 37 °C and specific binding determined
as in panel A. K
and B
values are the mean ± S.E. of three
independent experiments performed in duplicate. A representative
saturation curve is shown in the inset (
, total binding;
, nonspecific binding;
, specific
binding).
Since ARK binding to G protein subunits has been
shown in vitro (Pitcher et al., 1992; Koch et
al., 1993; Kim et al., 1993b), and the presence of
heterotrimeric G proteins in intracellular organelles is mentioned in
an increasing number of reports (see references in Balch(1992),
García-Higuera et al. (1994a),
and Neubig(1994)), we investigated the presence of endogenous G
proteins in the salt-stripped microsomal membranes used in our studies.
Specific
and
subunit
antibodies detect major bands of
40- and 35-kDa, respectively (Fig. 2), thus confirming the presence of heterotrimeric G
proteins in this preparation. G proteins can also be detected in the
microsomal membranes by a
S-labeled GTP
S binding
assay (data not shown). The identity of the subunit isoforms has not
been investigated in detail, although additional protein bands in the
40-46 kDa range can be detected with the
antibody at longer times of exposure. With regard to
subunits, comparison of the signal obtained in microsomal membranes
with that of a known amount of
subunits purified from bovine
brain (Fig. 2, lane 1) allows an approximate
quantitation of at least 10 pmol of microsomal
per mg of
stripped membrane protein, in the same order of magnitude as the B
detected for
ARK binding (see Fig. 1B).
Figure 2:
Presence of and
subunits
of heterotrimeric G proteins in rat liver stripped microsomal
membranes. Stripped microsomal membranes (60 µg of protein, lanes 1 and 3) or 20 ng of purified
subunits obtained from bovine brain (lane 2) were resolved by
11% SDS-PAGE, blotted to nitrocellulose membranes, and probed with
antibodies AS8 (
subunit antibody, 1/300, lane 3) or M14 (
subunit antibody, 5 µg/ml, lanes
1 and 2). Results are representative of three
experiments.
We next tested the ability of different
compounds to modulate ARK association with microsomal membranes
using the direct binding assay. Particularly, we investigated whether
ARK interaction would be modulated by known G protein activators.
In initial experiments, preincubation of stripped microsomal membranes
with the selective heterotrimeric G protein activator
AlF
(Finazzi et al., 1994) in
the presence of excess Mg
and GDP promoted a marked
increase in subsequent
ARK binding (Fig. 3A).
However, this effect seems to be due only to the presence of
Mg
and not to G protein activation, since a similar
increase in
ARK interaction is observed when only Mg
is added during the preincubation (Fig. 3A); GDP
alone has no effect on
ARK binding (data not shown). A more
detailed investigation on the effect of Mg
was
performed. Fig. 3B shows a dose-dependent effect of
submillimolar concentrations of Mg
on
ARK
association with the microsomal membranes, which reaches an
4-fold
increase over control binding at
1 mM Mg
. Such fold-increase is similar to that
observed in Fig. 3A at 2.5 mM Mg
, thus suggesting that the Mg
effect attains a maximum in such concentration range. The
presence of Mg
promotes an increase in the apparent B
(65 pmol/mg of protein with 1 mM
Mg
) without changing the affinity of
ARK for its
binding sites. The divalent cation Mn
can substitute
for Mg
(70% of Mg
effect at 1
mM Mn
). In order to optimize
ARK
binding and to approach physiological intracellular concentrations, 1
mM MgCl
was routinely included in all subsequent
experiments.
Figure 3:
Modulation of S-labeled
ARK binding to microsomal membranes. A, stripped
microsomal membranes (0.75 µg of protein/µl) were preincubated
for 15 min at 37 °C in buffer A in the absence(-) or presence
(+) of 12.5 mM MgCl
and/or
AlF
(5 mM NaF, 50 µM AlCl
, 1 mM GDP) and then incubated for 2 min
at 37 °C in the presence of
S-labeled
ARK (20
nM,
500 cpm/pmol) in a final volume of 300 µl.
Specific
ARK binding was determined as in Fig. 1. Data are
mean ± S.E. of six independent experiments. B, effect
of magnesium on
ARK binding:
S-labeled
ARK (20
nM,
500 cpm/pmol) was incubated for 5 min at 37 °C in
the presence of stripped microsomal membranes (0.75 µg of
protein/µl) in a buffer containing 20 mM Tris-HCl, pH 7.5,
in the absence or presence of the indicated concentrations of
MgCl
. Specific binding was determined as detailed in Fig. 1and under ``Experimental Procedures.'' Bound
ARK in the absence of MgCl
(1.14 ± 0.2 pmol of
ARK/mg of protein) was taken as the basal value to which all the
other experimental conditions were referred. Data are mean ±
S.E. of two independent experiments performed in
duplicate.
