(Received for publication, March 30, 1995)
From the
Interaction of human C5a anaphylatoxin with cell surface
receptors mediates cell activation and receptor desensitization.
Treatment of differentiated HL60 cells or transiently transfected COS-7
cells with C5a or phorbol 12-myristate 13-acetate (PMA) results in
rapid hyperphosphorylation of the C5aR. In an attempt to gain more
insight into the function of phosphorylation in the desensitization of
C5aR, we have initiated experiments to identify phosphoacceptor sites
at the amino acid level after stimulation of cells with either C5a or
PMA. In this report we show that C5aR is phosphorylated exclusively on
serine residues in both differentiated HL60 and transfected COS-7 cells
irrespective of the stimulus used. Peptide mapping after cyanogen
bromide cleavage of phosphorylated C5aR indicates that despite the
presence of a protein kinase C consensus motif the third cytoplasmic
loop is not phosphorylated when cells are challenged with either C5a or
PMA. Thus, whether the cells are stimulated with C5a or PMA, the
phosphorylation sites appear to be restricted to serine residues in the
carboxyl tail. Phosphoamino acid analysis of a series of mutants in
which an individual serine residue was replaced by a threonine residue
indicates that the C5aR undergoes C5a-dependent phosphorylation to the
maximal stoichiometry of 6 mol of PO/mol of receptor at
Ser
, Ser
, Ser
,
Ser
, Ser
, and Ser
.
Simultaneous substitution of serine residues by alanine at positions
332, 334, and 338 affected neither the binding of C5a nor the cell
surface expression of the mutant, but resulted in a dramatic reduction
(more than 80%) of both C5a- and PMA-mediated phosphorylation as
compared to the wild type receptor. This result suggests that
phosphorylation on the segment extending from Ser
to
Ser
is required for the subsequent phosphorylation of the
carboxyl-terminal tail of C5aR.
Leukocytes respond to activating signals generated by
chemotactic factors, such as N-formyl peptides, C5a, ()interleukin-8, or platelet-activating factor (PAF), with a
complex array of biochemical events that culminate with the production
of superoxide and the release of lysosomal enzymes(1) . The
biological responses are initiated by binding of chemotactic factors to
receptors that are all members of the G protein-coupled receptor
family(2, 3, 4, 5, 6, 7) .
Despite the persistent presence of chemotactic factors, the cellular
responses are transient and cells become rapidly refractory to further
stimulation with the same agonist(8, 9) . This
phenomenon, termed homologous desensitization, is common to many
hormonal and neurotransmitter signaling
systems(10, 11) . Receptor phosphorylation appears to
be a key mechanism by which many G protein-coupled receptors are
regulated (12, 13, 14) .
It has been shown
recently that the C5aR is phosphorylated rapidly upon binding of C5a,
even in presence of staurosporine, a potent inhibitor of protein kinase
C(15, 16) . Moreover, disruption of the signaling
pathway by ADP-ribosylation of G proteins with pertussis
toxin does not reduce phosphorylation mediated by saturating doses of
agonist(49) . It is therefore postulated that a specific G
protein-coupled receptor kinase(s) (GRK) is involved in the process.
Phosphorylation of C5aR is correlated with an attenuation of
agonist-mediated GTPase activity in membranes prepared from
agonist-treated cells and may explain the homologous desensitization of
C5a mediated-responses(16) . In addition, C5aR but not the N-formyl peptide receptor (FPR), is phosphorylated in absence
of agonist in cells stimulated with phorbol 12-myristate 13-acetate
(PMA), a potent activator of protein kinase C(15) . However,
although the difference in phosphorylation between C5aR and FPR
correlates with the attenuation of C5a-dependent GTPase activity in
PMA-treated cells(16) , it is unclear whether protein kinase C
has a functional role in the heterologous desensitization of C5aR in
leukocytes stimulated with other chemoattractants. In differentiated
HL60 cells, although N-formyl-Met-Leu-Phe is thought to
activate protein kinase C it is unable to induce phosphorylation of
unoccupied C5aR(15) , whereas in transfected RBL-2H3 cells
thrombin and antigen have the capacity to induce phosphorylation of
unoccupied C5aR(16) .
