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(Received for publication, October 12, 1994; and in revised form, December 8, 1994) From the
G protein-coupled receptor-mediated signaling is attenuated by a
process referred to as desensitization, wherein agonist-dependent
phosphorylation of receptors by G protein-coupled receptor kinases
(GRKs) is proposed to be a key initial event. However, mechanisms that
activate GRKs are not fully understood. In one scenario,
G protein-coupled receptors (GPRs) ( Until now, six
GRKs have been identified by molecular cloning (GRK1 to
GRK6)(5, 6, 7, 8, 9, 10, 11) .
The molecular mechanisms that govern the activity of these kinases,
both in their interaction with receptor substrates and in regulation of
their function, are not completely understood. An important step in GRK
activation is translocation, whereby the cytosolic enzyme is targeted
to its substrate (the agonist occupied form of the GPR) in the plasma
membrane. It appears that different GRKs have evolved distinct
mechanisms of membrane localization. Rhodopsin kinase (GRK1) is unique
among GRKs in that it is modified at its carboxyl terminus by a
geranylgeranyl isoprenoid moiety, which allows the kinase to be
directly anchored to the plasma membrane(12, 13) .
GRK5 nonspecifically binds lipids which stimulate its
autophosphorylation and may help target it to cell
membranes(14) . In contrast, GRK2 and GRK3 ( In the present effort, we evaluated
the role of various classes of membrane lipids in regulating
Figure 1:
A, lipid regulation
of agonist-dependent phosphorylation of mAChRs by
Various membrane lipids were found to modulate the ability of
In order to assess whether regulation of We
wondered if PS and PIP
Figure 2:
Competitive interactions of PS and
PIP
PS increased the initial rate
and extent (Fig. 3A) of receptor phosphorylation by
Figure 3:
Comparison of PS and G protein
G
Figure 4:
A,
modulation of lipid regulation by GST-
It is unclear whether the effect of the
GST- The current theory that
membrane-anchored G The present
results support a mechanism of modulation of The G The
resolution of the solution structure of the PH domain of pleckstrin by
nuclear magnetic resonance spectroscopy has suggested that PH domains
may be lipid interaction sites in vivo(19) . In this
regard, PIP
Volume 270,
Number 11,
Issue of March 17, 1995 pp. 5742-5747
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-subunits of G proteins (G
) activate
certain GRKs (-adrenergic receptor kinases 1 and 2, or GRK2 and
GRK3), via a pleckstrin homology domain in the COOH terminus. This
interaction has been proposed to translocate cytosolic -adrenergic
receptor kinases (ARKs) to the plasma membrane and facilitate
interaction with receptor substrates. Here, we report a novel finding
that membrane lipids modulate ARK activity in vitro in a
manner that is analogous and competitive with G.
Several lipids, including phosphatidylserine (PS), stimulated, whereas
phosphatidylinositol 4,5-bisphosphate inhibited, the ability of these
GRKs to phosphorylate agonist-occupied m2 muscarinic acetylcholine
receptors. Furthermore, both PS and phosphatidylinositol
4,5-bisphosphate specifically bound to ARK1, whereas
phosphatidylcholine, a lipid that did not modulate ARK activity,
did not bind to ARK1. The lipid regulation of ARKs did not
occur via a modulation of its autophosphorylation state. PS- and
G-mediated stimulation of ARK1 was compared
and found strikingly similar; moreover, their effects together were not
additive (except at initial stages of reaction), which suggests that PS
and G employed a common interaction and activation
mechanism with the kinase. The effects of these lipids were prevented
by two well known G-binding proteins, phosducin
and GST-ARK-(466-689) fusion protein, suggesting that the
G-binding domain (possibly the pleckstrin homology
domain) of the GRKs is also a site for lipid:protein interaction. We
submit the intriguing possibility that both lipids and G proteins
co-regulate the function of GRKs.