The fact that ARK binding to microsomal membranes
is not modulated by known G protein activators is further confirmed by
the lack of effect on
ARK association of mastoparan, a
tetradecapeptide which stimulates guanine nucleotide exchange and
activates heterotrimeric G proteins (Higashijima et al., 1988;
Colombo et al., 1992) or GTP
S, a non-hydrolyzable GTP
analog which is a general activator of G proteins (Colombo et
al., 1992; Tsai et al., 1992) (data not shown). The
presence of other nucleotides such as GDP, ATP, GTP, or App(NH)p
(0.5-1 mM) did not have any effect on
ARK binding,
and did not promote the release of previously bound
ARK from the
microsomal membranes (data not shown). Other possible modulatory
compounds, such as cAMP or heparin, a
ARK inhibitor (Benovic et al., 1989a), do not significantly modulate
ARK
interaction. The association of the kinase with the stripped microsomal
membranes is reduced by
50% when tested in the presence of 100
mM potassium gluconate, an ionic condition similar to the
intracellular medium (not shown, see also
García-Higuera et al. (1994a)).
In summary, our data indicate that despite its presence in microsomal
membranes and the previously reported interaction with
ARK in
vitro (Pitcher et al., 1992) G protein activation was not
required and did not modulate
ARK binding in our model.
We have
previously shown (García-Higuera et
al., 1994a) that ARK association to microsomal membranes is
reversible, i.e. that previously extracted or recombinant
ARK1 can interact with salt-stripped microsomes, thus indicating
that the microsomal component involved in the interaction is not
removed during the mild stripping procedure. Fig. 4A shows that when salt-stripped membranes are further treated with
0.1 M Na
CO
at pH 11, a commonly used
method for removing strongly-attached peripheral membrane proteins
(Fujiki et al., 1982; Yu et al., 1992),
ARK
association (as assessed by an activity assay) is not affected, thus
suggesting that
ARK interacts with a microsomal component which
behaves as an integral membrane protein. A more detailed and
quantitative analysis of the interaction of
ARK with
Na
CO
-stripped membranes was performed.
Immunoblot analysis indicates that the interaction of recombinant
ARK with a defined amount of salt-stripped microsomal membranes (Fig. 4B, lane 1) is not altered after removal of
additional peripheral proteins with the Na
CO
pH
11 treatment (lane 2); the association with the
Na
CO
-stripped membranes is blocked in the
presence of 200 mM NaCl (lane 3). Direct binding
studies using 50 nM
S-labeled
ARK show that
a given amount of microsomal membranes retains 86 ± 9% (mean
± S.D. of two independent experiments performed in triplicate)
of specific kinase binding after the Na
CO
pH 11
treatment, thus leading to an
1.5-fold increase in binding
specific activity (up to 82 ± 5 pmol/mg protein). Similar
results were obtained in other sets of assays using 25 nM labeled
ARK. It is worth noting that the
Na
CO
treatment leads to the loss of
50% of
the G protein
subunits from the membranes, as assessed by
immunoblot analysis (Fig. 4C). The fact that such
50% decrease in G protein subunits does not alter
ARK
association suggests that G proteins may not be the main anchor for
ARK in the microsomal membranes (also see below).
Figure 4:
Influence of microsomal membrane
pretreatment with NaCO
on
ARK association
and on the presence of G protein
subunits. A, study of
ARK association by using an activity assay. Stripped microsomal
membranes were resuspended in buffer A or in 100 mM
Na
CO
, pH 11, and incubated for 30 min at 4
°C in order to extract remaining peripheral proteins. After one
wash in buffer A, control and treated membranes were incubated for 10
min at 37 °C with a similar amount of a soluble extract containing
ARK activity exactly as described
(García-Higuera, et al., 1994).
Soluble and membrane-associated
ARK were separated by
centrifugation and
ARK activity in the supernatant (S)
and microsomal membrane (M) fractions was determined by using
a rhodopsin phosphorylation assay as detailed under ``Experimental
Procedures.'' The arrow indicates the position of
phosphorylated rhodopsin. B, immunoblot analysis of
ARK
association. Aliquots of salt-stripped microsomal membranes were
treated in the absence (lane 1) or presence (lanes 2 and 3) of Na
CO
pH 11 as indicated
under ``Experimental Procedures'' and extracted proteins
separated by high-speed centrifugation. The same volume of membrane
pellets (corresponding to 50 µg of protein in the initial, non
Na
CO
-treated membranes) were resuspended in 20
mM Tris-HCl, pH 7.5, 2 mM MgCl
and
incubated for 5 min at 37 °C with 30 nM recombinant
ARK in the absence (lanes 1 and 2) or presence (lane 3) of 200 mM NaCl. After centrifugation at
250,000
g for 10 min, the amount of bound
ARK in
each sample was assessed by immunoblot analysis as detailed under
``Experimental Procedures.'' 80 ng of recombinant
ARK
were directly resolved and analyzed in the same gel as a control (lane 4). Results are representative of two independent
experiments. C, presence of G protein
subunits in
microsomal membranes subjected to different stripping procedures.
