(Received for publication, September 13, 1994; and in revised form, November 11, 1994)
From the
Attenuation of receptor-mediated signal amplification in
response to external stimuli, an essential step in the balance of
cellular activation, may be mediated by receptor phosphorylation. We
have recently shown that the carboxyl-terminal cytoplasmic domain of
the N-formyl peptide receptor (FPR) interacts with G proteins
and demonstrate here that this same region of the FPR is specifically
phosphorylated by a neutrophil cytosolic kinase with properties similar
to the G protein-coupled receptor kinase, GRK2. Both kinase activities
show a lack of sensitivity toward protein kinase A, protein kinase C,
and tyrosine kinase inhibitors but demonstrate almost identical
sensitivity toward the kinase inhibitor heparin. Kinetic studies
demonstrated that GRK2 has a K for the
carboxyl-terminal domain of the FPR of approximately 1.5 µM and that denaturation of the substrate results in an almost
complete loss of phosphorylation. Comparative studies reveal that GRK3
has approximately 50% of the activity of GRK2 toward the FPR carboxyl
terminus, whereas GRK5 and GRK6 have no detectable activity.
Site-directed mutagenesis of numerous regions of the FPR carboxyl
terminus demonstrated that, whereas Glu
/Asp
and Asp
are critical for phosphorylation, the
carboxyl-terminal 10 amino acids are not required. Simultaneous
substitution of Thr
, Thr
,
Ser
, and Thr
resulted in an
50%
reduction in phosphorylation, whereas simultaneous substitution of the
upstream Ser
, Thr
, Thr
, and
Ser
or merely the Ser
and Thr
residues resulted in an
80% reduction in phosphorylation.
The introduction of negatively charged glutamate residues for
Ser
and Thr
or Thr
and
Ser
resulted in marked stimulation of phosphorylation.
These results suggest a hierarchical mechanism in which phosphorylation
of amino-terminal serine and threonine residues is required for the
subsequent phosphorylation of carboxyl-terminal residues. These results
provide the first direct evidence that an intracellular domain of a
chemoattractant receptor is a high affinity substrate for GRK2 and
further suggest a role for GRK2 or a closely related kinase in the
attenuation of receptor-mediated activation of inflammatory cells.
Neutrophils respond to a large number of structurally diverse
ligands with functions such as chemotaxis, superoxide production, and
degranulation. Ligands are recognized through their binding to cell
surface receptors, which results in the stimulation of phospholipases
and the mobilization of intracellular calcium(1) . Many of
these chemotactic receptors have recently been cloned, including the
receptors for N-formyl peptides(2) , complement
component C(3) , platelet-activating
factor(4) , IL-8 (5, 6) and homologous
receptors for which the ligand has not yet been determined (7) . These receptors are all members of the class of receptors
coupled to GTP-binding regulatory proteins (G proteins
; for
a review, see Refs. 8 and 9). The precise regulation of neutrophil
responses is crucial due to the cytotoxic effects of superoxide and
degradative enzymes on host tissues. Following an initial exposure to
ligand, neutrophils rapidly but reversibly become unresponsive or
desensitized to subsequent stimulation with either the same or
unrelated chemoattractant(10, 11) . Multiple
mechanisms may contribute to desensitization, including receptor
phosphorylation, endocytosis, and the down-regulation of receptor
expression(12) .
G protein-coupled receptor kinases (GRKs)
are a rapidly growing family of protein kinases which phosphorylate
their respective ligand-bound receptors on Ser/Thr-containing
intracellular
domains(13, 14, 15, 16) . Once
phosphorylated, these receptors may bind accessory proteins, termed
arrestins, which further uncouple the receptor from the G
protein(17) . G proteincoupled receptor kinases were originally
implicated in the light-stimulated phosphorylation of rhodopsin (18) and the agonist-dependent phosphorylation of the
-adrenergic receptor (19) and are presumed to
be an essential component in the homologous desensitization of multiple
receptors. GRK2 (also termed
-adrenergic receptor kinase) has a
relatively broad tissue distribution, but has been shown to be
particularly abundant in peripheral blood leukocytes as well as myeloid
and lymphoid cell lines (20) . Furthermore, platelet-activating
factor was able to stimulate GRK2 translocation from the cytosol to the
membrane of mononuclear cells(20) , where it may be anchored by
the
subunits of G proteins(21, 22) . This
is proposed to be an initial step in the process of GRK2-dependent
desensitization and to provide a mechanism for increasing the
selectivity of GRK2 for its receptor substrates. Recently, the FPR has
been demonstrated to become rapidly phosphorylated in a dose-dependent
manner(23, 24) . Pretreatment of the cells with
staurosporine, a protein kinase C inhibitor, failed to prevent the
ligand-stimulated phosphorylation of the receptor, suggesting that a
kinase other than protein kinase C was responsible for the observed
phosphorylation.