)represent a
superfamily of plasma membrane receptors that recognize an eclectic
variety of environmental stimuli and transduce their signals to the
internal milieu of cells(1) . GPR-activated cellular signaling
rapidly wanes due to regulatory processes collectively known as
desensitization. An important component to desensitization, especially
underlying its rapid phase, is an uncoupling of the GPR from its G
protein by phosphorylation. Two classes of protein kinases mediate this
phosphorylation: second messenger-dependent kinases (e.g. PKC
and PKA) mediate agonist-independent phosphorylation of GPRs and
initiate heterologous desensitization, whereas a unique class of
serine-threonine protein kinases, namely G protein-coupled receptor
kinases (GRKs), mediate agonist-dependent phosphorylation of GPRs and
initiate homologous
desensitization(2, 3, 4) .ARK1 and
ARK2) are not modified by isoprenylation. Instead, in vitro studies suggest that membrane-associated subunits of
heterotrimeric G proteins (G) aid both in the
membrane targeting and activation of
ARKs(15, 16, 17, 18) .
G binds ARK1 and ARK2 in
vitro(16) , and the site of this interaction has been
mapped to a region partly within the pleckstrin homology (PH) domain in
the carboxyl terminus of ARK1 and ARK2(18) . Recent
studies in which the solution structure of the PH domain of pleckstrin
was solved suggested that PH domains may contain lipid-binding
domains(19) . Subsequently, Harlan et al.(20) reported that phosphatidylinositol-4,5-bisphosphate
(PIP
) directly binds to the PH domains of several proteins,
including ARK1 (GRK2); however, the functional consequence of
lipid binding was not assessed.ARK1
and ARK2 activity, by measuring their ability to mediate
agonist-dependent phosphorylation of human m2 (hm2) muscarinic
acetylcholine receptors (mAChRs) in vitro. We report here
novel results demonstrating that these GRKs bound to and were dually
regulated either in a positive or negative manner by charged
phospholipids. Lipid-mediated regulation of ARK1 and 2 activity
did not appear to occur through a mechanism that involves
autophosphorylation, in contrast to what was recently reported for
GRK5(14) . Furthermore, analogous to G,
the lipids appeared to interact with ARKs via the
carboxyl-terminal region which contains the
G-binding domain and PH domain.
Materials
Spodoptera frugiperda (Sf9)
insect cells were obtained from Invitrogen. Sf-900 II insect cell media
and gentamicin were purchased from Life Technologies, Inc. Recombinant
baculoviruses encoding the human m2 muscarinic acetylcholine receptors
were kindly provided by Drs. Elliot Ross and E. M. Parker, University
of Texas (Dallas). G purified from bovine brain
was a generous gift from Dr. Pat Casey, Duke University (Durham, NC).
Phosducin purified from bovine retina was kindly provided by Dr. Y. K.
Ho, University of Illinois (Chicago).
[-
P]ATP and
1-quinuclidinyl[phenyl-4-
H]benzilate
were purchased from Amersham Corp. Lipids were either purchased from
Sigma or Avanti Polar Lipids.Cell Culture
Sf9 cells were cultured as described (21) , either in monolayers or in suspension using Sf-900 II
serum-free media supplemented with gentamicin (50 µg/ml).Purification and Reconstitution of hm2 mAChRs
Sf9
cells (at a density of 2 
10
cells/ml for suspension
culture) were infected with recombinant baculovirus expressing the hm2
mAChRs at a multiplicity of infection of 5. At 65-72 h
post-infection, cells were harvested and membranes prepared essentially
as described in Richardson et al.(16) . The hm2 mAChRs
were purified from Sf9 cell membranes and reconstituted into chick
heart lipids (16) . The specific activity of the purified
receptors was determined by
[H]1-quinuclidinyl[phenyl-4-[H]benzilate
ligand binding assays(22) .Phosphorylation of the Purified and Reconstituted hm2
mAChRs
The GRKs, ARK1, and ARK2 were expressed in Sf9
cells using recombinant baculovirus encoding the various GRKs and
purified (>95%) as described previously(23) . The
GST-ARK-(466-689) fusion protein was prepared as
reported(18) . All phosphorylation reactions were carried out
with purified and reconstituted hm2 mAChRs (0.2-0.4 pmol) and
ARKs (30 nM) in 20 mM Tris-HCl, pH 7.4, 5 mM MgCl, 2 mM EDTA, in the presence of 50
µM [-
-P]ATP (specific activity
of 1000 cpm/pmol), for 1 h at 37 °C in a total reaction volume of
100 µl. Reactions were terminated with SDS sample buffer, and
samples were resolved on 8% polyacrylamide gels(24) .
