(Received for publication, June 12, 1995)
From the
The -adrenergic receptor kinase (
ARK) is a member of
growing family of G protein coupled receptor kinases (GRKs).
ARK
and other members of the GRK family play a role in the mechanism of
agonist-specific desensitization by virtue of their ability to
phosphorylate G protein-coupled receptors in an agonist-dependent
manner.
ARK activation is known to occur following the interaction
of the kinase with the agonist-occupied form of the receptor substrate
and heterotrimeric G protein
subunits. Recently, lipid
regulation of GRK2, GRK3, and GRK5 have also been described. Using a
mixed micelle assay, GRK2 (
ARK1) was found to require phospholipid
in order to phosphorylate the
-adrenergic receptor. As
determined with a nonreceptor peptide substrate of
ARK, catalytic
activity of the kinase increased in the presence of phospholipid
without a change in the K
for the
peptide. Data obtained with the heterobifunctional cross-linking agent N-3[
I]iodo-4-azidophenylpropionamido-S-(2-thiopyridyl)cysteine
([
I]ACTP) suggests that the activation by
phospholipid was associated with a conformational change in the kinase.
[
I]ACTP incorporation increased 2-fold in the
presence of crude phosphatidylcholine, and this increase in
[
I]ACTP labeling is completely blocked by the
addition of MgATP. Furthermore, proteolytic mapping was consistent with
the modification of a distinct site when GRK2 was labeled in the
presence of phospholipid. While an acidic phospholipid specificity was
demonstrated using the mixed micelle phosphorylation assay, a notable
exception was observed with PIP
. In the presence of
PIP
, kinase activity as well as
[
I]ACTP labeling was inhibited. These data
demonstrate the direct regulation of GRK2 activity by phospholipids and
supports the hypothesis that this effect is the result of a
conformational change within the kinase.
The molecular mechanisms involved in signal transduction of G
protein-coupled receptors are best understood in the visual system
where rhodopsin serves as the ``receptor'' for light (1) and the -adrenergic pathway in which the
-adrenergic receptor (
AR) (
)binds
catecholamines(2, 3) . A feature common to both model
systems as well as many other G protein receptors is the diminished
responsiveness with time to a signal of equal intensity. This
phenomenon is known as desensitization (4) and exhibits both an
agonist-specific and nonspecific pattern. Rapid, agonist-specific
desensitization of rhodopsin and the
-adrenergic
receptor (
AR) occurs in response to the
phosphorylation of the receptor by the enzymes rhodopsin kinase and the
-adrenergic receptor kinase (
ARK)(5) . Rhodopsin
kinase and
ARK are members of a family known as G protein-coupled
receptor kinases (GRKs). A common feature to the GRK family of kinases
is multi-site phosphorylation of receptor substrates in response to
agonist occupancy(6) . The relationship between agonist
occupancy and receptor phosphorylation by GRKs is key to the
specificity of the desensitization process, while other kinases such as
protein kinase A and C play a role in nonspecific or heterologous
desensitization. Two possible mechanisms could explain the enhanced
phosphorylation of the activated form of the receptor by kinases of the
GRK family. First, receptor occupancy may induce a conformational
change exposing potential phosphorylation sites previously sequestered
from the kinase. Alternatively, interaction of the kinase with the
agonist-bound form of the receptor could result in enhanced catalytic
activity of the kinase. The bulk of the experimental evidence supports
the latter hypothesis(7, 8, 9) . In addition
to the enhanced catalytic activity of GRKs in the presence of
agonist-occupied receptor, GRK2 and GRK3 activity is also increased by
heterotrimeric G protein
subunits(10, 11, 12, 13) . The
potential for finely controlled desensitization by the interplay of
receptors and
subunits is an exciting possibility given the
evidence for dual regulation of GRK2 and GRK3 by these
proteins(14) .
While G protein-coupled receptors serve as
substrates for the kinase after reconstitution into phospholipid
vesicles, only recently has specific lipid requirements for GRKs been
described. GRK5 was reported to require phospholipid for maximal
catalytic activity(15) . In this case, phospholipid-stimulated
autophosphorylation of GRK5 was necessary for phosphorylation of the
AR and rhodopsin. In addition, GRK2 and GRK3 were
regulated by phospholipids via the interaction with the
carboxyl-terminal portion of the kinase known as the pleckstrin
homology domain(16, 17) . In the initial report, the
incorporation of negatively charged lipids into phospholipid vesicles
resulted in a physical interaction of GRK2 or GRK3 with the vesicle.