By analogy with the visual and
-adrenergic systems(10, 17) , one can speculate
that chemoattractant receptor phosphorylation impairs coupling with the
G proteins. On the other hand, the rapid disappearance of
agonist-occupied chemoattractant receptors from the cell surface and
the apparent segregation of occupied receptors from the G protein
partner (18, 19, 20, 21) suggest
that the rapid loss of membrane receptors might represent an additional
mechanism whereby signal transduction is rapidly attenuated in
leukocytes. A definitive functional link between phosphorylation of the
C5aR and uncoupling from the G protein is yet to be established. It is
currently unknown whether a complete inhibition of the signal
transduction requires a molecular interaction between the
phosphorylated C5aR and arrestin-like molecules (22) , and
whether there is some relationship between the phosphorylation of
serine and/or threonine residues and internalization/sequestration of
occupied C5aR.
Prior to a clear demonstration of the role played by the phosphorylation of C5aR in uncoupling from G protein(s) and internalization/sequestration of occupied C5aR, a precise localization of phosphorylated sites is essential. Based on the hydropathy plot and the predicted topology of C5aR, several putative PKC phosphorylation sites can be identified in the cytoplasmic domain. The third intracellular loop between TMD5 and TMD6 contains the motif LRTWSRRA that conforms to the consensus sequence (R/K)XX(S/T)X(R/K)X for potential phosphorylation by protein kinase C. In addition, the predicted COOH-terminal tail contains 11 serine and threonine residues with several less favorable phosphorylation motifs for PKC.
In this
report, we identify phosphorylation sites of C5aR in vivo. We
show that the putative PKC phosphorylation motif in the third
intracellular loop is not modified even when phosphorylation is
mediated by PMA; both PMA-induced and C5a-induced phosphorylation of
the C5aR map in the COOH-terminal cytoplasmic tail and are restricted
to serine residues. In presence of agonist C5aR is phosphorylated with
the maximal stoichiometry of 6 mol of PO/mol of receptor.
We also examine the effect of the simultaneous replacement of
Ser
, Ser
, and Ser
by alanine
residues and determine that these residues are the main phosphorylation
sites in both PMA- and C5a-induced phosphorylation.
Figure 1:
Immunoprecipitation and phosphoamino
acid analysis of P-labeled C5aR. A,
phosphorylated C5aR was immunoprecipitated with affinity-purified IgG
from
P-labeled dHL-60 cells or transfected COS-7 cells
after stimulation with either 50 nM C5a or 1 µM PMA. Immunoprecipitates were analyzed by SDS-PAGE under reducing
conditions, and autoradiography. Immunoprecipitation of C5aR was
completely blocked by addition of competing C5aR COOH-terminal peptide.
All immunoprecipitates used in this study presented a similar pattern. B,
P-labeled C5aR was eluted from Sepharose
beads, hydrolyzed, and electrophoresed on cellulose TLC plates, as
described under ``Experimental Procedures.'' Positions of
phosphoserine (P-S), phosphothreonine (P-T),
and phosphotyrosine (P-Y) were assessed by ninhydrin staining
of the standards that were coelectrophoresed with the radioactive
phosphoamino acids. Radioactive spots that migrate below
phosphotyrosine are phosphopeptides arising during partial acid
hydrolysis. Cellulose TLC plates were then overexposed to Fuji x-ray
films at -80 °C. The figure is representative of at least
three independent experiments.
To identify
the nature of the phosphorylated residues, we subjected the P-labeled C5aR, eluted from the protein A-Sepharose beads,
to limited acid hydrolysis. The phosphoamino acids were resolved on
thin layer cellulose plates by two-dimensional electrophoresis and
visualized by autoradiography (Fig.1B). From these
experiments (n = 3), we observed that the
P-labeled C5aR proteins contained exclusively
phosphoserine residues whether they were purified from dHL-60 or from
transfected COS-7 cells stimulated with either C5a or PMA. This result
was quite unexpected in view of the elevated number of threonine
residues present in the putative third intracellular loop and the
cytoplasmic tail, which have been proved to be the main targets for
kinases in numerous receptors.
Figure 2:
Mapping of the phosphorylated regions of
C5aR after stimulation with either C5a or PMA. A, positions of
methionine residues and putative phosphorylation sites in the primary
sequence of the C5aR are indicated. The blackrectangles represent the putative TMDs. The serine/threonine-rich cytoplasmic
tail and the putative PKC motif in the third intracellular loop between
TMD5 and TMD6 have been expanded. The theoretical masses of the
peptides generated by CNBr cleavage are indicated for the wild type and
the mutant receptor Met
Leu (M
L). B,
phosphopeptide analysis after CNBr cleavage of
P-labeled
C5aR.