Salt-stripped purified microsomal membranes (800 µg of protein)
were treated in the absence (lanes 2 and 3) or
presence (lanes 4 and 5) of Na
CO
pH 11 as indicated under ``Experimental Procedures'' in
a final volume of 800 µl. After high-speed centrifugation to
separate membranes and extracted proteins, equivalent aliquots of the
membrane pellets (60 µg of proteins, lanes 2 and 4) and extracted supernatants (60 µl, lanes 3 and 5) were resolved by 11% SDS-PAGE, blotted to nitrocellulose
membranes, and probed with a
subunit antibody (M14, Santa Cruz
Biotechnology, 5 µg/ml) as described under ``Experimental
Procedures.'' 60 µg of unstripped microsomal membranes (lane 1) and 20 ng of purified
subunits obtained
from bovine brain (lane 6) were resolved and blotted in the
same gel and probed as above.
We next
investigated the functional consequences of ARK binding to the
microsomal membranes by comparing the activity of the free and bound
kinase toward exogenous substrates. Fig. 5shows that, compared
to free
ARK (lane 1), a marked inhibition of
light-dependent rhodopsin phosphorylation is observed in the presence
of increasing amounts of microsomal membranes (lanes
3-5), which do not show any residual
ARK activity (lane 2). This result indicates that bound
ARK is less
able to interact with rhodopsin and/or to phosphorylate this
membrane-bound specific substrate. It is worth noting that the same
effect is observed when a synthetic peptide substrate is used to test
the activity of
ARK (Fig. 7A), thus showing that
the observed effect is not restricted to the rhodopsin phosphorylation
assay. Interestingly, it has been reported that rhodopsin
phosphorylation by the homologous, if not identical, muscarinic
acetylcholine receptor kinase was strongly inhibited by heterotrimeric
G proteins in the absence of guanine nucleotides in a dose-dependent
way (Haga and Haga, 1992).
Figure 5:
Effect of the presence of different
concentrations of microsomal membrane proteins on the phosphorylation
of rhodopsin by ARK. Recombinant
ARK (20 nM) was
preincubated alone or in the presence of the indicated amounts of
stripped microsomal membranes in 20 mM Tris-HCl, pH 7.5, 1
mM MgCl
in a final volume of 50 µl for 10 min
at 37 °C. The phosphorylation reaction (30 min, 30 °C) was
initiated by the sequential addition of phosphorylation buffer and
purified rod outer segments to give the final concentrations detailed
under ``Experimental Procedures'' (final volume, 60 µl).
Phosphorylated rhodopsin was resolved by SDS-PAGE and visualized by
autoradiography. Results are representative of three independent
experiments.
Figure 7:
The activity of ARK toward a
synthetic peptide substrate is modulated by the presence of stripped
microsomal or plasma membrane fractions and by heterotrimeric G protein
activators. Stripped microsomal (panel A) or plasma (panel
B) membrane fractions (0.6 µg of protein/µl) were
preincubated for 15 min at 37 °C in the presence of 20 nM
ARK in 20 mM Tris-HCl, pH 7.5, 1 mM
MgCl
, alone or in the presence of
AlF
(5 mM NaF, 50 µM AlCl
, 100 µM GDP) or mastoparan (20 or
100 µM) and GTP
S (50 µM). The activity
of bound
ARK toward the synthetic peptide substrate RRREEEEESAAA
was subsequently assessed for 30 min at 30 °C, after the sequential
addition of peptide substrate and phosphorylation buffer to give the
final concentrations detailed under ``Experimental
Procedures.'' After determination of the radioactivity
incorporated into the peptide, the activity of
ARK in the presence
of stripped microsomal membranes (0.7 ± 0.17 pmol of phosphate
incorporated) or stripped plasma membranes (0.6 ± 0.28 pmol of
phosphate incorporated) alone were taken as the basal values to which
all the other experimental conditions were referred. Data are mean
± S.E. of two to four independent experiments performed in
duplicate.
