(Received for publication, August 10, 1995; and in revised form, October 2, 1995)
From the
The binding of small peptide ligands to high affinity
chemoattractant receptors on the surface of neutrophils and monocytes
leads to activation of heterotrimeric G-proteins, stimulation of
phosphatidylinositol-phospholipase C (PI-PLC), and subsequently to the
inflammatory response. It was recently shown (Amatruda, T. T., Gerard,
N. P., Gerard, C., and Simon, M. I.(1993) J. Biol. Chem. 268,
10139-10144) that the receptor for the chemoattractant peptide
C5a specifically interacts with G, a G-protein
subunit of the G
class, to trigger ligand-dependent
stimulation of PI-PLC in transfected cells. In order to further
characterize this chemoattractant peptide signal transduction pathway,
we transfected cDNAs encoding the formylmethionylleucylphenylalanine
receptor (fMLPR) into COS cells and measured the production of inositol
phosphates. Ligand-dependent activation of PI-PLC was seen in COS cells
transfected with the fMLPR and G
and stimulated with
fMLP but not in cells transfected with receptor alone or with receptor
plus G
. Chimeric receptors in which the N-terminal
extracellular domain, the second intracellular domain, or the
intracellular C-terminal tail of the fMLP receptor was replaced with
C5a receptor domains (Perez, H. D., Holmes, R., Vilander, L. R., Adams,
R. R., Manzana, W., Jolley, D., and Andrews, W. H.(1993) J. Biol.
Chem. 268, 2292-2295) were capable of ligand-dependent
activation of PI-PLC when co-transfected with G
. A
chimeric receptor exchanging the first intracellular domain of the
fMLPR was constitutively activated, stimulating PI-PLC in the absence
of ligand. Constitutive activation of PI-PLC, to a level 233% of that
seen in cells transfected with wild-type fMLP receptors, was dependent
on G
. Site-directed mutagenesis of the first
intracellular domain of the fMLPR (amino acids 54-62) reveals
this to be a domain necessary for ligand-dependent activation of
G
. These results suggest that different receptors
which mediate similar biochemical responses may utilize distinct
mechanisms to activate G-proteins. Differences among the signaling
pathways triggered by chemoattractant factor receptors suggest an
opportunity for pharmacologic modifications of the inflammatory
response.
The responses of neutrophils and monocytes to bacterial
infection are regulated by chemoattractant or proinflammatory factors,
small peptide or lipid molecules that are ligands for high affinity
receptors on the surface of inflammatory cells(1) . G-proteins,
or heterotrimeric GTP-binding regulatory proteins, have been implicated
in signal transduction of the response to chemoattractant ligands
through biochemical studies of the effects of GTP analogues on
chemoattractant factor binding and activation of phospholipases in
permeabilized cells and membranes(2) . The ability of pertussis
toxin (Bordetella pertussis islet-activating protein) to
inhibit signaling by many chemoattractant ligands in neutrophils and
monocytes implies a major role for pertussis toxin-sensitive G-protein
heterotrimers in chemoattractant factor
signaling(1, 3) . Two G-protein subunit isotypes
that are modified by pertussis toxin, G
and G
, are expressed in inflammatory
cells and have been proposed to regulate the pertussis
toxin-inhibitable activation of PI-PLC (
)observed in
neutrophils and monocytes(1, 4) .
Chemoattractant
peptides and other G-protein-coupled ligands also activate PI-PLC
through pertussis toxin-resistant signal transduction pathways in
inflammatory cells(5, 6) . We and others recently
determined that G, a G
class G-protein
that is expressed in hematopoietic cells, specifically and potently
interacts with the receptors for the chemoattractant peptide C5a to
trigger ligand-dependent activation of PI-PLC in transfected cells (7, 8) . In the studies presented here we further
explore signal transduction by this pathway. First, we examined the
activation of PI-PLC by the receptor for the chemoattractant peptide
fMLP in transfected cells. Then, we examined the role of specific
domains of the fMLP receptor in signaling through the use of chimeric
and mutagenized chemoattractant factor receptors.
Heterologous cells transfected with the receptor for the
chemoattractant ligand fMLP have ligand binding properties similar to
those seen in inflammatory cells(10, 11) . Although
rapid activation of PI-PLC is seen in inflammatory cells after exposure
to this ligand, COS cells expressing the fMLP receptor show only a
slight ligand-dependent activation of PI-PLC (31 ± 9% increase
in IP production (S.E., n = 8 experiments) (Fig. 1). In cells transfected with G or with
the fMLP receptor and G
, a 2-fold increase in
base-line IP release was seen. Subsequent addition of fMLP ligand
resulted in a dose-dependent increase in IP production, up to a maximal
level of 349 ± 25%, the level seen in unstimulated cells (S.E., n = 17) (Fig. 1). As in the case of the C5a
receptor(7) , activation of PI-PLC by the fMLP receptor was
dependent on expression of G
, and no ligand-dependent
activation of PI-PLC was seen after co-transfection of G
with the fMLP receptor (Table 1).