Phosphorylated receptors were visualized and the amount of incorporated P quantified by phosphorimaging with a Bas2000
Fuji-bioimager. The extent of phosphorylation of the receptors by
ARK1 observed in the presence of carbachol, and the absence of
added lipid, was taken as 100% (
4-5 mol of phosphate/mol of
receptor). Purified lipids were lyophilized and sonicated and used at
the concentrations indicated. In reactions that contained lipids,
lipids were preincubated with the receptors and ARKs for
5-10 min at room temperature before reactions were started with
[--P]ATP.Peptide Mapping of Phosphorylated hm2
mAChRs
Phosphorylated receptors were incubated in 0.05%
digitonin and centrifuged in a Centricon device to remove excess
[-P]ATP. Subsequently, the receptors were
electrophoresed on an 8% polyacrylamide gel and then electroblotted
onto an Immobilon-CD filter. The phosphorylated receptors were
visualized by phosphorimaging, and the corresponding band was excised
and minced into small pieces. The receptors were digested with Staphylococcus aureus V8 protease (50 µg/ml) at 37 °C
for 16 h in buffer containing 50 mM NH
CO, 7 mM EDTA, pH 8.0. The
filters were washed and phosphopeptides were eluted by adding 50 µl
of 4 M guanidine HCl, 0.1% Triton X-100 and sonicating for 10
min(25) . This process was repeated twice and resulted in the
recovery of 60-80% of applied phosphoprotein. The peptides were
lyophilized, resuspended in 125 mM Tris-HCl, pH 6.8, and
electrophoresed on 25% acrylamide gels prepared as in (26) .
The gels were dried, and peptide maps were analyzed by phosphorimaging.
Lipids
of desired composition were mixed and dried together under high purity
NARK Binding to Phospholipid Vesicles and then redissolved in phosphate-buffered saline buffer.
The lipid mixtures (25 mg/ml) were sonicated in short bursts for
1-2 min, chilled on ice for 1 min, and re-sonicated. Lipid
vesicles (concentrations as shown in the legend to Fig. 1, C and D) were added to purified ARK1 (100 ng) in a
total volume of 50 µl in a Beckman TLA-100 centrifuge tube and
incubated for 5-15 min (or until equilibrium was attained) at
room temperature. The tubes were spun at 100,000 g for
30 min. The vesicles pelleted with this treatment effectively separated
lipid-bound ARK1 from the unbound ARK in the supernatant. The
ARK1 content in both the pellet and supernatant fractions was
determined using Western blot analysis using an anti-ARK
polyclonal antibody. Visualization was performed using the Renaissance
Chemiluminescence Kit and Reflection Autoradiography film (DuPont NEN).
NIH Image 1.52 software was used to quantitate the films.
ARK1. The figure
is a graphic representation of phosphorylation of purified
reconstituted hm2 mAChRs by ARK1, in the presence of muscarinic
agonist carbachol (1 mM), with (open bar) or without (closed bar; control at 100%) the addition of various lipids.
The results are means from two to five experiments with similar
results. The maximal effects of lipids (observed with 50 µg for all
lipids, except for phosphatidic acid, whose effects were maximal with
0.5-5 µg), whether stimulatory or inhibitory, are shown. B, concentration-dependent stimulation by phosphatidylserine
and inhibition by PIP of ARK1 activity. The graph
shows the percent change in receptor phosphorylation by ARK1 with
increasing amounts of PS or PIP (1.0 ng to 100
µg/100-µl reaction), when compared with receptor
phosphorylation in the absence of lipid (100%). Experiments were
performed two to three times with similar results. The insets are representative phosphorimages. Concentration-dependent effects
of the other lipids were also assessed (data not shown). C and D, direct binding of ARK1 to phospholipids.
Centrifugation assays to study ARK1 binding to phospholipids were
performed as described under ``Experimental Procedures.''
Binding reactions were performed with ARK1 (100 ng) and
phospholipid vesicles that were made of varying concentrations of
either PC alone (C, D), PC and PS (C), or PC and
PIP (D). The amount of ARK1 that bound to
lipid vesicles was assessed by quantifying ARK1 immunoreactivity
in pellet fractions and is expressed as percent of the total ARK1
in the supernatant and pellet combined. In control experiments with PC
vesicles, ARK immunoreactivity was negligible at 0.05% (500
µM PC; Fig. 1C), and 0.0-0.1%
(180-900 µM PC; Fig. 1D).