In order to investigate whether ligand-dependent
FPR phosphorylation and desensitization could be a result of
phosphorylation of the Ser/Thr-rich carboxyl terminus of the FPR, we
utilized a fusion protein containing the carboxyl-terminal 47 amino
acids of the FPR, which we have previously shown interacts specifically
with G proteins(25) . In this report, we provide the first
direct evidence that 1) both serine and threonine residues within the
carboxyl terminus of the FPR are phosphorylated by a neutrophil
cytosolic kinase with properties similar to GRK2; 2) GRK2 has an
affinity for the cytoplasmic tail of the FPR approaching that of the
liganded -adrenergic receptor; 3) phosphorylation by GRK2 likely
proceeds in a sequential manner with phosphorylation of residues
between 334 and 339 being dependent on prior phosphorylation of
residues between 328 and 329; and 4) introduction of negatively charged
residues in the native receptor sequence stimulates subsequent
phosphorylation of downstream residues.
Figure 1:
Amino acid
similarity between the carboxyl-terminal domains of three
chemoattractant receptors. The amino acid sequences of the
carboxyl-terminal domains of the FPR, C receptor, and IL8
receptor B are aligned so as to optimize similarity with the
introduction of a minimal number of gaps. The following groups of amino
acids were considered similar in overall properties: Ser and Thr; Asp
and Glu; Lys, Arg, and His; Ala and Gly; Ala and Val; Leu, Ile, and
Val.
Phosphorylation was assayed as follows. Purified, eluted fusion
protein, at the indicated concentration as determined with the BCA
reagent (Pierce), was incubated with purified GRK in assay buffer (20
mM Tris-Cl, pH 7.2, 2 mM EDTA, 7 mM MgCl) containing 0.1 mM [
-
P] ATP (1.5 fmol/cpm) in a final
volume of 30 µl at 30 °C for the indicated time.
Phosphorylation reactions with glutathione-agarose-bound fusion
proteins were performed as follows. Fusion protein bound beads
(containing 5-10 µg of protein as judged by Coomassie Blue
staining of polyacrylamide gels) were incubated with either neutrophil
cytosol extract (from 10
cell equivalents) or purified GRK
(
100 ng) in a final volume of 300 µl of assay buffer on ice
for 60 min. The beads were centrifuged, washed three times with assay
buffer, and brought to 50 µl with assay buffer.
[
-
P]ATP (1-2 µCi) was added, and
the sample was incubated at 30 °C for 20 min. All reactions were
quenched with SDS sample buffer followed by electrophoresis on a 12.5%
polyacrylamide gel. Gels were stained with Coomassie Blue and
subsequently exposed to x-ray film for visualization of the
P-labeled proteins. Relative determinations of protein and
P content were performed with a Molecular Devices
Densitometer and PhosphorImager, respectively. Absolute determinations
of
P were accomplished by cutting out the bands from the
polyacrylamide gels and counting by liquid scintillation.
Phosphoamino acid content was determined as described(33) .
Briefly, following transfer of the SDS gel to Immobilon P membrane
(Millipore), the fusion protein band was excised and hydrolyzed in 6 M HCl for 1 h at 100 °C. The sample was dried and
resuspended with 0.5 µg each of phosphoserine, phosphothreonine,
and phosphotyrosine and spotted onto a cellulose thin layer plate (100
µm, EM Laboratories). Phosphoamino acids were separated by
chromatography in 5:3 isobutyric acid, 0.5 M ammonium
hydroxide. Unlabeled phosphoamino acid standards were visualized with
ninhydrin, whereas P-labeled phosphoamino acids were
visualized with a Molecular Devices PhosphorImager.
Chemoattractant receptors undergo a functional uncoupling from G proteins following exposure to agonist, likely contributing to the process of desensitization(10, 11) . The recent cloning of numerous chemoattractant receptors reveals that they possess strong structural homology, belonging to the class of seven transmembrane-spanning G protein-coupled receptors(8) . Furthermore, these receptors appear to belong to a subclass of G protein-coupled receptors of small size containing approximately 350 amino acids(34) . This compares to the well characterized adrenergic and muscarinic receptors whose larger size (from approximately 400 to almost 600 amino acids) is primarily due to larger intracellular domains. Related chemoattractant receptors share higher homology in their intracellular domains as compared to their extracellular domains, suggesting that they may share common mechanisms of signal transduction and receptor regulation(4) . A comparison of the carboxyl-terminal tail of three chemoattractant receptors is presented in Fig. 1. The overall homology between any pair of these receptors is approximately 50% with regions as high as 75% over stretches of 17-19 amino acids. The highest homology is in the central third of this domain, whereas the lowest is in the last third of the carboxyl-terminal tail. Of note is the almost complete lack of serine and threonine residues in the first half of the tail sequences and preponderance of these residues in the second half of the tail. Preceding and within the Ser/Thr-rich region are numerous conserved acidic residues, less common in the other portions of the tail.