With the exception of PIP
, this resulted in enhance
phosphorylation of the human m2 muscarinic acetylcholine receptor. The
addition of PIP
resulted in inhibition of phosphorylation
of the receptor in a competitive manner with respect to other
phospholipids. Purified heterotrimeric G protein
subunits
were able to reverse this inhibition. Furthermore, the lack of
additivity suggested a common site of interaction on the kinase for the
lipids and G protein
subunits. This hypothesis was further
supported by the finding that two previously characterized G protein
subunit binding proteins, phosducin and glutathione S-transferase-
ARK(466-689) fusion protein,
prevented the effects of the phospholipids. In a subsequent report,
similar effects in terms of PIP
-enhanced binding of GRK2 to
phospholipid vesicles was described. In contrast to the previous
manuscript, data are presented that demonstrate increased GRK2 activity
when coincubated with both PIP
and G protein
subunits. Additionally, the remaining lipids previously reported to
increase kinase activity in the absence of
subunits in this
case required the addition of G protein
subunits to enhance
GRK2 activity. The interpretation by these authors was that effective
membrane localization of
ARK, which enhanced both the rate and
extent of phosphorylation of receptor substrates, required the
simultaneous presence of two pleckstrin homology domain ligands.
In
this manuscript, we provide evidence of an acidic phospholipid
requirement of GRK2 based on the phosphorylation of dodecyl
maltoside-solubilized receptors and the direct activation of the kinase
toward peptide substrates by the addition of various phospholipids.
Additionally, lipids that failed to enhance kinase activity did not
increase labeling of GRK2 with the the heterobifunctional reagent
[I]ACTP. Finally, data obtained in the absence
of G protein
subunits agree with the original report in
which PIP
promotes kinase binding to phospholipid vesicles
but inhibits enzymatic activity(16) . Thus, we provide evidence
for catalytic activation as well as a conformational change in GRK2
following the interaction of the kinase and phospholipid. These data
raise the possibility of a third level of regulation of GRK2 activity
within the cell and suggest that the mechanism of phospholipid is more
complex than simply targeting of the kinase to the plasma membrane.
For reconstitution studies, the purified receptor was reinserted
into phosphatidylcholine vesicles, pelleted by centrifugation, and
resuspended in 20 mM Tris-HCl, pH 7.4, 2 mM EDTA as
described previously(22) . The concentration of receptor was
determined using the -adrenergic receptor antagonist
[
I]iodocyanopindolol.
For studies in mixed
detergent-lipid micelles, the purified receptor was diluted in 1 mM dodecyl maltoside, 100 mM NaCl, 10 mM Tris-HCl,
pH 7.4, 5 mM EDTA and concentrated on a YM-100 membrane using
a centricon device (Amicon). Alternatively, receptor purified in
digitonin underwent detergent exchange on a G-50 column equilibrated in
0.5 mM dodecyl maltoside, 100 mM NaCl, 10 mM Tris-HCl, pH 7.4, 5 mM EDTA and concentrated on a YM-100
membrane using a centricon device. Crude phosphatidylcholine vesicles
were produced using a tip sonicator with three 1-min bursts on ice. The
desired amount of AR was added to various amounts of
phospholipid in 20 mM Tris-HCl, pH 7.4, 2 mM EDTA.
Other phospholipids, stored as stock solutions in chloroform, were
first dried under a stream of N
. The desired amount of
phospholipid was mixed with dodecyl maltoside-solubilized
AR, from which alprenolol had been removed as
described above, prior to use in the phosphorylation assay.
Phosphorylation
of dodecyl maltoside-solubilized AR was performed in
the presence or absence of crude phosphatidylcholine or various
purified phospholipids in a buffer of 20 mM Tris-HCl, pH 7.4,
2 mM EDTA, 7.5 mM MgCl
, 0.1 mM [
-
P]ATP (200-1,000 cpm/pmol).
The final volume was 50 µl, and the phosphorylation reaction was
carried out at 30 °C for various times as indicated. The reaction
was stopped, and the phosphate incorporation was determined as detailed
above.