Since purified subunits or
stimulators of G protein activation and dissociation such as GTP
S
have been shown to stimulate
ARK activity toward different
substrates (Haga and Haga, 1992; Pitcher et al., 1992:
Müller et al., 1993; Pei et al.,
1994), we next investigated whether preincubation of stripped
microsomal membranes with specific agents that would activate
endogenous heterotrimeric G proteins may modulate the activity of bound
ARK. Fig. 6A shows that the marked inhibition of
ARK activity observed in the presence of microsomal membranes
(compare lanes 1 and 3) is relieved when the
microsomes were previously preincubated with the G protein activators
AlF
(lane 4) or mastoparan plus
GTP
S (lane 5). This effect cannot be simply ascribed to a
release of bound
ARK, since such compounds do not show any effect
on
ARK binding to the microsomal membranes ( Fig. 3and data
not shown), and the same result is obtained when the microsomal
membranes are pelleted and then resuspended in phosphorylation buffer
in the presence of rhodopsin (data not shown), thus indicating a direct
effect on bound
ARK as a consequence of G protein activation and
subunit dissociation. The same effect can be observed when
ARK
functionality is assessed using a synthetic peptide substrate (Fig. 7A). It is interesting to note that similar
results were apparent when we used stripped plasma membranes instead of
microsomal membranes for these experiments: bound
ARK activity
toward either rhodopsin or a peptide substrate was marked inhibited and
was only relieved in the presence of heterotrimeric G protein
activators (Fig. 6B and 7B). These data
suggest a general feature of
ARK interaction with cellular
membranes and indicate, for the first time using physiological membrane
preparations, that the activity of
ARK is regulated by endogenous
G proteins in different intracellular locations.
Figure 6:
Activation of endogenous heterotrimeric G
proteins present in stripped microsomal or plasma membrane fractions
modulate the phosphorylation of rhodopsin by ARK. Recombinant
ARK (20 nM) was preincubated in the presence of 0.7
µg of protein/µl of either stripped microsomal (A) or
stripped plasma membrane fractions (B) as detailed under
``Experimental Procedures'' and in Fig. 5, in the
absence or presence of AlF
(5 mM NaF, 50 µM AlCl
, 100 µM GDP)
or mastoparan (100 µM) plus GTP
S (50
µM), as indicated. The activity of bound
ARK was
assessed by the rhodopsin phosphorylation assay as described in the
legend to Fig. 5. Results are representative of three
independent experiments.
The fact that G
protein activation can modulate the activity of bound ARK clearly
demonstrates a functional link between these proteins in the microsomal
membranes. Such a functional link could be the consequence of a direct
association of
ARK to G proteins under basal conditions, which
would result in kinase activation in the presence of specific G protein
stimulators. Alternatively, or in addition,
ARK could be binding
to a different protein in the stripped microsomes, but its activity may
be modulated by additional interactions with G protein
subunits released upon G protein activation.
Since attempts to
directly identify the microsomal protein(s) involved in ARK
interaction using different cross-linkers were unsuccessful, we tried
another approach based on investigating the ability of fusion proteins
containing different
ARK domains to displace
S-labeled kinase association with microsomal membranes. It
has been described that the
binding site domain of
ARK
is localized in the COOH-terminal portion of the kinase (Koch et
al., 1993; Kameyama et al., 1993) and GST fusion proteins
containing the last 222 amino acids of
ARK have been recently used
to characterize
ARK interactions with
subunits (Pitcher et al., 1992; Inglese et al., 1994; Touhara et
al., 1994). Therefore, we prepared a similar fusion protein
construct (FP2) containing amino acids 437-689 of bovine
ARK1 and an additional construct containing amino acids
50-145 of the kinase (FP1) (see Fig. 8A), which
contains a highly charged region that could be involved in
electrostatic interactions (Benovic et al., 1989b). The
ARK fusion proteins were expressed in E. coli and
purified by affinity chromatography on gluthatione-Sepharose columns;
purified GST alone was also prepared as a negative control. As
expected, FP2 (encompassing the COOH-terminal portion of
ARK), was
able to associate with purified G protein
subunits isolated
from bovine brain, whereas GST alone or FP1 did not associate or showed
a very weak interaction (Fig. 8B). Consistently, FP2
blocked
subunit activation of rhodopsin phosphorylation by
ARK, whereas FP1 had no an inhibitory effect (Fig. 8C). We next investigated whether these proteins
could inhibit
ARK association with stripped microsomal membranes
by measuring the binding of
S-labeled kinase to microsomes
preincubated under control conditions or in the presence of different
concentrations of GST or GST-
ARK fusion proteins (Fig. 8D). Interestingly, GST-FP2 showed only a very
slight effect on
ARK binding over a range of concentrations that
have been shown to strongly inhibit
ARK association to purified G
protein
subunits in vitro or to block
subunit modulation of type II adenylyl cyclase in reconstituted or
permeabilized systems (IC
in the range 10-20
µM) (Pitcher et al., 1992; Koch et al.,
1993; Touhara et al., 1994; Inglese et al., 1994).