Given the recent observations of ligand-dependent
phosphorylation of the FPR (23, 24) and the
determination of high expression levels of human GRK2 in peripheral
blood leukocytes and HL60 cells(20) , we asked whether the
Ser/Thr-rich carboxyl terminus of the FPR could serve as an effective
substrate for an endogenous neutrophil kinase. For this purpose we
utilized a fusion protein containing the carboxyl-terminal 47 amino
acids of the FPR expressed at the carboxyl-terminal end of glutathione S-transferase (GST-FPR). We have previously shown that such a
fusion protein is capable of interacting with G proteins
and that the amino-terminal two-thirds of this region of the FPR is of
critical importance(25) .
GST-FPR bound to
glutathione-agarose beads was incubated with a neutrophil cytosol
extract followed by extensive washing and subsequent incubation in the
presence of [-
P]ATP (Fig. 2, panel B). Analysis by SDS-polyacrylamide gel electrophoresis
demonstrated the presence of a phosphorylated protein (lane 1)
corresponding to the position of the Coomassie Blue-stained fusion
protein (Fig. 2, panel A, lane 1). The
specificity of the phosphorylation (Fig. 2, panels B and C) was demonstrated by the negligible phosphorylation
of the glutathione S-transferase protein lacking the 47-amino
acid FPR tail (but containing the sequence IVTD in place of the FPR
tail, lane 2). We next attempted to determine the class of
kinase responsible for the observed phosphorylation through the use of
specific kinase inhibitors. Phosphorylation of GST-FPR by the
neutrophil extract kinase activity (Fig. 2, panel B)
was carried out in the presence of staurosporine to inhibit protein
kinase C (lane 3), H9 to inhibit protein kinase A (lane
4), heparin to inhibit G protein-coupled receptor kinases (lane 5), and herbimycin A to inhibit tyrosine kinases (lane 6). Of these, the only inhibitor to have an effect was
heparin, suggesting the involvement of a G protein-coupled receptor
kinase. To test this possibility directly, purified GRK2 was
substituted for the crude neutrophil extract in the phosphorylation
assay (Fig. 2, panel C). The results demonstrate an
identical pattern of phosphorylation as observed with the crude
extract, namely specific phosphorylation of the GST-FPR fusion protein
as compared with the GST-only construct and inhibition by heparin but
not the other kinase inhibitors.
Figure 2:
Phosphorylation of the immobilized FPR
carboxyl terminus. Fusion protein, either containing or lacking the
carboxyl-terminal 47 amino acids of the FPR (lanes 1, 3, 4, 5, and 6 or lane 2,
respectively), was bound to glutathione-agarose beads and purified by
extensive washing. Bound fusion protein was then incubated with either
a crude neutrophil extract (panel B) or GRK2 (panel
C), in a mixture containing 20 mM Tris-Cl, pH 7.2, 2
mM EDTA, 7 mM MgCl and washed
extensively. The beads were then incubated with 1-2 µCi of
[
-
P]ATP in a final volume of 50 µl of
the same buffer at 30 °C for 60 min. Prior to the addition of
[
-
P]ATP, the following additions were made:
staurosporine (300 nM final, lane 3); H9 (20
µM final, lane 4); heparin (1 µM final, lane 5), and herbimycin A (2 µM final, lane 6). Reactions were quenched with SDS sample
buffer followed by electrophoresis on a 12.5% polyacrylamide gel. The
gel was stained with Coomassie Blue (panel A) and subsequently
exposed to x-ray film for visualization of the
P-labeled
proteins (panels B and C). The two arrows to
the right of each panel indicate the positions of the
GST-FPR fusion protein (upper arrow) and the GST fusion
protein lacking the FPR carboxyl terminus (lower arrow). The
data are representative of three
experiments.
To compare the activity found in
the neutrophil extract with that of a purified G protein-coupled
receptor kinase, we determined the sensitivity of these two activities
to the polyanionic inhibitor heparin. GST-FPR bound to agarose beads
was assayed for phosphorylation in the presence of increasing
concentrations of heparin (Fig. 3). Both activities were
inhibited by heparin with IC values of 0.1-1
µM. These values are very similar to the IC
for heparin previously reported using light-activated rhodopsin
as the substrate (35) and suggest that the activity observed in
the neutrophil extract may be due to a G protein-coupled receptor
kinase. We next determined if the characteristics of the
glutathione-agarose-bound fusion proteins extended to the eluted,
soluble fusion proteins. Following elution with reduced glutathione,
the GST-FPR and GST proteins were dialyzed extensively to remove free
glutathione and exchange the buffer. Fig. 4shows that the
GST-FPR protein is also a substrate for GRK2 in an entirely soluble
system (lane 1). As with the glutathione-agarose-bound fusion
protein, the protein lacking the carboxyl terminus of the FPR was not
significantly phosphorylated (Fig. 4, lane 3).