A unique feature of the GRK family is the ability to
phosphorylate the agonist-occupied form of a variety of G
protein-coupled receptors. In order for a receptor to serve as a
substrate for ARK, the protein is typically purified and
reinserted into phospholipid vesicles. If phosphorylation of the
receptor in detergent is attempted, no significant incorporation of
phosphate is observed. Based on binding data, the receptor exhibits
appropriate binding properties and is not degraded. While the
possibility exists that detergents inhibit the kinase or reconstitution
into bulk lipid provides a conformational structure required for
interaction with GRKs, only recently have specific lipid requirement
for GRKs been described(15, 16, 17) . Fig. 1demonstrates the phosphorylation of the
AR in dodecyl maltoside by GRK2. The phosphorylation
of the receptor requires the addition of crude phosphatidylcholine and
is stimulated by the addition of
-adrenergic agonist. The
stoichiometry of phosphorylation is maximal at 4-5 mol of
phosphate/mol of receptor. In data not shown, the effect of
isoproterenol is blocked by the addition of the
-adrenergic
receptor antagonist alprenolol. Also, there is a linear dependence
between the amount of
AR added to the reaction mixture
and the phosphate incorporation observed after resolving the receptor
by SDS-PAGE with the maximal stoichiometry remaining
4 mol
phosphate/mol receptor. Finally, the phosphorylated receptor is not
pelleted by a 300,000
g centrifugation step in
contrast to reconstituted receptor.
Figure 1:
Phosphorylation of
detergent-solubilized -adrenergic receptor by GRK2.
AR is expressed in Sf9 cells, solubilized in
dodecyl maltoside, and purified by affinity chromatography using an
alprenolol-Sepharose column. Alprenolol is removed by size-exclusion
chromatography on a G-50 column in 100 mM NaCl, 10 mM Tris-HCl, pH 7.4, and 1 mM dodecyl maltoside. The
receptor is then concentrated using a Centricon-100 ultrafiltration
device prior to phosphorylation. Receptor phosphorylation is carried
out as described in the text, and the reaction is quenched by the
addition of SDS sample buffer. The receptor is resolved by 9%
SDS-polyacrylamide gel electrophoresis followed by autoradiography. The
phosphorylation reaction is performed in the presence (lanes1 and 2) or absence (lane3)
of crude phosphatidylcholine (50 µg). Isoproterenol (50
µM) is added (lanes2 and 3) to
demonstrate the agonist dependent nature of receptor phosphorylation by
GRK2. The stoichiometry of phosphorylation is determined by excising
the receptor band, quantitating the
P and expressing the
data as mol of phosphate/mol of
AR.
In order to define the
phospholipid specificity of the GRK2 phosphorylation reaction,
solubilized AR is added to a variety of neutral,
acidic, and basic phospholipids. As shown in Fig. 2, only lipids
with a net negative charge including cardiolipin, phosphatidylglycerol,
phosphatidic acid, phosphatidylserine, and phosphatidylinositol support
the phosphorylation of the
AR by GRK2. The addition of
crude, but not purified, phosphatidylcholine results in receptor
phosphorylation. This suggests that a phospholipid other than
phosphatidylcholine is responsible for the activation of GRK2 observed
above. Fig. 3compares the effects of phosphatidylinositol to
those of PIP
. While phosphatidylinositol enhanced receptor
phosphorylation by GRK2, there is no significant
AR
phosphorylation in mixed micelles containing PIP
.
Figure 2:
Phospholipid specificity of
AR phosphorylation by
ARK. 2.5 µl of
a phospholipid (10 mg/ml stock in chloroform) is taken to dryness with
a stream of nitrogen gas and rehydrated with 10 µl of 20 mM Tris-HCl, pH 7.2, 2 mM EDTA, and 1 mM dodecyl
maltoside at 30 °C. Mixed micelles are formed by the addition of
Alprenolol-free
AR. The final volume of the
phosphorylation reaction is 50 µl and contained 2.8 pmol of
receptor. The phosphorylation reaction is begun by the addition of
kinase and ATP at 30 °C and stopped after 60 min with 10 µl of
the SDS-PAGE stop solution. 20-µl aliquots were loaded on a 12%
polyacrylamide gel, and the phosphorylated receptor was localized by
autoradiography. The samples are as follows: lane 1, no lipid
(incubation performed in the absence of 10 µM isoproterenol); lane 2, no lipid; lane 3, crude
phosphatidylcholine (incubation performed in the absence of 10
µM isoproterenol); lane 4, crude
phosphatidylcholine; lane 5, cardiolipin; lane 6,
lysophosphatidylcholine; lane 7, palmitic acid; lane
8, phosphatidyl-DL-glycerol; lane 9, purified
phosphatidylcholine; lane 10, phosphatidylethanolamine; lane 11, sphingomyelin; lane 12, phosphatidic acid; lane 13, phosphatidyl-L-serine; lane 14,
phosphatidylinositol.