P-Labeled C5aR was immunoprecipitated with
affinity-purified IgG from COS-7 cells that were transfected either
with the wild type C5aR (Wt) or with the Met
Leu mutant form (M
L). Cells were stimulated with either 50
nM C5a (left panel) or with 1 µM PMA (right panel). The phosphorylated receptor was eluted from the
Sepharose beads, treated with CNBr for 16 h, and the CNBr-peptides were
separated by SDS-PAGE under reducing conditions using the multiphasic
buffer system described by Wiltfang et
al.(28) .
As illustrated in Fig.2(lower part), cleavage of wild type C5aR immunoprecipitated from C5a-stimulated COS-7 cells yielded a single phosphorylated fragment that migrated with an apparent molecular mass of 8-9 kDa on SDS-polyacrylamide gels. Uncleaved C5aR migrated in this electrophoretic system at the interface between the 10% separating gel and the 20% resolving gel. The lack of phosphorylated species in the range of 16 kDa suggests that the third intracellular loop is not phosphorylated after C5a stimulation. Surprisingly, the same pattern of phosphorylation was observed and a 16-kDa phosphorylated species was not detected after cleavage of wild type receptor immunopurified from PMA-treated cells. To rule out the possibility that the 8-9-kDa phosphorylated band is in fact the 16-kDa peptide with an aberrant electrophoretic behavior, we generated a mutant receptor in which the methionine residue at position 120 was replaced by leucine. With this mutant receptor, only two CNBr fragments with molecular masses of 8.8 and 29 kDa were expected (see Fig.2, upper part). As with the wild type receptor, the mutant receptor yielded a phosphorylated cyanogen bromide fragment in the range of 8-9 kDa whether the phosphorylation was mediated by C5a or by PMA. Lack of phosphorylated 16-kDa species could have resulted from an adsorption of this peptide on the glassware during freeze-drying. To rule out this possibility, experiments were performed in which the cleavage of C5aR was carried out in the gel slice. After washing, the gel slice was further loaded on a gel and phosphorylated peptides were resolved by SDS-PAGE. The same pattern of phosphorylated bands was observed (not shown). Thus, this strongly suggests that the third intracellular loop is not phosphorylated, despite the presence of an amino acid sequence that conforms to a PKC phosphorylation motif.
Together the results indicate that the carboxyl-terminal tail of unoccupied C5aR is the exclusive target for a PMA-stimulated kinase, presumably PKC, and that the same domain is also phosphorylated by a staurosporine-insensitive kinase(15, 16, 49) , presumably a member of the GRK family, which specifically recognizes the agonist-occupied state of C5aR.
Figure 3:
Phosphoamino acid analysis of P-labeled C5aR mutants. Individual serine residues in the
carboxyl tail of the C5aR were replaced with a threonine residue, and
each mutant was expressed in COS-7 cells. Three days post-transfection,
the cells were metabolically labeled with
P-labeled
orthophosphoric acid and stimulated with 50 nM C5a. After
immunoprecipitation,
P-labeled C5aR mutants were processed
for phosphoamino acid analysis, as described under ``Experimental
Procedures.'' Cellulose TLC plates were then exposed to Fuji x-ray
films at -80 °C or analyzed with a PhosphorImager. For each
mutant, the autoradiograph is representative of at least two
independent experiments. P-S, phosphoserine; P-T, phosphothreonine; P-Y,
phosphotyrosine.
To further characterize the region
of the carboxyl terminus of C5aR that is phosphorylated in the presence
of PMA, we constructed a mutant in which Ser,
Ser
, and Ser
were replaced with alanine
residues, leaving only Ser
, Ser
, and
Ser
as potential phosphorylation sites. This mutant
proved to be correctly transported to the surface of transfected COS-7
cells, as evidenced by an intense surface immunostaining of
permeabilized cells (data not shown). The level of surface expression
of the mutant receptor was further assessed by binding of
I-labeled C5a to the transfected COS-7 cell monolayers.