Phosphorylation carried out under these conditions was also sensitive
to heparin (lane 2), but not the other protein kinase
inhibitors characterized in Fig. 2(data not shown). Prolonged
phosphorylation with increased amounts of GRK2 yielded GST-FPR fusion
protein which was phosphorylated to an extent of 2-3 mol of
phosphate/mol of fusion protein. This compares to the maximal levels of
phosphorylation observed with the
-adrenergic receptor using
similarly isolated GRK2, assayed in the absence of additional
stimulatory factors, such as G protein
subunits(22) .
Figure 3:
Sensitivity of phosphorylation of the FPR
carboxyl terminus to heparin. Fusion protein bound to
glutathione-agarose beads was assayed for phosphorylation by either the
crude neutrophil extract () or GRK2 (
) as described in the
legend to Fig. 2. Heparin was added at the indicated
concentration prior to the addition of
[
-
P]ATP. Quantitation of the
P-labeled proteins was carried out with a Molecular
Devices PhosphorImager. The data represent the mean and standard error
from three experiments.
Figure 4:
Phosphorylation of the soluble GST-FPR by
GRK2. Fusion protein (0.5 µM), either containing or
lacking the carboxyl-terminal 47 amino acids of the FPR (GST-FPR or
GST, respectively) was incubated with GRK2 (62.5 nM) and
heparin (1 µM) as indicated in a mixture containing 20
mM Tris-Cl, pH 7.2, 2 mM EDTA, 7 mM MgCl, and 0.1 mM [
-
P]ATP (1.5 fmol/cpm) in a final
volume of 30 µl at 30 °C for 60 min. Reactions were quenched
with SDS sample buffer followed by electrophoresis on a 12.5%
polyacrylamide gel. The gel was stained with Coomassie Blue (upper
panel) and subsequently exposed to x-ray film for visualization of
the
P-labeled proteins (lower panel). The
positions of molecular mass standards (in kilodaltons) is indicated on
the left.
The expressed portion of GST itself contains a total of 14 serine and threonine residues, 4 of which exist in an environment resembling the preferred site for phosphorylation of peptides by GRK2, being located 1-3 amino acids carboxyl-terminal to an acidic residue(36) . Despite this abundance of potential GRK2 phosphorylation sites in the primary sequence of GST, none of these sites serves as a substrate for the kinase (Fig. 4, lane 3). This suggests that the serine and threonine residues in the carboxyl terminus of the FPR are presented in a unique fashion, with phosphorylation being dependent on the three-dimensional structure of the protein. We tested this hypothesis by assaying phosphorylation of GST-FPR fusion protein which had been denatured by boiling and rapid cooling. Denaturation resulted in a reduction in the level of phosphorylation by about 90% (Fig. 5A). This confirms that more than simply the primary amino acid sequence of the FPR is involved in the recognition by GRK2. In fact, this result indicates that the three-dimensional structure is absolutely required and that the FPR carboxyl terminus in the incorrect conformation cannot be phosphorylated by GRK2. Results similar to those with GST were obtained when comparing maltose-binding protein to a fusion protein of the FPR carboxyl terminus to maltose-binding protein (not shown), further attesting to the specificity of the reaction. Examination of the sequence of the carboxyl terminus of the FPR reveals that both serine and threonine residues are in a position to be potential sites of phosphorylation. Analysis of the phosphoamino acid content of the phosphorylated GST-FPR protein by acid hydrolysis followed by thin layer chromatography revealed that 65% of the phosphoamino acids consisted of phosphothreonine, whereas 35% consisted of phosphoserine (Fig. 5B). As expected, no phosphotyrosine was detected. These results indicate that phosphorylation occurs in proportion to the content of serine and threonine in the carboxyl terminus of the FPR (see Fig. 1).
Figure 5:
Characteristics of phosphorylation of the
soluble GST-FPR by GRK2. A, effect of denaturation on GST-FPR
phosphorylation. GST-FPR was either incubated at room temperature (lane 1) or heated to 100 °C for 5 min followed by rapid
cooling on ice (lane 2) and analyzed for phosphorylation by
GRK2 as described under ``Experimental Procedures.''
Reactions were terminated with SDS sample buffer followed by
electrophoresis on a 12.5% polyacrylamide gel. The gel was stained with
Coomassie Blue (upper panel) and subsequently exposed to x-ray
film for visualization of the P-labeled fusion proteins (lower panel). B, analysis of phosphoamino acid
content of phosphorylated GST-FPR. GST-FPR, phosphorylated by GRK2, was
transferred from an SDS gel to Immobilon P membrane, and the fusion
protein band was excised and acid-hydrolyzed. The sample was
resuspended with unlabeled phosphoserine, phosphothreonine, and
phosphotyrosine, spotted onto a cellulose thin layer plate, and
separated by chromatography. Unlabeled phosphoamino acid standards were
visualized with ninhydrin, whereas
P-labeled phosphoamino
acids were visualized by exposure to x-ray film. Phosphoserine (PS), phosphothreonine (PT), and phosphotyrosine (PY) migrated 4.2 cm, 5.1 cm, and 5.8 cm, respectively, from
the origin. Relative quantitation of the
P-labeled
phosphoamino acids was carried out with a Molecular Devices
PhosphorImager.