Figure 3:
Effect of PIP on GRK2
phosphorylation of the
AR.2.5
µl of phosphatidylinositol, PIP
(10 mg/ml stock in
chloroform), or chloroform are taken to dryness with a stream of
nitrogen gas and rehydrated with 10 µl of 20 mM Tris-HCl,
pH 7.2, 2 mM EDTA, and 1 mM dodecyl maltoside at 30
°C.
AR phosphorylation by GRK2 in mixed micelles
is performed as described above in the presence or absence of 10
µM isoproterenol.
To
further investigate the effect of phospholipid on GRK2 activity, a
nonreceptor peptide substrate (RRREEEEESAAA) previously shown to serve
as a ARK1 substrate is used(25) . The time course of
phosphorylation of the peptide by GRK2 is linear for 2 h in the absence
or presence of phospholipid (Fig. 4). However, there is a
substantial increase in phosphate incorporation observed with the
addition of crude phosphatidylcholine to the reaction mixture. The
effect of phosphatidylcholine is not due to protection of the kinase
from degradation or other nonspecific effects as Western blotting
reveals equal amounts of the 80,000 M
kinase band
without evidence of proteolytic cleavage (data not shown).
Figure 4:
Time course of synthetic peptide
phosphorylation by GRK2. The synthetic peptide substrate RRREEEEESAAA
(1 mM) is incubated for various times as indicated in the
presence () or absence (
) of crude phosphatidylcholine (50
µg). The reaction is stopped by spotting the sample on a square of
P-81 ion-exchange paper and washing in phosphoric acid as described.
Each sample is performed in triplicate, and the results shown are that
of a typical experiment.
The
kinetic parameters of phosphorylation are determined in the presence of
varying amounts of the peptide substrate. As shown in Table 1,
the effect of phospholipid is to increase the V of the phosphorylation reaction approximately 3-fold
(8.7-25.4 nmol/min
mg of
ARK) without a change in the K
for the peptide substrate. In data
shown in Table 2, phosphatidylinositol increased phosphorylation
of the peptide substrate 6-fold while PIP
decreased GRK2
activity to 30% of the control level.
As GRK5, a member of the GRK
family related to ARK, has been shown to undergo
phospholipid-stimulated autophosphorylation and association with
phospholipid vesicles(15) , we examine GRK2 to determine if a
similar mechanism may be responsible for the phospholipid activation of
the kinase. GRK2 does not autophosphorylate to any significant degree
in the presence or absence of crude phosphatidylcholine (Fig. 5). At 1 h, the maximal amount of autophosphorylation is
observed with a stoichiometry of 0.1 mol phosphate/mol kinase. A
Western blot of GRK2 incubated with vesicles prepared from purified
lipids demonstrates a significant amount of immunoreactivity associated
with the pellet (Fig. 6). 10-20% of the immunoreactive
GRK2 did pellet with phosphatidylinositol, and
5% pelleted with
PIP
. These data stand in contrast to that seen with GRK5 (15) and suggest different mechanisms of lipid activation of
the two kinases. Moreover, the demonstration of GRK2 association with
vesicles containing either phosphatidylinositol or PIP
is
in agreement with the previously published
findings(16, 17) .
Figure 5:
Autophosphorylation of GRK2.
Autophosphorylation of GRK2 is determined under conditions identical to
receptor phosphorylation studies containing 80 ng (1 pmol) of purified
GRK2 with () or without (
) added crude phosphatidylcholine
(50 µg). The kinase is resolved by SDS-PAGE and localized by
autoradiography. The gel slice containing the GRK2 band is excised and
counted to determine the stoichiometry of phosphorylation, which is
expressed as mol of phosphate/mol of GRK2.