ARK1 to phosphorylate agonist-activated hm2 mAChRs (Fig. 1A), which are known substrates for ARK1 and
ARK2(15, 16, 27) . Of the lipids tested,
phosphatidic acid, phosphatidylinositol (PI), phosphatidylserine (PS),
phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) enhanced
ARK1-mediated receptor phosphorylation by 2-3-fold, compared
with reactions in the absence of added lipid (control; 100%).
Phosphatidylcholine (PC) had no significant effect on ARK1
activity. In direct contrast, a key phospholipid, PIP,
markedly inhibited receptor phosphorylation by ARK1 by as much as
90%. No effects were observed with three other classes of membrane
lipids: cholesterol, sphingolipids (sphingosine and sphingomyelin), and
glycolipids (cerebroside). The effects of PS and PIP were
further characterized and shown to occur in a concentration-dependent
manner (Fig. 1B). Parallel experiments with ARK2
and lipids produced similar results (data not shown). These results are
the first demonstration that agonist-dependent phosphorylation of a G
protein-coupled receptor by GRKs may be governed by membrane lipids. ARK activity by lipids
was a consequence of direct interaction between lipid and kinase, we
employed a phospholipid binding assay utilizing ultracentrifugation,
similar to that developed by Harlan et al.(20) , and
measured the appearance of ARK immunoreactivity in pellets
consisting of various mixtures of phospholipids. Phospholipid vesicles
consisting of 500 µM PC alone bound ARK1 poorly
(<1% of ARK1 bound to these vesicles) and were used as controls (Fig. 1, C and D). In studies with PS, 37% of
the total added ARK1 was detected in pellets that contained
phospholipid vesicles of 60 µM PS and 500 µM PC (Fig. 1C). ARK1 immunoreactivity in these
vesicles increased to 55% when the PS content in PC/PS vesicles was
increased to 100 µM (Fig. 1C). Similarly,
in studies with PC and PIP we observed specific binding of
ARK1 to PIP (Fig. 1D). Increased
PIP content in PC/PIP vesicles resulted in
higher amounts of ARK1 immunoreactivity in pellet fractions (Fig. 1D). These results demonstrated that lipids which
regulated ARK activity toward the hm2 mAChRs (PS and
PIP) specifically bound ARK1; in contrast, PC, which
did not regulate ARK-mediated phosphorylation of hm2 mAChRs, bound
ARK1 minimally. Although the exact mechanism by which lipids
regulate ARKs is unknown, our data strongly suggest that lipids
directly interact with ARKs to modulate their function. interacted with GRKs at independent
or similar sites. When both PIP or PS were added, PS
effectively alleviated the PIP-mediated inhibition of
ARK1 phosphorylation of agonist-occupied hm2 receptor in a
concentration-dependent manner (Fig. 2). Similarly, PIP inhibited PS stimulation of ARK1 in a
concentration-dependent fashion (Fig. 2). These results suggest
that PS and PIP regulate ARK1 in a competitive manner
and, despite their opposing actions on the kinase, might share common
sites of interaction with ARK1.
with ARK1. The figure depicts agonist-dependent
phosphorylation of the hm2 mAChRs by ARK1 either in the presence
of different amounts of PS and PIP or control reactions in
the absence of either lipid (100%; solid bar). PIP strikingly inhibited PS stimulation of BARK1 to levels of
receptor phosphorylation observed in presence of atropine
(10-20%). PS effectively alleviate PIP inhibition of
ARK1 to 80% of control. The inset is a representative
phosphorimage. Reactions were performed twice with similar
results.
ARK1. These results resembled the effects of G to increase both the rate and extent of ARK1-mediated
phosphorylation of hm2 mAChRs ( (15) and (16) and Fig. 3A), suggesting that PS might activate ARK1
in a manner analogous to G. To address this
possibility, we first asked if the stimulatory effects of PS and
G were additive. When PS and G were tested together, their effects on receptor phosphorylation
by ARK1 were additive during the initial stages of the reactions (Fig. 3B), but nonadditive at 60 min (Fig. 3C). Moreover, addition of G alleviated PIP inhibition of ARK1 in a
concentration-dependent manner (data not shown). To further assess
whether PS and G activated ARKs in a similar
fashion, peptide maps of ARK1-phosphorylated hm2 mAChRs
(phosphorylated either in the presence of either PS or G or both) were compared. The results revealed that both PS and
G enhanced the phosphate content of a set of
similar peptides in a non-additive manner, when compared with peptides
obtained in the absence of either lipid or G (carbachol control, Fig. 3D). Collectively, these
results suggest that lipids and G either share
common sites of interaction with ARK or that their sites are
proximally located and binding of one allosterically modulates the
binding of the other.