We next examined the
kinetic parameters of the phosphorylation of the GST-FPR fusion
protein. Phosphorylation proceeded linearly for 3-4 h after which
no further phosphorylation was observed (data not shown). Incubation of
the fusion protein with increased amounts of GRK2 resulted in
proportional increases in the level of phosphorylation (as described
above), suggesting that under our assay conditions phosphorylation
ceased due to exhaustion of the kinase after about 4 h. We also
determined the effect of concentration of the fusion protein on the
rate of phosphorylation (Fig. 6A). Phosphorylation by
GRK2 reached a plateau with about 5 µM GST-FPR. Detailed
analysis of the kinetic parameters revealed that the carboxyl terminus
of the FPR is phosphorylated with a K of 1.5
± 0.5 µM and a V
of 3.0
± 0.7 nmol/min/mg of GRK2 (Fig. 6B). This K
value is approximately 6-fold higher than that
obtained for the agonist-bound state of the
-adrenergic receptor
and approximately 4-fold lower than that determined for the
phosphorylation of activated rhodopsin by GRK2(15) . In
addition to GRK2, we also tested the ability of three other purified G
protein-coupled receptor kinases to phosphorylate the FPR carboxyl
terminus. GRK3, which bears strong homology to GRK2, was active to
approximately 50% the level (similar K
, reduced V
) of GRK2, whereas GRK5 (37) and GRK6 (32) displayed no significant activity. The fact that two
related G protein-coupled receptor kinases were incapable of
phosphorylating the FPR carboxyl terminus further attests to the
specificity of the reaction between the GST-FPR and GRK2.
Figure 6:
Kinetics of phosphorylation of the FPR
fusion protein by G protein-coupled receptor kinases. A,
phosphorylation of GST-FPR by related G protein-coupled receptor
kinases. Fusion protein containing the carboxyl terminus of the FPR (at
the indicated concentration) was incubated with one of the following
GRKs (at a final concentration of 10 nM): , GRK2;
,
GRK3;
, GRK5; or
, GRK6 in a mixture containing 20 mM Tris-Cl, pH 7.2, 2 mM EDTA, 7 mM MgCl
, and 0.1 mM [
-
P]ATP (1.5 fmol/cpm) at 30 °C
for 30 min. Following electrophoresis on a 12.5% polyacrylamide gel and
exposure to x-ray film for visualization, the
P-labeled
proteins were cut out of the dried gel and quantitated by liquid
scintillation counting. Data are representative of two experiments. B, Scatchard analysis of the kinetic parameters of
phosphorylation by GRK2. Fusion protein containing the
carboxyl-terminal 47 amino acids of the FPR was incubated with GRK2
(62.5 nM) in the above buffer with 0.1 mM [
-
P]ATP (1.5 fmol/cpm) in a final
volume of 30 µl at 30 °C for 30 min. Reactions were quenched
with SDS sample buffer and analyzed as above. Data are plotted as v/F versus V, where v is the velocity of the
reaction (nmol/min/mg of GRK2) and F is the free concentration
of GST-FPR (µM). Data are representative of six
experiments with similar
results.
GRK2 has been shown to exhibit preferential phosphorylation of serine and threonine residues in an environment containing acidic residues (36) . Examination of the sequence of the carboxyl terminus of the FPR (Fig. 1) suggested that the serine and threonine residues at positions 328, 329, 331, 332, 334, 336, 338, and 339 were candidates for phosphorylation and that the 2 acidic residues at positions 326 and 327 may play a role in the interaction with GRK2. We therefore created mutations at these and the other sites shown in Fig. 7. The mutant GST-FPR fusion proteins were purified and analyzed for their ability to be phosphorylated by GRK2. The results are summarized in Table 1.
Figure 7: Sequences of mutant GST-FPR fusion proteins. The amino acid sequences of the wild type carboxyl terminus and mutant carboxyl termini are shown using the one-letter codes. Only the changed amino acids are indicated for the mutant proteins. The asterisk (*) represents the introduction of a stop codon to generate a truncated form of the protein.
Mutants 4, 5, 6, 8, and 9 were
designed to determine which serine and threonine residues were involved
in the phosphorylation by GRK2. The K values for
these mutants are not significantly different from that of the wild
type, suggesting that they do not contribute significantly to the
binding interaction between the carboxyl terminus of the FPR and GRK2.
The effects of these mutations are evident in the V
values. Mutant 9, which replaces Thr
,
Thr
, Ser
, and Thr
with
alanine residues, undergoes approximately 50% of the phosphorylation
compared to the wild type fusion protein. Mutant 8, which arose
spontaneously during the mutagenesis to produce mutant 9, retains the
threonine residue at position 336 and displays an increase in
phosphorylation (
15%) compared to mutant 9 suggesting that in
total these four potential sites represent approximately 50% of the
phosphorylation sites within the carboxyl terminus of the FPR sequence.