The binding capacity and the affinity of mutant and wild type receptors
were very similar (Fig.4). The dissociation constant (K
) was about 10 nM for wild
type and mutant receptors, and the maximal binding values (B
) were comparable (about 10
sites/cell for the wild type and 8.7
10
sites/cell for the mutant). The number of binding sites per cell
was consistently slightly lower in cells transfected with the mutant
cDNA, but the reduction never exceeded more than 15% (n = 3). However, when phosphorylation of the mutant was
assayed in COS-7 cells, we observed a dramatic reduction in the level
of phosphorylation although the mutant receptor behaved as a wild type
receptor with respect to surface expression and binding of C5a. It
exhibited no detectable basal phosphorylation and only a very weak
phosphorylation was observed when cells were challenged with either C5a
or PMA, as compared with the wild type receptor (Fig.5). Taking
into account the lower level of expression of the mutant receptor, we
estimated that the incorporation of phosphate represented less than 15%
of that found in the wild type receptor (n = 2),
although three additional phosphorylation sites (at positions 314, 317,
and 327) were present in the COOH-terminal cytoplasmic tail. These data
provide strong supporting evidence that the mutated serine residues are
primary phosphoacceptor sites in the basal phosphorylation and when
cells are stimulated with either PMA or C5a. In addition, they suggest
that the mechanism of phosphorylation proceeds in a hierarchical
manner. The lack of phosphorylation at Ser
,
Ser
, and/or Ser
may prevent the kinase(s)
to efficiently proceed further.
Figure 4:
Surface expression of Ser
Ala C5aR mutant. Identical amounts of COS-7 cells (2
10
cells) were transfected with the vector alone (mock-transfected), 10 µg of wild type (WT) or
mutant cDNA (A
), and 3 days
post-transfection, cell monolayers were assayed for their ability to
bind
I-labeled C5a in the presence or absence of 250
nM unlabeled C5a as described previously(4) . Binding
parameters were calculated by computer fitting using the iterative
nonlinear regression program Ligand(27) . The figure is
representative of three experiments.
Figure 5:
C5a- and PMA-induced phosphorylation of
wild type C5aR and Ser
Ala C5aR mutant.
Equal amounts of COS-7 cells (2
10
cells) were
transfected with 10 µg of wild type or mutant
(A
) cDNA. The ability of C5a or PMA to
induce phosphorylation of the wild type and the mutant receptors was
assayed after prelabeling cells with
[
P]orthophosphoric acid. Three days
post-transfection, cells were stimulated with either the buffer alone,
50 nM C5a, or 1 µM PMA for 15 min at 37 °C,
lysed, and then treated for receptor immunoprecipitation as described
under ``Experimental Procedures''. Immunoprecipitates were
analyzed by 10% SDS-PAGE under reducing conditions followed by
autoradiography or PhosphorImager analysis for quantification. Results
are representative of two independent
experiments.
To our knowledge this study represents the first attempt to
localize phosphorylation sites in C5aR in vivo. It
demonstrates that the Ser/Thr-rich carboxyl terminus of C5aR is the
main target for kinases in both C5a- and PMA-dependent phosphorylation.
Surprisingly, only phosphoserine residues were found to constitute the
phosphoacceptor sites, despite the existence of five threonine residues
within the cytoplasmic tail. A similar pattern of phosphorylation has
been described recently for another receptor, the luteinizing
hormone/CG receptor, in which the human chorionic gonadoptropin- and
PMA-stimulated phosphorylation occurs exclusively on serine residues of
the COOH-terminal cytoplasmic tail(30) . Such a situation is
nevertheless not a rule among G protein-coupled receptors. In the case
of rhodopsin, the photolyzed receptor is sequentially phosphorylated at
multiple serine and threonine residues in the COOH-terminal
region(31) . ()More recently, it has been shown with
a fusion protein containing the last 47 amino acids of FPR that the
carboxyl tail of FPR is sequentially phosphorylated by GRK2 on multiple
serine and threonine residues(32) . However, it remains to be
established in this latter case that the pattern of phosphorylation and
the kinase selectivity are identical in vivo.
One could
argue that the exclusive detection of phosphoserine is inherent to our
methodology that used a rabbit polyclonal antibody directed against the
COOH-terminal peptide (DTMAQKT
QAV) to
immunopurify C5aR. Indeed, phosphorylation of threonine residues at
positions 342 and 347 may impair the ability of the antibody to
precipitate C5aR and thereby lead to the exclusive immunoprecipitation
of a population of receptors that are not phosphorylated on these two
residues. However, this possibility is unlikely because identical
amounts of [
S]methionine-pulse-labeled C5aR were
immunoprecipitated from COS-7 cells whether cells were treated or not
treated with C5a (data not shown). This indicates that none of the
threonine residues present in the antigenic epitope are phosphorylated
or that their phosphorylation does not impair the formation of an
immune complex (if so, phosphothreonine should have been detected).