Figure 6:
Effect of lipids on GRK2 binding to
phospholipid vesicles. Lipids are dried under a stream of nitrogen and
resuspended in 20 mM Tris-HCl, pH 7.2, 2 mM EDTA with
three bursts of a tip sonicator. GRK2 (1 pmol) is added, and the
mixture is incubated on ice for 15 min. The phospholipid vesicles are
collected by centrifugation at 15,000 g for 30 min.
The supernatants and corresponding phospholipid pellets analyzed by
SDS-PAGE and GRK2 were detected by immunoblotting. GRK2 binding to
phosphatidylcholine and PIP
vesicles as well as a control
lane representing the total amount of kinase used in each incubation is
shown.
The heterobifunctional
cross-linking reagent, N-3-[I]iodo-4-azidophenylpropionamido-S-(2-thiopyridyl)cysteine
has been used to map the molecular structure of transducin's
subunit(24) . Under mild, nondenaturing conditions,
[
I]ACTP derivatizes reduced sulfhydryls to form
a mixed disulfide easily cleaved by the addition of excess reducing
agents. When GRK2 is incubated for 2 h in the dark with a 100-fold
molar excess of [
I]ACTP relative to kinase,
there is incorporation of
1 mol
[
I]ACTP/mol kinase. The addition of
phospholipid vesicles to the reaction results in a 2-fold enhancement
of incorporation to a stoichiometry of 2 mol of
[
I]ACTP/mol of
ARK1 (Fig. 7). The
additional [
I]ACTP incorporation observed in
the presence of phospholipid vesicles is blocked by the addition of
MgATP at concentrations identical to those used in the phosphorylation
assay. The effect of MgATP was specific for
[
I]ACTP in response to phospholipid as there is
no effect observed with [
I]ACTP incorporation
in the absence of phospholipid. In all cases, the
[
I]ACTP incorporation is sensitive to reducing
agents, indicating the presence of a mixed disulfide and not covalent
attachment via the azide moiety. When a variety of lipids are examined,
only the acidic phospholipids previously shown to enhance GRK2 activity
led to an increase in [
I]ACTP labeling of the
kinase (Fig. 8). Of note is the observation that PIP
not only failed to increase the labeling of GRK2, but decreased
[
I]ACTP incorporation to a level below that
seen in the basal state. A preliminary mapping experiment demonstrates
that the
I associated with GRK2 resulted in a unique
proteolytic map when cleaved with V-8 protease. The appearance of
proteolytic bands of 14 and 6 kDa are observed when the kinase is
labeled in the presence of the activating lipid phosphatidic acid (Fig. 9). These cleavage products are greatly diminished by
co-incubation of lipid and MgATP or with the omission of the
phospholipid to the labeling reaction (data not shown).
Figure 7:
[I]ACTP labeling
of GRK2. A 100-fold excess of [
I]ACTP
relative to kinase is incubated in the dark on ice for 2 h. The
labeling is stopped by the addition of 2% SDS and 40 mMN-ethylmaleimide (final concentrations). SDS sample buffer
without
-mercaptoethanol is added and the GRK2 resolved under
nonreducing conditions by SDS-PAGE. Fig. 7is a autoradiograph
under basal (lane1), 50 µg crude
phosphatidylcholine (lane2), MgCl
(7.5
mM) and ATP (0.1 mM) (lane3), and
phosphatidylcholine and MgATP (lane4) conditions.
The GRK2 band is excised and [
I]ACTP
incorporation determined by
counting. The specific activity of
the [
I]ACTP varied with each preparation and is
determined separately. These results are representative of
[
I]ACTP labeling performed a minimum of 4
times.
Figure 8:
Lipid
profile of [I]ACTP incorporation into GRK2. GRK2 is incubated with a 100-fold molar excess of
[
I]ACTP and the addition of 50 µg of
various lipids as described above. The reaction is terminated by the
addition of N-ethylmaleimide and SDS, and the kinase band is
resolved by electrophoresis. An autoradiograph of labeled GRK2 is
shown.
Figure 9:
Proteolytic digest of
[I]ACTP labeled GRK2. GRK2 is labeled
is the absence(-) or presence (+) of phosphatidic acid (50
µg) as described above. The reaction is stopped by the addition of N-ethylmaleimide (40 mM) and
[
I]ACTP-labeled GRK2 digested with V-8 protease
(1:10 ratio by weight) for the times indicated at 30 °C. The digest
is stopped by the addition of SDS-sample buffer without
-mercaptoethanol. The peptide fragments are resolved on a
10-20% SDS-PAGE gradient. [
I]ACTP-labeled
fragments are identified by autoradiography. The majority of
unincorporated [
I]ACTP is removed by desalting
on a G-50 column in 20 mM Tris-HCl, pH 7.2, 2 mM
EDTA, 0.02% Triton X-100 before
proteolysis.