The remaining likely sites of phosphorylation, Ser
,
Thr
, Thr
, and Ser
, were
replaced in mutant 4. Although we expected to see only at most a
45-50% reduction in the extent of phosphorylation (i.e. the complement of that observed with mutant 9), mutant 4 showed
almost no phosphorylation (an 83% reduction in the V
as compared to the wild type). We further analyzed this region by
subdividing mutant 4 into mutants 5 and 6, in which either only the
first two mutations or second two mutations were made. Mutant 5, in
which only Ser
and Thr
were replaced,
exhibited phosphorylation almost equal to that of mutant 4, in which 4
residues were replaced. We would then expect that phosphorylation might
not occur at residues Thr
and Ser
. However,
when only these 2 residues were replaced (mutant 6), a level of
phosphorylation (37%) intermediate between mutant 5 (23%) and mutant 9
(53%) was observed. We believe these results are most consistent with a
mechanism in which phosphorylation occurs in a sequential manner
beginning with residues Ser
and Thr
,
proceeding through residues Thr
and Ser
,
before reaching residues Thr
, Thr
,
Ser
, and Thr
.
To investigate this
possibility further, we attempted to mimic the phosphorylation of
certain serine and threonine residues by mutating them to negatively
charged glutamate residues, which spatially resemble phosphoserine and
phosphothreonine. Mutants 11, 12, and 13 correspond to mutants 5, 6,
and 4, respectively, with glutamine replacing the original serine or
threonine. Again, there is no significant effect on the K of the phosphorylation reaction. There is,
however, in all three cases, an increase in the V
of the reaction, indicating a stimulation of phosphorylation.
These values are not corrected for the fact that the number of
potential phosphorylation sites is reduced by 2 in the case of mutant
11 and 12 and by 4 in the case of mutant 13. If the level of
phosphorylation of mutant 13 is compared to that of the corresponding
alanine- and glycine-substituted mutant (mutant 4), then the increase
in phosphorylation due to the presence of the negatively charged
residues is 6-fold. If a similar comparison is made between mutants 11
and 5, the increase in phosphorylation due to the presence of the
negatively charged residues is 8-fold. Thus, we can speculate that when
either or both Ser
and Thr
become
phosphorylated by GRK2, the resulting intermediate is a superior
substrate for further phosphorylation events. This is consistent with
the sequential phosphorylation suggested by the results described
above.
Given the apparent importance of negatively charged residues
in the activation of GRK2, we also examined endogenous charged residues
within the carboxyl terminus of the FPR. Mutants 1, 2, 3, and 7 replace
charged residues with alanine and glycine residues, whereas mutant 10
removes the terminal 10 amino acids from the fusion protein. Whereas
mutant 1 displayed K and V
values almost identical with that of the wild type fusion
protein, mutant 2 possessed an increased K
and
somewhat decreased V
. At low substrate
concentrations (
K
), the combined effect would
result in significantly lower rates of phosphorylation as compared to
the wild type protein. Mutants 3 and 7, which replace acidic residues
within the phosphorylated region, were severely impaired in their
ability to be phosphorylated. Both mutants exhibited approximately
5-fold decreases in their V
values. The K
of mutant 3 was similar to that of the wild type
fusion protein, whereas the K
of mutant 7 was
lower than that of the wild type fusion protein. These results confirm
that acidic residues in the vicinity of the phosphorylation sites play
a critical role in determining the activity of the kinase. The deletion
construct, mutant 10, displayed an increased K
,
similar to mutant 2, but, in contrast to mutant 2, displayed a V
greater than that of the wild type protein.
This suggests that residues within the terminal 10 residues contribute
to the recognition of the substrate but may also sterically interfere
with catalysis, resulting in a higher V
upon
their removal.
Based on results described above which suggest that
the activity in the neutrophil extract may be due to a G
protein-coupled receptor kinase, such as GRK2, we compared the profile
of activity of the neutrophil kinase to that of purified GRK2 using the
collection of mutants described in Fig. 7and Table 1. In
addition to the 13 mutants, the wild type GST-FPR and control GST
fusion proteins were bound to glutathione-agarose beads and purified.
Phosphorylation was assayed on the fusion proteins bound to the beads
as described in Fig. 2. The results indicate a high degree of
correlation between the relative amounts of phosphorylation of the
fusion proteins by the two sources of kinase (Fig. 8). Since
protein amounts could only be estimated by densitometry of the
Coomassie Blue-stained gels and only a single concentration of fusion
protein was used, the results do not take into account the effects of K on the level of phosphorylation. This will
affect comparing the levels of phosphorylation between mutants to some
extent, but not comparing phosphorylation of a given mutant by the
neutrophil extract versus purified GRK2. In each case, very
similar levels of phosphorylation were observed for the two kinase
sources. The exceptions were mutants 11, 12, and 13 which showed
greater phosphorylation by the neutrophil extract as compared to the
purified GRK2. However, phosphorylation of these mutants by the GRK2
under these conditions did produce considerably greater phosphorylation
than seen with the wild type fusion protein, as observed with kinetic
analyses of the soluble fusion protein mutants 11, 12, and 13.