subunit stimulation of ARK1. A compares the time course
(0-90 min) of agonistdependent phosphorylation of hm2 mAChRs by
ARK1 in the presence of either PS (50 µg) or G (subunits) purified from bovine brain (100 nM), with
control reactions carried out in the absence of added lipid and
G (carbachol). B shows that the PS and
G effects on receptor phosphorylation by ARK1
were additive within the first 2 min of the reaction, while C shows that PS and G were nonadditive at 60
min. The data shown are means from two to three independent experiments
with similar results. D compares phosphopeptide maps obtained
from hm2 mAChRs that were phosphorylated with ARK1 and
subsequently proteolyzed with S. aureus V8 protease. Reactions
were carried out in the presence of either atropine (1 mM) (lane 1) or carbachol (1 mM) (lanes
2-5) and contained PS (lane 3), G (lane 4), or both (lane
5).
is known to bind
ARK1 in vitro and the G-binding
domain is localized within the carboxyl terminus of
ARK1-(467-689)(17, 18) . A glutathione S-transferase (GST)-ARK1-(466-689) fusion protein,
which is known to bind G and thus prevent its
ability to stimulate
ARK(17, 18, 28, 29) ,
strikingly reduced the ability of PS to stimulate ARK1-mediated
phosphorylation of the hm2 mAChRs by 80% (Fig. 4).
GST-ARK(466-689) contains the entire PH domain of ARK1,
which has been shown to directly bind PIP(20) . The
present result suggested to us that the GST-ARK fusion protein
served as a sink for lipids, and therefore bound PS, and prevented its
ability to stimulate receptor phosphorylation by ARK1. In support
of this hypothesis, the effects of the GST-ARK fusion protein were
competitively eliminated in a dose-dependent fashion when increasing
amounts of PS were added to the phosphorylation reaction (Fig. 4B).
ARK by
GST-ARK-(466-689) fusion protein and phosducin.
Agonist-dependent phosphorylation of the hm2 mAChRs by ARK1 was
carried out in the presence of added lipid (50 µg of either PS or
PIP), with or without GST-ARK-(466-689) fusion
protein (7 µM) or phosducin purified from bovine retina (3
µM). Receptor phosphorylation by ARK1 in the absence
of added lipid was taken as 100% (solid bar).
GST-ARK-(466-689) fusion protein and phosducin inhibited PS
stimulation of ARK1 by 80 and 75%, respectively; furthermore,
phosducin alleviated PIP inhibition of ARK1 to 90% of
control. The data shown are means from two to three independent
experiments. B, concentration-dependent elimination of the
effects of the GST-ARK fusion protein by PS. Increasing amounts of
PS (50-250 µg) were added to phosphorylation reactions that
contained purified reconstituted hm2 mAChRs (0.3 pmol), ARK1, PS
(25 µg), carbachol, and GST-ARK (7 µM). Control
reactions (solid bar) did not contain either GST-ARK or
PS and receptor phosphorylation observed under these conditions were
taken as 100%.
ARK fusion protein to prevent the effect of lipids on ARK
occurred as a result of lipid binding to the PH domain or
G domain or both. To further determine whether
other proteins with G-binding domains could modify
lipid regulation of ARK1, the ability of retinal phosducin, a
G-binding protein(30, 31) , to
prevent PS stimulation of ARK1 was assessed. Here, in studies with
mAChR and ARK1, phosducin markedly inhibited PS stimulation of
ARK1 by 75% (Fig. 4); the effects of phosducin were
concentration-dependent (data not shown). Furthermore, phosducin also
alleviated PIP inhibition of ARK1 (Fig. 4).