Thus, the phosphoacceptor sites are likely to be confined to serine
residues of the COOH-terminal cytoplasmic tail for agonist- and
PMA-dependent phosphorylation. Interestingly, four of the
phosphoacceptor sites of C5aR, i.e. Ser
,
Ser
, Ser
, and Ser
, are
conserved between human(3, 4) , mouse(33) ,
and dog C5aR(34) , whereas Ser
is replaced by an
alanine residue in the dog receptor and Ser
is replaced
by a threonine residue in the mouse receptor. High degree of
interspecies conservation of hydroxy amino acids at the former
positions provides additional support for a key role of these residues
in the regulation of C5aR.
The observation that the third
cytoplasmic loop is not phosphorylated with either C5a or PMA, despite
the presence of a putative PKC consensus motif, is not surprising if
one considers that the relatively short third cytoplasmic loop of
chemoattractant receptors is probably not a crucial region for coupling
to the G proteins. Mutagenesis approaches(35) , competition
strategies with fusion protein (36) or synthetic peptides
representing intracellular regions of FPR (37) were recently
used to probe the sites of interaction with the G protein(s). In all
the cases, it was concluded that the third cytoplasmic loop of FPR, and
presumably that of other members of the chemoattractant receptor
subfamily, was not critical for receptor/G protein coupling, whereas
the second intracellular loop and carboxyl-terminal tail of FPR
appeared to serve as the major contact sites with the G protein(s). Therefore, it is not unexpected to find the
incorporation of phosphate in the COOH terminus of C5aR. However, the
region encompassing residues 332-338, which contains the main
phosphorylation sites, is apparently not critical for binding and G
protein coupling as suggested by a previous report demonstrating that
deletion of the COOH-terminal region distal from position 327 does not
affect signal transduction in Xenopus oocytes(38) . In
contrast, the third intracellular loops of other members of the G
protein-coupled receptor superfamily, such as adrenergic and muscarinic
receptors, are much longer and include a sequence rich with
serine/threonine residues. In these receptors, the third loop has been
implicated as being of major importance in signal transduction and G
protein selectivity(39, 40) . Moreover, the
phosphorylation sites of the human mAChR m2 subtype expressed in Sf9
cells were assigned to serine and threonine residues in the central
part of the third intracellular loop(41) , and the agonist
phosphorylation of
-adrenergic receptor is
dramatically reduced if part of the third cytoplasmic loop is deleted (42) . In the latter case, four consecutive serine residues in
the third intracellular loop have been described as the sites for
GRK2-mediated phosphorylation and desensitization(43) .
The present study provides convincing evidence that the phosphoacceptor sites in the carboxyl tail of the C5aR expressed in COS-7 cells, and it is tempting to speculate that the phosphorylation of C5aR follows the same rules in myeloid cells. Several reasons lead us to believe that the results reflect, at least in part, the positions of the phosphorylation sites in receptors constitutively expressed in dHL60 cells. 1) Phosphoamino acid analysis of C5aR immunopurified from myeloid cells clearly indicates that serine residues are the main phosphorylated sites, and 2) cyanogen bromide cleavage of C5aR immunopurified from C5a- or PMA-treated dHL-60 cells yielded the same pattern of phosphopeptides as that of C5aR purified from COS-7 cells (data not shown). In dHL60 cells, as in COS-7, both PMA-mediated phosphorylation of unoccupied C5aR and C5a-mediated phosphorylation are restricted to serine residues of the carboxyl-terminal tail. However, we are unable to demonstrate whether in neutrophils or neutrophil-like cells the occupied C5aR is phosphorylated on the same serine residues, because presently we know very little about the kinase(s) that phosphorylates C5aR in myeloid cells. Although protein kinase C is likely to be involved in the basal phosphorylation of non-liganded receptor, protein kinase C is definitely not the chief enzyme involved in the phosphorylation of agonist-occupied C5aR. Indeed, C5a-dependent phosphorylation is mainly resistant to the action of PKC inhibitors, such as staurosporine(15, 16) , and is not abolished after disruption of the signal transduction pathway by pertussis toxin(49) . Therefore, it is postulated that the main kinase(s) involved in the phosphorylation of C5aR is a member of the G protein-coupled receptor kinase family.
The observation that C5aR is
equally well phosphorylated in dHL60 cells, C0S-7 cells, or rat
insulinoma cells (RINm5F) suggests that the kinase(s) involved in the
process is either widely distributed or that occupied C5aR is
efficiently phosphorylated by more than one member of the GRK family.