Together, these results provide still greater evidence that GRK2 or a
very closely related kinase may be responsible for the phosphorylating
activity observed in the neutrophil extract.
Figure 8:
Comparison of the phosphorylation by
neutrophil extract kinase and GRK2 of wild type and mutant FPR carboxyl
termini. Fusion proteins representing the wild type or mutant fusion
proteins described in Fig. 7were bound to glutathione-agarose
beads and assayed for phosphorylation as described in the legend to Fig. 2using either a crude neutrophil extract (cross-hatched
bars) or purified GRK2 (open bars). Reactions were
quenched with SDS sample buffer followed by electrophoresis on a 12.5%
polyacrylamide gel and subsequently exposed to x-ray film for
visualization. Quantitation of the P-labeled proteins was
carried out with a Molecular Devices PhosphorImager. Data are from
three preparations of fusion protein with experiments performed in
duplicate.
The results presented in this paper demonstrate for the first time that the carboxyl terminus of the FPR serves as an effective substrate for a neutrophil kinase, possibly the G protein-coupled receptor kinase, GRK2. Six results address the specificity of this phosphorylation.
First, the phosphorylation of the fusion protein lacking the carboxyl terminus of the FPR was negligible compared to that of the protein containing the FPR tail, despite the existence of numerous potential phosphorylation sites for GRK2 within the primary sequence of the GST. This indicates that the recognition of the phosphorylation site(s) within the FPR tail by the kinase is unique under our assay conditions.
Second, of the protein kinase inhibitors
tested, only the G protein-coupled receptor kinase specific inhibitor,
heparin, was able to prevent phosphorylation. Furthermore, the kinase
activity of the neutrophil extract and purified GRK2 exhibited similar
sensitivity to heparin (IC
100-300
nM), suggesting the former activity may be due to GRK2. Casein
kinase II, GRK5, and GRK6, which are also sensitive to heparin, exhibit
dissimilar IC
values of approximately 10, 1, and 15
nM, respectively.
Third, the tertiary structure of the FPR carboxyl terminus is critical for its recognition by GRK2. This is evidenced by the large reduction in activity of the GRK2 toward denatured GST-FPR as compared to the native protein. The residual phosphorylation observed could either be a result of a low affinity interaction with the denatured GST-FPR or a result of incomplete denaturation of the GST-FPR.
Fourth, phosphorylation of GST-FPR is specific to GRK2 and the closely related GRK3, but does not occur with the more distant G protein-coupled receptor kinases, GRK5 and GRK6.
Fifth, the K of the phosphorylation reaction
(1.5 µM) approached the affinity of GRK2 for the
-adrenergic receptor itself, further indicating that
the interaction of GRK2 with the carboxyl terminus of the FPR is highly
specific.
Sixth, mutagenesis revealed that even the alteration of a single amino acid (e.g. D333A) can result in almost complete loss of phosphorylation.
Studies with synthetic peptides of lengths
between 20 and 30 amino acids (approximately one-half of the size of
the carboxyl-terminal tail of the FPR) corresponding to regions of the
adrenergic receptor that serve as substrates for GRK2 demonstrate K
values of 1-5 mM(38) .
This difference in K
between
-adrenergic
receptor and receptor-derived peptides may reflect the requirement for
multiple interactions with numerous domains of the
-adrenergic
receptor which occurs only with the liganded receptor. Our results
indicate that the carboxyl terminus of the FPR contains sufficient
structural information to allow a high affinity interaction with GRK2,
although additional intracellular domains may further increase the
affinity of GRK2 for the FPR. The low observed V
for this reaction, as compared to that of the intact
-adrenergic receptor (V
= 78
nmol/min/mg(15) ), may suggest that additional interactions are
required to stimulate maximal activity of the kinase. Such a conclusion
is supported by recent experiments demonstrating that agonist-activated
receptors can increase the V
of phosphorylation
of synthetic peptides by GRK2, suggesting that the overall topology of
the activated receptor is more important than the substrate's
specific primary amino acid sequence in the stimulation of GRK2
activity(39) . One possible explanation of our results is that,
whereas the
-adrenergic receptor upon binding agonist
``assembles'' a recognition site for GRK2 out of multiple low
affinity sites, the FPR contains a pre-existing high affinity site that
is presented upon the binding of ligand to the receptor. The presence
of a pre-existing high affinity site for phosphorylation could also
explain the unique form of class desensitization displayed by
chemoattractant receptors where, for example, exposure of neutrophils
to C
results in the desensitization of the cell to fMLP
and other chemoattractants but not to unrelated
agonists(10, 40) .