These results support the concept that G and
lipids interact with ARK1 in a common region. The results also
suggest that phosducin contains lipid-binding sites that allow it to
compete with ARK1 for both PIP and PS. The results
observed with phosducin are interesting. Hekman et al.(32) recently demonstrated that phosducin inhibits
phosphorylation of purified and reconstituted ARs by
ARK1 only in the presence of G proteins (or
G); in the absence of G proteins, phosducin had no
effect on ARK activity. It is thought that phosducin has no direct
interaction with ARKs, but rather it regulates the ability of
ARKs to bind G(32) . The results we
observed with phosducin and lipids were strikingly similar and suggest
to us that the effect of phosducin was to regulate the ability of
ARKs to interact with lipids. subunits aid the targeting of
ARKs to their membrane-bound substrates is both attractive and
well supported(2, 17, 18) . Based on our
observations, we propose that both G proteins and lipids participate in
the translocation and activation of ARKs. A critical question
regarding GRK function has been whether GRKs are constitutively active
or are normally inhibited or activated by other unidentified regulatory
molecules in the cell. The observation that different lipids may either
enhance or suppress kinase activity is revealing, since it provides a
candidate molecule (PIP) that may directly inhibit GRKs and
others that may stimulate GRKs. Furthermore, the apparent specificity
of charged phospholipids to produce these effects complements the fact
that the intracellular face of the plasma membrane is enriched with
such lipids. Binding of GRKs to G and/or charged
lipids in vivo might direct these enzymes to the plasma
membrane, where they may phosphorylate agonist-activated GPRs and
initiate receptor desensitization. In support of this speculation,
recent reports have suggested that a small, but significant, population
of ARK is normally localized to the plasma membrane in cells and
that other pools of ARKs also exist in intracellular
membranes(33, 34) . However, the relative
contributions of G and lipids in regulation of
ARKs need to be dissected. It also remains unclear why some lipids
stimulate ARKs, whereas other lipids inhibit their activity.
Furthermore, we cannot preclude the possibility of a supplementary
effect of lipids in directly modulating the reconstituted receptors.
Further studies will address these questions and elucidate the exact
domains in GRKs involved in their regulation by lipids.ARK1 and ARK2
that is quite distinct from a previous finding concerning effects of
lipids on another member of the GRK family. Kunapuli et al.(34) demonstrated that lipids increased
autophosphorylation of GRK5 and suggested a role for
autophosphorylation in regulating GRK5 by demonstrating that an
autophosphorylation-deficient mutant had decreased GRK5 activity. It is
to be noted however that the previous study did not demonstrate that
the lipid-mediated increase in autophosphorylation had a functional
effect on the ability of GRK5 to phosphorylate receptor substrates. The
lipid effects on ARK reported here occur via a mechanism that does
not involve autophosphorylation. In contrast to what may be the case
for GRK5, autophosphorylation appears to be unimportant, or less
important, in regulating ARK1 and ARK2. In the present study,
we observed autophosphorylation of both ARKs, but at very low
substoichiometric levels. In studies with ARKs and mAChR, the
stoichiometry of ARK1 autophosphorylation was 0.03 mol of
phosphate/mol of ARK or less; this increased to no more than 0.1
mol phosphate/mol of protein in the presence of PS and decreased with
PIP. Thus, these substoichiometric levels of
autophosphorylation do not support a role for autophosphorylation of
ARK in the lipid-mediated regulation of activity. A second
important difference that should be noted in terms of the regulation of
the ARK isozymes and other members of the GRK family, including
GRK5, is that the other GRKs (rhodopsin kinase, GRK4, GRK5, and GRK6)
lack a G-binding domain and do not possess PH
domains.-binding domain of ARK1 and
ARK2 is partially contained within a PH
domain(28, 35, 36) . PH domains have only
recently been recognized as sites for interaction between proteins (28, 35, 36) and have been subsequently found
in an increasing number of diverse
molecules(35, 37, 38, 39, 40, 41, 42) ;
however, their function(s) remain uncertain. In one proposal, proteins
with PH domains have recently been suggested to be either effectors of
G or molecules similar to
G(28). An alternate suggestion has been made that
PH domains are interaction sites for phosphate groups(42) .