Similar results were obtained in the presence of G protein activators
(data not shown). On the contrary, the fusion protein GST-FP1,
containing an amino-terminal region of the kinase, markedly decreased
labeled
ARK interaction with the microsomal membranes, with a
potency (
60% of binding inhibition at 15 µM) similar
to that of the COOH terminus on
-related functions.
Interestingly, similar results were obtained when the effects of these
fusion proteins on
S-labeled
ARK binding were tested
in Na
CO
-stripped membranes, which are partially
devoid of G proteins (Fig. 4C and Kehlenbach et
al.(1994)) but retain the ability to interact with the kinase (60
± 5% and 106 ± 8% of control binding of 40 nM
S-labeled
ARK in the presence of 20 µM GST-FP1 and GST-FP2, respectively). Taken together, our data
indicate that the mechanism of
ARK association with microsomal
membranes is different from that of its interaction with G protein
subunits, and suggest that
ARK is primarily interacting
with an additional protein component of the membranes.
Figure 8:
Effect of GST-ARK fusion proteins on
S-labeled
ARK binding to microsomal membranes. A, domain structure of bovine
ARK1 indicating the regions
from which fusion protein containing amino acids 50-145 (FP1) or
437-689 (FP2) were derived; the proposed location of the
pleckstrin homology domain (PH) and the
subunits
binding region are also indicated. B,
binding
properties of GST-
ARK fusion proteins. Purified bovine brain
subunits were incubated with identical concentrations of
GST, FP1, and FP2 and protein complexes pelleted using
glutathione-Sepharose beads as indicated under ``Experimental
Procedures.'' Bound proteins were resolved by SDS-PAGE,
transferred to nitrocellulose, and detected with a specific antibody;
purified brain
subunits (G
) were used as a
positive control. C, effect of GST-
ARK fusion proteins
FP1 and FP2 on rhodopsin phosphorylation by
ARK in the presence of
G protein
subunits. The rhodopsin phosphorylation assay was
performed as described under ``Experimental Procedures'';
after incubation for 40 min at 30 °C in the presence of 20 nM recombinant
ARK and 80 nM
subunits alone
or in the presence of 10 µM GST or the indicated
GST-
ARK fusion proteins the reaction was stopped by addition of
SDS-PAGE buffer, and phosphorylated rhodopsin was resolved by 12%
SDS-PAGE followed by autoradiography. D, dose-dependent
inhibition of
S-labeled
ARK binding to microsomal
membranes by GST-
ARK fusion proteins. Stripped microsomal
membranes (0.75 µg of protein/µl) were preincubated for 30 min
at 37 °C in 20 mM Tris-HCl, pH 7.5, 2 mM MgCl
with the indicated concentrations of GST or the
fusion proteins FP1 and FP2 or an equivalent amount of bovine serum
albumin in the same vehicle (control conditions). Labeled
ARK was
then added at a final concentration of 15 nM. After 5 min at
37 °C
ARK specific binding was determined as detailed in Fig. 1and under ``Experimental Procedures.'' Data are
expressed as percentage of the binding detected in control conditions,
and are means ± S.E. of 3-4 independent experiments
performed in duplicate.
In order to
confirm these results and start to delineate the region of ARK
involved in the association with microsomal membranes, we tested the
effects of additional fusion proteins. In these experiments, microsomal
membranes were incubated with recombinant
ARK in the presence of
different GST-
ARK constructs (Fig. 9A) and either
GST alone or 200 mM NaCl as controls. Membrane-bound and
soluble
ARK were separated by high-speed centrifugation, and the
ARK activity remaining in the supernatant was quantitated. Control
experiments were performed to show that remaining fusion proteins per se did not have any effect on the phosphorylation assay
(data not shown). Fig. 9B show that, as expected, NaCl
strongly inhibits
ARK binding to the membranes (i.e. high
ARK activity present in the supernatant), whereas binding is high
in the presence of GST (i.e. low
ARK activity remaining
in the supernatant), which does not inhibit
ARK association (see Fig. 8D). The presence of GST-
ARK fusion proteins
comprising amino acids 1-147 or 50-145 (FP1) clearly
inhibits
ARK binding to microsomal membranes, whereas a construct
encompassing amino acids 1-88 does not, thus suggesting that the
main determinants of
ARK association are located in the region
88-145. Taken together, these results indicate that the
interaction of this regulatory kinase with the microsomes
preferentially involves a domain located within the amino-terminal
portion of
ARK, and suggest that G protein
subunits are
not the main anchor for
ARK in the microsomal membranes.