Our results indicate that the
serine and threonine residues within both regions encompassed by mutant
4 and mutant 9 are phosphorylated by GRK2. However, if the
phosphorylation of these serine and threonine residues were random and
independent, it would be expected that mutants 4 and 9 would both show
approximately 50% of the level of phosphorylation compared to the wild
type sequence. The fact that replacement of the upstream serine and
threonine residues resulted in a greater reduction in phosphorylation
of the remaining serine and threonine residues suggests that a
hierarchy of phosphorylation exists, in which phosphorylation of
carboxyl-terminal serine and threonine residue(s) is dependent on the
prior phosphorylation of amino-terminal residue(s). Further evidence in
favor of this mechanism is provided by mutants 5 and 6, which subdivide
the serine and threonine residues of mutant 4. Here, replacement of the
2 amino-terminal-most serine and threonine residues results in a
similar reduction in phosphorylation as mutant 4, in which 4 serine and
threonine residues are replaced (80% reduction). Replacement of
the next 2 serine and threonine residues results in a level of
phosphorylation (
63% reduction) between those of mutants 4 and 9
(
47% reduction). The simplest explanation of these results invokes
a sequential mechanism of phosphorylation beginning with residues 328
and/or 329, followed by residues 331 and/or 332, and finally residues
334 through 339.
Our results also indicate that the acidic residues at positions 326, 327, and 333 play a critical role in the stimulation of GRK2 activity by the receptor. Since acidic residues appear important in the interaction between GRK2 and synthetic peptides(36) , it is possible that phosphorylation of serine and threonine residues juxtaposed to asparagine and glutamine residues creates a new acidic phosphoserine or phosphothreonine recognition sequence for the phosphorylation of a subsequent carboxyl-terminal serine or threonine. Such a mechanism has been proposed for glycogen synthase kinase-3, which appears to use newly generated phosphoserine residues as recognition sites to phosphorylate subsequent serine residues in a hierarchical manner(41) . We attempted to test whether the introduction of a negative charge at potential sites of phosphorylation would stimulate the activity of the GRK2. For this purpose, mutants equivalent to mutants 4, 5, and 6 were created substituting glutamate residues for the original serine and threonine residues. The greatest effect was seen with the replacement of the first 2 serine and threonine residues with glutamine. This resulted in a 2-fold increase relative to the wild type fusion protein and an 8-fold increase relative to the alanine-substituted mutant. These results provide very strong supporting evidence that the first phosphorylation step greatly enhances the subsequent phosphorylation events. Sequential phosphorylation has also recently been shown to occur in the phosphorylation of rhodopsin by rhodopsin kinase, but it was not clear whether other GRKs possess a similar catalytic mechanism (42, 43, 44) . Our results suggesting a conserved mechanism of phosphorylation between multiple members of the class of GRKs are of particular significance in light of the amino acid sequence homology between members of the class of G protein-coupled receptor kinases (31) .
We and others have previously shown
that the carboxyl terminus of the FPR interacts with G
proteins(25, 45) . We utilized both a fusion protein,
similar to that used here, and synthetic peptides to map regions of the
FPR involved in the interaction with G proteins. Our results showed
that the second intracellular loop and portions of the carboxyl
terminus could interrupt binding of the FPR to G proteins. Within the
carboxyl terminus of the FPR, the most effective region comprised the
first third of the carboxyl terminus
(Phe-Arg
), the region juxtaposed to
the membrane as predicted from receptor modelling. Also effective was a
region representing the central portion of the carboxyl terminus
(Ser
-Leu
), corresponding to the
region demonstrated here to be phosphorylated. A peptide representing
the terminal 12 amino acids of the FPR was ineffective in disrupting
the FPR-G protein interaction. Our results presented here provide for a
simple mechanism whereby phosphorylation of a region of the FPR
involved in binding to G proteins could prevent this interaction and
result in an uncoupling of the activated FPR from G protein. As with
rhodopsin and the
-adrenergic receptor systems, the subsequent
binding of an arrestin-like protein could further potentiate the
uncoupling.
In conclusion, the novel findings presented in this paper expand the family of known substrates for G protein-coupled receptor kinases to include a member of the chemoattractant class of hematopoietic cell receptors. We also obtained evidence that phosphorylation of the FPR carboxyl terminus by GRK2 is the result of a high affinity interaction and proceeds in a hierarchical manner. Together, these results provide a basis for further experiments to examine the mechanism of action of G protein-coupled receptor kinases using intact chemoattractant receptors and to determine the exact involvement of G protein-coupled receptor kinases in the desensitization and regulation of the entire class of chemoattractant receptors.
Addendum-At the time of submission of
this manuscript, Takano et al.(46) published a report
describing a mutant of the platelet-activating factor receptor in which
Ser/Thr residues in the carboxyl-terminal tail were replaced with Ala.
This mutant more potently activated multiple signal transduction
pathways, leading to the conclusion that desensitization had been
affected. Furthermore, a synthetic peptide corresponding to this region
was phosphorylated by -adrenergic receptor kinase 1 (GRK2). These
results provide corroborating evidence for our contention that GRKs may
be involved in the desensitization of chemoattractant receptors.