However, neither phosphoserine nor phosphothreonine appeared to
regulate the ability of ARK1 to phosphorylate the hm2 mAChRs (data
not shown). In contrast, our results indicate that, at least in
ARK1 and ARK2, the G-binding domain
and/or PH domain can also interact with phospholipids. has been shown recently to directly bind the PH
domain in ARK1 and various other PH domain-containing proteins (20) . It is proposed that the NH terminus of PH
domains contain the lipid-binding motif, and this has been specifically
demonstrated in the PH domain in pleckstrin(20) . In ARK1
and ARK2, the G-binding domain overlaps with
the COOH-terminal end of the PH domain. Therefore, it is possible that
the lipid binding (presumably NH-terminal) and
G-binding regions are separate modules within the
PH domain in ARK. Yet, the close proximity of these sites in
ARK may allow for allosteric regulation between lipids and
G in their interactions with ARK. Our data
provide initial evidence to support this hypothesis.
)AR, -adrenergic receptor; ARK,
-adrenergic receptor kinase; G,
subunits of G proteins; G protein, guanine nucleotide-binding protein;
GRK, G protein-coupled receptor kinase; GST, glutathione S-transferase; mAChR, muscarinic acetylcholine receptors; PC,
phosphatidylcholine; PG, phosphatidylglycerol; PS, phosphatidylserine;
PIP, phosphatidylinositol 4,5-bisphosphate; PE,
phosphatidylethanolamine; PI, phosphatidylinositol; PH, pleckstrin
homology.
We thank Roger Bodine for excellent technical
assistance and Robin Rylaarsdam for helpful discussions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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R. Sterne-Marr, J. J. G. Tesmer, P. W. Day, R. P. Stracquatanio, J.-A. E. Cilente, K. E. O'Connor, A. N. Pronin, J. L. Benovic, and P. B. Wedegaertner G Protein-coupled Receptor Kinase 2/Galpha q/11 Interaction. A NOVEL SURFACE ON A REGULATOR OF G PROTEIN SIGNALING HOMOLOGY DOMAIN FOR BINDING Galpha SUBUNITS J. Biol. Chem., February 14, 2003; 278(8): 6050 - 6058. [Abstract] [Full Text] [PDF]
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 S. Chakrabarti, M. Oppermann, and A. R. Gintzler Chronic morphine induces the concomitant phosphorylation and altered association of multiple signaling proteins: A novel mechanism for modulating cell signaling PNAS, March 27, 2001; 98(7): 4209 - 4214. [Abstract] [Full Text] [PDF]
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C. V. Carman, L. S. Barak, C. Chen, L.-Y. Liu-Chen, J. J. Onorato, S. P. Kennedy, M. G. Caron, and J. L. Benovic Mutational Analysis of Gbeta gamma and Phospholipid Interaction with G Protein-coupled Receptor Kinase 2 J. Biol. Chem., March 31, 2000; 275(14): 10443 - 10452. [Abstract] [Full Text] [PDF]
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G. S. Kroog, X. Jian, L. Chen, J. K. Northup, and J. F. Battey Phosphorylation Uncouples the Gastrin-releasing Peptide Receptor from Gq J. Biol. Chem., December 17, 1999; 274(51): 36700 - 36706. [Abstract] [Full Text] [PDF]
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A. N. Pronin, C. V. Carman, and J. L. Benovic Structure-Function Analysis of G Protein-coupled Receptor Kinase-5. ROLE OF THE CARBOXYL TERMINUS IN KINASE REGULATION J. Biol. Chem., November 20, 1998; 273(47): 31510 - 31518. [Abstract] [Full Text] [PDF]
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C. V. Carman, T. Som, C. M. Kim, and J. L. Benovic Binding and Phosphorylation of Tubulin by G Protein-coupled Receptor Kinases J. Biol. Chem., August 7, 1998; 273(32): 20308 - 20316. [Abstract] [Full Text] [PDF]
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G. Wu, J. L. Benovic, J. D. Hildebrandt, and S. M. Lanier Receptor Docking Sites for G-protein beta gamma Subunits. IMPLICATIONS FOR SIGNAL REGULATION J. Biol. Chem., March 27, 1998; 273(13): 7197 - 7200. [Abstract] [Full Text] [PDF]
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J. L. Sui, J. Petit-Jacques, and D. E. Logothetis Activation of the atrial KACh channel by the beta gamma subunits of G proteins or intracellular Na+ ions depends on the presence of phosphatidylinositol phosphates PNAS, February 3, 1998; 95(3): 1307 - 1312. [Abstract] [Full Text] [PDF]
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