Figure 9:
Analysis of the effect of amino-terminal
GST-ARK fusion proteins on
ARK association with microsomal
membranes. A, schematic representation of the GST-
ARK
constructs used in the experiments. B, effect of GST-
ARK
fusion proteins on
ARK binding to microsomal membranes. Stripped
microsomal membranes (0.75 µg of protein/µl) were preincubated
for 30 min at 37 °C as detailed in Fig. 8D with the
indicated fusion proteins (15 µM) or NaCl 200 mM,
and recombinant
ARK was then added at a final concentration of 10
nM. After 5 min at 37 °C, bound and soluble
ARK were
separated by high-speed centrifugation as detailed under
``Experimental Procedures,'' and unbound
ARK was
quantitated in 5 µl of the supernatants using the rhodopsin
phosphorylation assay. Results are representative of five independent
experiments.
An early step in the regulation of AR is the
phosphorylation of the agonist-occupied receptor by
ARK, which was
initially described as a soluble enzyme that transiently translocates
to the plasma membrane upon receptor activation (Lefkowitz, 1993;
Inglese et al., 1994; Haga et al., 1994a). Recent
data have shown that
ARK can interact with purified
subunits or whole heterotrimeric G proteins in vitro (Pitcher et al., 1992; Kim et al., 1993b), thus suggesting
possible anchors for the kinase in the plasma membrane. In addition, we
have recently reported that
ARK associates with internal
microsomal membranes both in vitro and in situ (García-Higuera et al.,
1994a). These data indicate that several
ARK pools
(microsome-bound, plasma membrane-bound, and cytosolic) exist inside
the cell and suggest that complex mechanisms (probably involving
specific interactions with different proteins) may operate in order to
regulate the subcellular distribution and activation of
ARK. In
this report, we have further characterized the association of
ARK
with microsomal membranes using a direct
S-labeled
ARK binding assay, and investigated the modulation of the
functionality of the microsome-bound kinase.
Our data indicate that
ARK binds very rapidly to stripped microsomal membranes with a K
of
20 nM. The kinase appears to
associate with only one population of high affinity binding sites,
although the possibility of more than one population of sites with
similar binding affinities cannot be completely ruled out. Such binding
sites are suggested to be protein components of the microsomal
membrane, since previous data from our laboratory showed that protease
or heat pretreatment of the microsomes strongly inhibits
ARK
association (García-Higuera et
al., 1994a). Furthermore, the lack of effect on
ARK binding
of Na
CO
pH 11 treatment shown in the present
report indicates that the kinase binding sites behave as integral
membrane proteins. It is worth noting that the affinity of
ARK
interaction with microsomal membranes is higher than that reported for
its association with agonist-activated
AR (K
> 100 nM) and similar to that
obtained with purified G proteins (K
58
nM) or isolated
subunits (K
32 nM) (Kim et al., 1993b), consistent
with a functionally relevant role. The present study provides the first
report of a high affinity interaction of
ARK with a physiological
membrane preparation. Association with microsomal membranes is not
unique for
ARK since a number of other proteins
(pp60
, PKC-
, ADP-ribosylation factors, kinesin, and
dynein) have been reported to interact in different ways with such
types of preparations (Resh, 1989; Chida et al., 1994; Tsai et al., 1992; Yu et al., 1992; Thissen and Casey,
1993).
Given the previously reported interaction of ARK with
purified G proteins and
subunits and the stimulation of
ARK activity by free
subunits (see above), these
proteins were obvious candidates for participating in
ARK binding
and for modulating the activity of microsomal
ARK, since their
presence in intracellular membranes has been increasingly appreciated
(see references in García-Higuera et
al. (1994a), Neubig(1994), and Nuoffer and Balch, 1994). We
confirmed by [
S]GTP
S binding and immunoblot
analysis the presence of heterotrimeric G proteins in our preparations,
and its preliminary quantitative analysis was compatible with a role in
ARK association, taking into account the apparent B
of
ARK binding. However, our results
clearly show that the interaction of
ARK with the microsomal
membrane is not dependent on heterotrimeric G protein activation, since
no significant changes can be observed under experimental conditions
routinely used for G protein stimulation (Colombo et al.,
1992; Tsai et al., 1992; De Almeida et al., 1993; Kim et al., 1993b). The only apparent modulators of
ARK
binding were low millimolar concentrations of divalent cations such as
Mg
. Since Mg
can modulate a variety
of enzymatic reactions (including phosphorylation/dephosphorylation
processes, although the involvement of proteins kinases as mediators of
its effect is unlikely in our experimental conditions), the mechanisms
of the Mg
effect on
ARK interaction and its
possible functional relevance remain to be established.