Figure 1:
Accumulation of
[H]inositol phosphates in COS-7 cells transfected
with the human fMLP receptor. COS-7 cells were transfected with the
fMLP receptor or with the fMLP receptor plus G
,
labeled, washed, and stimulated with medium alone (0) or fMLP ligand as
described under ``Materials and Methods.'' Levels of IP were
determined as described. Each shaded bar represents the mean
of three assays from a representative experiment. Clear bars represent standard error.
These findings support
previous reports that the signal transduction pathways triggered by
fMLP and C5a are similar(12, 13) . In order to
investigate the role of specific domains of these receptors in the
activation of PI-PLC via the G pathway, we
transfected COS cells with cDNA clones, which encode chimeric
chemoattractant factor receptors. The cell surface expression and
ligand binding properties of these receptors have been previously
described(10) . Fig. 2shows schematic diagrams of the
receptor chimeras. The chimeric receptor in which the extracellular
N-terminal domain of the fMLP receptor was replaced by the
corresponding region of the C5a receptor (Ch 15) was capable of
interacting with G
to stimulate hydrolysis of
phosphatidylinositol. Addition of ligand resulted in an increase in IP
production to a level 3.5 times that seen in unstimulated cells (Fig. 2). A similar pattern was seen in chimeric receptors in
which the second intracellular loop (Ch 18) or the C-terminal tail (Ch
7) of the fMLP receptor were replaced with corresponding domains of the
C5a receptor (Fig. 2). In cells transfected with the chimeric
receptors, fMLP ligand induced activation of PI-PLC in a dose-dependent
pattern but with an increased ED
when compared with
wild-type fMLP receptors expressed in COS cells (Fig. 3).
Ligand-dependent stimulation of IP production by these receptors was
dependent on co-expression of G
. As in the wild-type
fMLP receptor, only a minor activation of PI-PLC was seen in COS cells
transfected with chimeric receptors alone, and no ligand-dependent
activation of PI-PLC was seen in cells transfected with receptors and
with another G-protein of the G
class (Table 1).
Figure 2:
Accumulation of inositol phosphates in
COS-7 cells transfected with chimeric chemoattractant peptide
receptors. COS-7 cells were transfected with a control
-galactosidase plasmid (Lac) or with the G
cDNA and the following receptor clones: an fMLP receptor
construct (10) or the chimeric receptors Ch 7, Ch 15, Ch 18, or
Ch 16. Schematic diagrams of receptor clones show intracellular domains
below and extracellular domains above. C5a receptor domains are marked
in black. Cells were labeled and then stimulated with medium
alone (0) or with 500 nM fMLP (+), and levels of inositol
phosphate were measured. The figure shows results from a representative
experiment. Each shaded bar represents the mean of three
assays ± S.E. (clear bars).
Figure 3:
Dose-response analysis of activation of
PI-PLC by fMLP receptor chimeras. COS 7 cells were transfected with the
G cDNA and the fMLP receptor or the chimeric
receptors Ch 7, Ch 15, or Ch 18. Cells were labeled and stimulated with
fMLP ligand (0-500 nM), and then levels of IP production
were determined. The figure displays IP production for each receptor
clone relative to the maximal production seen when cells transfected
with that clone were stimulated with 500 nM fMLP. Maximal IP
production averaged 5,192 cpm. The figure represents results from three
independent experiments ± S.E. of the mean, normalized relative
to total incorporation of [
H]myoinositol into
membranes.
A
contrasting pattern was seen in cells transfected with a chimeric fMLP
receptor in which the first intracellular domain was replaced with the
corresponding domain of the C5a receptor. In this receptor, Ch 16, the
region of the wild-type fMLP receptor cDNA encoding the sequence
LVIWVAGFRMTHTVTTISYLNKAVA was replaced with a portion of the C5a
receptor cDNA which encoded LVVWVTAFEAKRTINAIWFLNLAVA. This chimeric
receptor triggered activation of PI-PLC in the absence of ligand (Fig. 2). The level of IP production in cells transfected with
this receptor and with G was 233 ± 18% of the
level seen in cells transfected with native fMLP receptors and
G
(S.E., n = 14; p <
0.0001). Addition of fMLP ligand (5-500 nM) to cells
expressing this receptor resulted in a further increase in IP
production. The constitutive activation of PI-PLC by this receptor
required co-expression of G
(Table 1).
The
studies with chimeric receptor Ch 16 suggest that the first
intracellular domain of the chemoattractant receptors plays a role in
the activation of G. Hydrophobicity plots predict
that the residues RMTHTVTTI comprise the first intracellular domain of
the fMLP receptor. In order to analyze the role of this region of the
receptor in the activation of G
, we studied the
signal transduction properties of fMLP receptors that were mutagenized
by cassette alanine mutagenesis(20) . The activation of PI-PLC
in cells transfected with G
and with the mutagenized
receptors is shown in Fig. 4. Mutant receptor clone p301,
encoding the sequence, RMTAAATTI, allowed ligand-dependent activation
of PI-PLC at ligand concentrations of 50-500 nM. In
contrast, the receptor p300 (AAAHTVTTI) triggered an attenuated
increase in PI-PLC activation when stimulated with fMLP, and receptor
p312 (RMTHTVAAA) did not activate PI-PLC when stimulated with ligand.