Regulation of G protein-coupled receptor function involves
the process of desensitization in which a cell exposed to an agonist
becomes less sensitive to subsequent stimulation. In the
AR-adenylyl cyclase system, nonselective, and
agonist-specific forms of desensitization occur and appear to be
related to phosphorylation of the receptor(26) . Kinases of the
GRK family are thought to play a role in rapid, agonist-specific
desensitization, as these enzymes phosphorylate the receptor in an
agonist-dependent fashion. Several lines of evidence support this
proposed role of GRKs in the desensitization process. First, cells that
express
ARs that have had the putative GRK2
phosphorylation sites deleted exhibit delayed
desensitization(27) . Second, a permeabilized cell system has
been used to demonstrate that heparin, a potent inhibitor of GRK2,
blocked both agonist-induced receptor phosphorylation and
desensitization(28) . Third, type-specific antibodies directed
toward GRK3 attenuated odorant-induced desensitization in olfactory
cells(29, 30) . Fourth, Ishii et al.(31) have shown that GRK3 blocks thrombin signaling when
the receptor and kinase are coexpressed in Xenopus oocytes.
Finally, overexpression of a GRK2 dominant negative mutant in airway
epithelial cells attenuates desensitization of the
AR(32) . At this time, these data are
consistent with a role of GRK-mediated receptor phosphorylation in the
process of agonist-specific desensitization.
The agonist-dependent
phosphorylation of receptors by GRK2 and other members of the GRK
family is a key feature of this class of enzymes. A conformational
change in the receptor could expose potential phosphate acceptor sites
to the kinase resulting in agonist-dependent phosphorylation of the
receptor. However, this does not appear to be the mechanism involved in
receptor-GRK interactions(7) . Alternatively, the kinase
appears to interact with the agonist-occupied form of the receptor,
which primarily results in an increase in the V of the enzyme(8, 9) . Presumably, a
conformational change occurs in GRK2 and other GRKs, which results in
enhanced catalytic efficiency. Recently, it has been shown that the
peptide mastoparan increases the activity of rhodopsin kinase (8) and a GRK isolated from porcine brain with properties
similar to
ARK1(13) . Since mastoparan activates G
proteins by mimicking a structure similar to agonist-occupied
receptors(33) , a similar mechanism would seem likely in the
stimulation of kinase activity.
In addition to the activation of the
kinase following the interaction with agonist-occupied receptors, GRKs
also interact with membranes via different mechanisms. Photostimulation
of rhodopsin results in the association of rhodopsin kinase with the
retinal membrane. The translocation to the membrane requires the
farnesylation of rhodopsin kinase, a post-translational modification
unique to this member of the GRK family(6) . GRK2 (ARK1),
GRK3 (
ARK2), and a related kinase from porcine brain all exhibit
enhanced phosphorylation of agonist-occupied receptors in the presence
of
-subunits from heterotrimeric G
proteins(10, 11, 12) . The effect of
exogenous
-subunits is to increase the rate and maximal
stoichiometry of phosphorylation(11, 12) . This effect
is synergistic with the activation of the kinase by agonist-occupied
receptor or mastoparan (13) . Similarly to rhodopsin kinase,
ARK1 activity has been shown to translocate from the cytosol to
the plasma membrane following stimulation of the target cell with a
wide variety of agonists including isoproterenol, PGE
,
somatostatin, and platelet activating
factor(34, 35, 36) . Unlike rhodopsin kinase,
GRK2 does not undergo isoprenylation(37, 38) .
However, the interaction of the kinase with the
-subunits
appears to target
ARK to the membrane(11) . Thus, two
different molecular mechanisms exist for localizing GRKs to a membrane
surface. Most recently, a third member of the GRK family (GRK5) was
cloned and found to lack the sequence required for either
isoprenylation or interaction with
-subunits(15) .