Messenger RNAs for both GRK2 and GRK6 are abundantly expressed in
myeloid cells, and both kinases might be involved in the regulation of
the chemoattractant receptors(44, 45) . GRK2 binds to,
and phosphorylates, the carboxyl terminus of FPR (32) and PAF
receptor in vitro(46) . However, presently there is
very little evidence that GRK2 phosphorylates the carboxyl terminus of
FPR and C5aR in vivo. The observation that PAF induces the
translocation of GRK2 from the cytosol to the plasma membrane of
neutrophils provides indirect evidence that PAF receptors interact with
and is probably regulated by GRK2 in neutrophils(44) . In
contrast, the lack of induction of GRK2 translocation by C5a suggests
that the C5aR does not interact with this kinase. However, this
conclusion is based on a single report and needs further confirmation.
It has been suggested previously on the basis of phosphorylation
studies with synthetic peptides that GRK2 is an acidotropic kinase,
which prefers acidic amino acids on the NH-terminal side of
serine or threonine residues(47) . In this context, it is worth
noting that in C5aR the sequences EES
VV and RES
could be potential phosphorylation sites for GRK2. Interestingly,
the amino acid alignment of the carboxyl-terminal sequence of C5aR with
that of FPR reveals that Ser
and Ser
in
C5aR correspond to Ser
and Thr
in FPR,
which are phosphorylated by GRK2 in vitro. In both receptors,
these residues are preceded by glutamic or a aspartic acid residues
(see Fig.6). Other serine residues that are in a neutral
(PS
LL) or an electropositive environment
(RKS
L, KS
F, and TRS
T) are
probably not the best substrates for GRK2. Localization of
phosphorylation sites in other G protein-coupled receptors has not been
delineated precisely, except in the case of rhodopsin, and presently it
is difficult to extract a consensus phosphorylation sequence for the
different GRKs. However, if one compares the carboxyl-terminal
sequences of C5aR, FPR, interleukin-8 receptor, PAF receptor, and
rhodopsin, it appears that the main phosphoacceptor sites of rhodopsin
and C5aR in vivo, as well as those of FPR in vitro,
are clustered in a region that presents striking similarities from one
receptor to the next (Fig.6).
Figure 6: Amino acid alignment of the COOH-terminal domains present in C5aR, FPR, rhodopsin, PAF receptor, and interleukin-8 receptor. Alignment was performed with DNAstar software using the J. Hein method. A consensus sequence was extracted when a residue was present in the same position in at least two sequences. Hydroxy amino acids for which phosphorylation has been demonstrated are boxed. Numbers refer to the positions of amino acids in C5aR.
On the basis of the lack of
phosphorylation of the mutant Ser
Ala, we
propose that C5aR is multiply phosphorylated in a sequential manner.
Ser
, Ser
, and/or Ser
are the
primary phosphoacceptor sites whose phosphorylation is essential to
allow the kinase to proceed further on other serine residues. A
sequential phosphorylation has been demonstrated previously in the case
of rhodopsin in which the light-dependent phosphorylation occurs first
on serine 338 and subsequently on serine 343 and threonine
336(31) . In the case of C5aR, an immediate question is whether
the basal phosphorylation plays any role in the phosphorylation
process. It is tempting to speculate that the basal phosphorylation of
C5aR on one of the key serine residues (332, 334, or 338) is important
to ``prime'' the agonist-occupied receptor as a good
substrate for a specific kinase, which will sequentially modify other
serine residues. Such a mechanism has been proposed for the
phosphorylation of glycogen synthase by glycogen synthase kinase-3 and
casein kinase II. In this system, a primary phosphorylation of glycogen
synthase by casein kinase II, is required to transform the protein into
a substrate for glycogen synthase kinase-3, which sequentially modifies
4 serine residues(48) . It will be interesting to test whether
the replacement of serine residues at positions 332, 334, and 338 of
C5aR with negatively charged residues could boost the C5a-mediated
incorporation of phosphate.
In conclusion, further studies are
required to establish how phosphorylation of the serine residues of the
COOH-terminal tail of C5aR contributes to the shut-down of signal
transduction. The observation that the mutant Ser
Ala is not phosphorylated further is of major importance
to examine the contribution of serine phosphorylation in homologous
desensitization and to investigate whether the agonist-induced
internalization of the C5aR is dependent on its phosphorylation status.
Examination of additional combinations of mutations to address this
issue is under way.