Our studies
on the modulation of the activity of microsome-bound ARK put
forward two interesting findings: (i) the bound kinase is in an
inactive state, as shown by its reduced ability to phosphorylate
rhodopsin or a synthetic peptide substrate; and (ii) although
ARK
binding is not affected by stimulators of heterotrimeric G proteins
such as mastoparan/GTP
S or AlF
,
these agents promote a marked increase in the activity of bound kinase,
indicating a functional link between endogenous microsomal G proteins
and
ARK.
It has been previously shown that the phosphorylation
of rhodopsin or the muscarinic acetylcholine receptor by rhodopsin
kinase or ARK-like kinases was strongly inhibited by phospholipid
vesicles containing purified, non-activated heterotrimeric G proteins
(Kelleher and Johnson, 1988; Haga and Haga, 1990, 1992), and that this
effect was relieved in the presence of GTP
S (i.e. subunit
dissociation) (Haga and Haga, 1992). It has been suggested that such an
inhibitory effect is due to a competition between trimeric G proteins
and receptor kinases for overlapping sites on the activated receptor.
However, the fact that we observed inhibition of
ARK activity
toward both an activated receptor (rhodopsin) and a synthetic peptide
substrate suggest that, in addition, the interaction of
ARK with
the membranes temporarily masks or alters functionally relevant domains
of the kinase. The activation of microsomal heterotrimeric G proteins
would favor the interaction of free
subunits with
ARK,
which will then adopt an active conformation. Interestingly, the same
pattern of modulation of
ARK activity can be detected when
stripped plasma membranes were used instead of microsomal membranes,
thus suggesting a general feature of
ARK interaction with cellular
membranes. Our results confirm, for the first time using physiological
membrane preparations, that
subunits activate
ARK-mediated phosphorylation (Haga and Haga, 1990, 1992; Pitcher et al., 1992; Kameyama et al., 1993; Kim et
al., 1993b). Furthermore, we show that
ARK activity can be
modulated by endogenous G proteins in different intracellular
locations. The fact that G protein stimulation also increases
ARK
activity toward a soluble peptide substrate is in line with recent in vitro results showing that phosphorylation of soluble
substrates by
ARK or related kinases is enhanced in the presence
of
subunits (Haga et al., 1994b; Kim et
al., 1993b). Interestingly, such results are obtained in the
presence of putative synergistic modulators of the kinase (activated
receptors, synthetic receptor fragments, mastoparan) but not in its
absence (Pitcher et al., 1992; Kim et al., 1993b).
Taken together, these data suggest that G protein
subunits
stimulate the enzymatic activity of
ARK, in addition to its
possible role as a membrane anchor for the kinase (Haga et al. 1994a, 1994b; also see below).
Our results using either plasma
or microsomal membranes support a clear distinction between the process
of ARK association with cellular membranes and that of kinase
activation. Since the association of
ARK with cellular membranes
does not require G protein stimulation, it could take place under
resting, basal conditions, whereas
ARK activation leading to
substrate phosphorylation would be dependent on the stimulation of G
proteins by specific signals. This is consistent with previous data
showing that
ARK associates equally well to the heterotrimeric G
protein or to the
dimer alone, thus leading to the
suggestion that
ARK targeting to the plasma membrane may take
place prior to receptor activation (Kim et al., 1993b).
A
key issue to be addressed is the identity of the protein(s) involved in
the association of ARK with the microsomal membranes. To date,
ARK1 has been reported to interact with the activated
AR and with
dimers, which have been
proposed to play a key role in the kinase targeting to the periphery of
the plasma membrane (Pitcher et al., 1992; Kim et
al., 1993b). The presence of
subunits in our
salt-stripped microsomal membrane preparation argued for a role of
these proteins in
ARK association. The fact that G protein
stimulators did not have any effect on
S-labeled
ARK
binding did not rule out its participation, since it has been
previously shown that the activation of purified G
/G
proteins does not modulate the kinase binding to heterotrimeric G
proteins, which could be an entity recognized by
ARK (Kim et
al., 1993b). On the other hand, the functional link between
membrane-bound
ARK and activated G proteins discussed above does
not necessarily imply G proteins as the only anchors of
ARK, since
(as mentioned under ``Results'')
ARK could also be
binding to a different protein in the stripped microsomes, and its
activity modulated by additional interactions with
subunits
released upon G protein stimulation.