Consistent with the later is the finding that
AR or
rhodopsin phosphorylation by GRK5 is not enhanced by the addition of
-subunits. However, phospholipid-stimulated
autophosphorylation of GRK5 at Ser-484 and Thr-485 increased receptor
phosphorylation
15-fold and represents yet a third mechanism for
membrane association of GRKs.
Despite the experimental data, which
demonstrate the importance of phospholipid associations between GRKs, a
clear effect of phospholipids is only now beginning to emerge. The
observation that G protein-coupled receptors serve as substrates of
GRKs following reconstitution into phospholipid vesicles or if
expressed in high numbers in the plasma membrane of cells such as Sf9
insect cells (39) suggests the importance of lipids in the
phosphorylation of receptor substrates. The traditional detergent for
the solubilization of ARs has been
digitonin(20) . While biologic activity is preserved, the
digitonin is difficult to remove due to its low critical micellar
concentration (CMC). Furthermore, digitonin tends to concentrate with
most ultrafiltration techniques. We have used dodecyl maltoside to
effect solubilization and purification of the
AR.
Dodecyl maltoside has a defined critical micellar concentration and
forms micelles of
50,000 Da. We have taken advantage of these
properties to concentrate the purified receptor using a YM-100
membrane. Under these conditions, detergent passes through while
detergent-receptor micelles are retained by the membrane. In this
manner, we could manipulate the receptor preparation without excessive
concentration of the detergent. In addition, enzymatic activity of
rhodopsin kinase (40) and
ARK1 (
)is minimally
affected by dodecyl maltoside while other detergents completely inhibit
kinase activity despite concentrations below the critical micellar
concentration of the detergent.
In the current study, we clearly
demonstrate that detergent-solubilized AR serves as a
substrate for GRK2, provided phospholipid is added to the
phosphorylation reaction. Under the conditions used in this study, the
receptor resides in a mixed detergent-lipid micelle. The concentration
of detergent used would not permit the formation of pure lipid vesicles
typical of previous reconstitution experiments. Furthermore, the
receptor under these conditions does not pellet following a 300,000
g centrifugation step adding support to the notion
that the receptor is present in mixed micelles. This data would suggest
that GRK2 has a phospholipid requirement for phosphorylation of
receptor substrates. Using a variety of neutral, acidic, and basic
phospholipids, we clearly demonstrate that negatively charged
phospholipids, including cardiolipin, phosphatidylglycerol,
phosphatidic acid, phosphatidylserine, and phosphatidylinositol, were
necessary for phosphorylation of the
AR by GRK2.
Previously, purified receptor was first inserted into crude
phosphatidylcholine vesicles in order to observe GRK2-dependent
phosphorylation. Therefore, we initially performed studies of
phospholipid requirements of GRK2 using the same preparation of crude
phosphatidylcholine. The fact that crude, but not purified,
phosphatidylcholine preparations resulted in kinase activity is
consistent with the notion that a phospholipid(s) other than
phosphatidylcholine is required by GRK2. Thus, the long recognized
requirement for reconstitution of the
AR into
phosphatidylcholine vesicles most likely serves to provide a source of
negatively charged phospholipid to the phosphorylation reaction.
As
mentioned above, the lipid profile demonstrates that phospholipids with
a net negative charge at physiologic pH enhance the phosphorylation of
the AR when studied in mixed detergent lipid micelles.
A notable exception is the effect of PIP
, as receptor
phosphorylation is not observed when this phospholipid is included in
the phosphorylation assay. Similar data has recently been reported when
phosphorylation of the m2 muscarinic acetylcholine receptor was studied
in reconstituted lipid vesicles (16) . In contrast, others
reported that PIP
enhanced GRK2 phosphorylation of the
AR only in the presence of added
subunits
of heterotrimeric G proteins(17) . While the stoichiometry of
phosphorylation is rather low compared with that previously reported
using crude phosphatidylcholine, qualitatively similar results were
noted for a variety of lipids tested. In contrast to our findings and
that of DebBurman et al.(16) , in which >4 mol of
phosphate/mol of receptor was achieved in the absence of
subunits, a recent manuscript (17) indicated the stoichiometry
was < 0.5 mol of phosphate/mol of
AR without the
addition of G protein
subunits.