Two main lines of evidence
support the notion of a binding site for ARK other than G protein
subunits. First, the lack of effect on
ARK association of membrane
pretreatment with Na
CO
pH 11, which leads to an
50% reduction in immunoreactive G proteins (Kehlenbach et
al.(1994) and Fig. 4C). Second, the effect of
different GST-
ARK fusion proteins on
ARK binding. It has been
described that the carboxyl-terminal portion of
ARK-1 is
responsible for the activation by G protein
subunits and
that the minimal
binding domain is localized to a 125-amino
acid stretch (Koch et al., 1993; Kameyama et al.,
1993). This region partially overlaps with a pleckstrin homology domain
present in
ARK (Touhara et al., 1994). It is worth noting
that, although pleckstrin homology domains have been recently reported
to interact with phospholipid vesicles rich in phosphatidylinositol
4,5-bisphosphate (Harlan et al., 1994), this does not appear
to play a predominant role in the interaction of
ARK with
microsomal membranes, since we have previously shown that the
association is heat and protease-sensitive and therefore involves a
protein component of the microsomes
(García-Higuera et al., 1994a).
Interestingly, our results show that GST-FP2, a fusion protein
containing the COOH terminus of
ARK which has been used by others
to characterize
ARK interactions with
subunits (Pitcher et al., 1992; Inglese et al., 1994; Touhara et
al., 1994), does not inhibit
ARK1 binding to microsomal
membranes, whereas it is able to interact with purified
subunits (Fig. 8B) and to inhibit the
effect
on rhodopsin phosphorylation by
ARK (Fig. 8C). In
agreement with these results, recent experiments (
)indicate
that purified phosducin, which is able to interact with G protein
subunits (DebBurman et al., 1995) does not inhibit
ARK binding to microsomal membranes in our experimental conditions
(94 ± 12% of control binding at 300-450 nM phosducin, mean ± S.E. of four experiments). On the
contrary, a fusion protein containing an NH
-terminal
portion of the kinase (residues 50-145) which does not bind
purified
subunits (FP1), strongly inhibits
ARK
association with a potency similar to other reported effects of
ARK fragments (Koch et al., 1993, 1994; Inglese et
al., 1994; Touhara et al., 1994). A similar inhibitory
effect on
ARK binding can be observed with a GST-
ARK fusion
protein comprising amino acids 1-147, whereas a 1-88
fragment is without effect, thus suggesting the
ARK anchoring
domain may reside within residues 88-145.
Although at present
we cannot totally exclude the possibility that intact G proteins or
specific dimers play a role in
ARK association with
microsomal membranes, we feel that our data strongly suggest that
ARK binding is preferentially mediated via a high affinity
interaction with a currently unidentified microsomal protein. As shown
in the model depicted in Fig. 10, such protein component (X) appears to interact with a region of the amino terminus of
the kinase, thus suggesting a new targeting domain of
ARK. Bound
ARK would be inactive until G protein stimulation leads to
additional interactions of the COOH terminus of
ARK with
-subunits. In line with other recent results, our data
suggest that the regulation of
ARK activity and subcellular
distribution will likely involve multiple interactions with G protein
subunits, phospholipids (DebBurman et al., 1995), different
domains of G protein-coupled receptors (Haga et al., 1994b;
Ruiz-Gómez et al., 1994), and additional
anchoring proteins. In this regard, the existence of anchor proteins
has been previously reported for other protein kinases such as protein
kinase A (Hausken et al.(1994) and references therein) and
protein kinase C (Ron et al., 1994), and emerges as a general
mechanism for regulating the subcellular distribution and activity of
protein kinases (Mochly-Rosen, 1995). Future research will strive to
identify the
ARK anchor protein in the microsomes, and to more
precisely localize the
ARK binding domain. Although it has been
suggested that the NH
terminus of GRKs may interact with
activated receptors (Palczewski et al., 1993), (
)there is little knowledge on the role of this domain,
which does not have significant sequence homology with other known
GRKs, including GRK1, GRK6, and GRK5, which have been shown to display
a different mechanism of membrane association (Kunapuli et
al., 1994). Further investigation using different experimental
approaches would be needed to ascertain the factors (expression of G
protein-coupled receptors, specific combinations of heterotrimeric G
proteins, kinase anchors . . . ) and mechanisms governing the complex
subcellular distribution of this key regulatory kinase and the possible
function(s) of
ARK in microsomal membranes
(García-Higuera et al., 1994a).
Figure 10:
Proposed model for the interaction of
ARK with microsomal membranes and the modulation of the activation
of the bound kinase. The domain structure of
ARK indicates the
regions from which fusion proteins GST-
ARK 50-145 (FP1) or
GST-
ARK 437-689 (FP2) are derived. X, putative
anchor protein;
,
, and
are heterotrimeric G protein
subunits and
* denotes G protein
activation.
This work is dedicated to the memory of the late Dr. Oscar Menéndez-Avello.