The initial step in the
mechanism of lipid regulation of GRK2 activity must involve the
interaction between lipid and the kinase or the receptor. Evidence of a
specific lipid-kinase interaction is provided by the finding that GRK2
becomes associated with vesicles provided they contain negatively
charged lipids such as phosphatidylserine, phosphatidylinositol, or
PIP. However, the phosphorylation data presented in this
manuscript as well as that previously reported (16) suggests
that the effects of lipids are more complex than simply targeting the
kinase to the membrane surface. This is evident by the the effect of
PIP
to cause membrane association in addition to inhibition
of receptor phosphorylation.
In order to test the hypothesis that
GRK2 activity is increased by phospholipids, we used a previously
characterized peptide substrate of ARK(25) . The advantage
of the peptide substrate was 2-fold. First, the peptide substrate was
designed to bind to ion exchange paper in 75 mM phosphoric
acid permitting a large number of phosphorylation reactions necessary
to obtain kinetic data. Second, the peptide substrate permits the
identification of direct effects of phospholipid upon the kinase in the
absence of any possible phospholipid-receptor interactions. The
catalytic activity increases 3-fold with respect to the peptide
substrate without a change in the K
in
the presence of crude phosphatidylcholine. We determined the kinetic
parameters using crude phosphatidylcholine as this was the source of
lipid that has been used for years in the reconstitution assay. Knowing
that the crude preparations were 20% phosphatidylcholine and the
results that demonstrate that crude but not purified
phosphatidylcholine resulted in receptor phosphorylation in the mixed
micelle system, we suspect that other phospholipids were responsible
for
ARK activity in the reconstitution assay. As further support,
we demonstrate that the inclusion of phosphatidylinositol to the
peptide assay results in a dramatic enhancement of phosphate
incorporation. Moreover, PIP
inhibited the ability of GRK2
to phosphorylate the synthetic peptide substrate, consistent with our
data and that of others(16) , in which receptor phosphorylation
was studied. In general, peptides are poor substrates for GRKs when
compared with reconstituted receptors based on their low affinity for
the kinase; however, they provide valuable data as to the mechanism of
GRK activation. In this situation, the simplest explanation of the data
is that GRK2 is directly activated following interaction with
phospholipid. This observation would explain in part the apparent
requirement for G protein-coupled receptors to be reconstituted into
phospholipid vesicles in order to serve as GRK2 substrates.
Finally,
we have used [I]ACTP as a probe to assess
conformational changes that may occur in the kinase. We have observed
that GRK2 will incorporate
1 mol of ACTP/mol of kinase under basal
conditions. In the presence of crude phosphatidylcholine, the
stoichiometry of [
I]ACTP labeling doubled. We
suggest that this represents a sulfhydryl group exposed following the
interaction of GRK2 and phospholipid. This hypothesis is further
supported by three observations. First, the increase in
[
I]ACTP incorporation secondary to phospholipid
exposure is completely blocked in the presence of MgATP. Second, the
V-8 proteolytic map of [
I]ACTP-labeled GRK2
identifies two unique bands in the presence of phospholipid that are
diminished under basal conditions or in the presence of MgATP. Finally,
lipids, which have been shown to activate GRK2, also increased the
[
I]ACTP incorporation. More importantly,
PIP
, which binds GRK2 but results in an inhibition of
catalytic activity completely abolished any
[
I]ACTP labeling, including what appears to be
the site labeled in the absence of lipids. At this time, our working
hypothesis is that the sulfhydryl group(s) exposed following
phospholipid interaction with GRK2 is near the ATP binding site and/or
catalytic groove of the kinase and protected from ACTP labeling by the
binding of MgATP. Furthermore, our ability to label this additional
site serves as a probe of a putative conformational change, which
occurs as the kinase is activated by regulatory lipids.
Data
presented in this manuscript provide the first direct evidence to
support the direct regulation of GRK2 by lipid. The effect on catalytic
activity, in addition to the membrane localization, which occurs via
the interaction between various lipids and the pleckstrin homology
domain of GRK2 and GRK3, have clear implications as to the regulation
of the kinase. Given the apparent association of several members of the
GRK family with phospholipid membranes, it is tempting to speculate
that many of the GRKs require phospholipid for maximal catalytic
activity. While several different types of interaction between various
GRKs and phospholipid membranes have been described, it will prove
valuable to test whether a common molecular mechanism exists among this
family of kinases. We are currently mapping the sites of ACTP
incorporation in GRK2 to permit such a study. Additional studies are
ongoing to define specific phospholipid interactions with ARK and
extend the current studies to other members of the GRK family.