None of the mutagenized receptors triggered constitutive activation of
PI-PLC. These receptors had diminished ligand binding capacity;
receptor p301 bound fMLP with a calculated K
of 50
nM, while receptors p300 and p312 bound ligand with K
of >50 nM, and equilibrium binding
could not be demonstrated (data not shown).
Figure 4:
Accumulation of inositol phosphates in
COS-7 cells transfected with fMLP receptor mutants. COS cells were
transfected with cDNA clones encoding -galactosidase (Lac), G
, fMLP receptor +
G
, mutant receptors p300 + G
,
p301 + G
, or p312 + G
.
Cells were then labeled with [
H]myoinositol and
stimulated with fMLP peptide, 500 nM (+), or
medium(-), and inositol phosphate production was measured as
described. Each shaded bar represents the mean of three assays
from a representative experiment. Clear bars represent
standard error.
Chemoattractant peptide
ligands can activate a G class G-protein,
G
, in transfected cells. The signal transduction
properties of fMLP receptors reported here are similar to those
described for the C5a receptor expressed in COS cells (7, 8) . The physiological role of G
in the response of inflammatory cells to these ligands has not
been defined. While inhibition of the chemoattractant factor responses
of inflammatory cells by PTX implies that the major signal transduction
pathway activated by chemoattractant ligands is through G-protein
heterotrimers including G
or
G
(4, 13) , G
could account for aspects of the chemoattractant response which
are not inhibited by
PTX(1, 5, 6, 7, 12) .
Studies with chimeric chemoattractant peptide receptors may allow
delineation of the functional roles of receptor domains in ligand
binding and signal transduction(10, 14, 15) .
Exchange of the N-terminal extracellular domain (Ch 15), second
intracellular loop (Ch 18), or C-terminal tail (Ch 7) from the C5a to
the fMLP receptor results in receptors that are capable of signal
transduction via G. These domains are proposed to be
similar in overall topology and to share regions of primary sequence
homology (13) . The chimeric receptors were previously shown to
be expressed on the cell surface and to bind fMLP ligand. We noted an
increase in the EC
of these chimeric receptors, compared
with wild-type fMLP receptors. This could reflect decreased ligand
binding affinity or cell surface expression of the chimeric receptors (10) or may indicate alterations in the interactions of
chimeric receptors with G
.
Our results suggest
that the second intracellular domains of the fMLP and C5a receptors
confer similar functional properties to these receptors. In earlier
studies, replacement of the second intracellular domain of the fMLP
receptor with the corresponding region of a receptor of similar
structure, the FPR2 receptor, allowed ligand binding and calcium
mobilization through a PTX-sensitive signaling pathway(14) .
Synthetic peptides corresponding to this region of the fMLP receptor
were found to interact with G and possibly
to inhibit the association of the fMLP receptor with G-protein
heterotrimers(16, 17) . Together with the present
findings, these results support the hypothesis that the second
intracellular domain of chemoattractant peptide receptors contains a
structural motif which is generally necessary for activation of
G-proteins by seven-transmembrane domain receptors. A hydrophobic
residue present in this domain both in the fMLP receptor (Leu-128) and
the C5a receptor (Phe-139) has been proposed to be essential for
activation of G
by muscarinic and adrenergic
receptors(18) .
Studies with chimeric receptor Ch 7 indicate
that the C-terminal tail of the C5aR can substitute for the homologous
region of the fMLPR in the activation of G. Note that
a region of the tail of the fMLPR (residues 323-339,
LTEDS
TQTSDTATNSTL) is conserved in the C5aR (LTEESVVRESKSFTRSTV) (13) . This chimeric receptor was previously shown to bind fMLP
ligand with high affinity. When expressed with G
,
this receptor is capable of activation of PI-PLC in a ligand-dependent
pattern and is not constitutively activated. Conservation of ligand
binding and ligand-induced calcium mobilization has previously been
demonstrated in chimeric receptors in which the C-terminal domain of
the fMLP receptor was replaced with the analogous region of the FPR2
receptor(14) . In addition, a functional role for this domain
of the fMLP receptor has been proposed from studies using synthetic
peptides, which interfere with the interaction of receptors with
G
i
or G
i
(16, 17) .
Our studies suggest that the first intracellular domain of
chemoattractant peptide receptors regulates the activation of PI-PLC
via the G pathway. The constitutive activation of
G
by chimeric receptor Ch 16 implies that the first
intracellular domains of the C5a and fMLP receptors are not
functionally equivalent, despite similar topology and some conservation
of primary sequence. Previous studies of the first intracellular domain
of the fMLP receptor have not indicated that it is involved in signal
transduction. A receptor chimera in which the first intracellular
domain of the fMLP receptor was replaced with a highly conserved
homologous region of the FPR2 receptor showed a decrease in ligand
binding affinity similar to that seen with fMLP
C5a receptor
chimera Ch 16(14) . However, the fMLP
FPR2 receptor
chimera did not trigger constitutive activation of calcium
mobilization. Possible mechanisms to account for constitutive
activation of the chimeric fMLP
C5a receptor Ch 16 include: (a) the first intracellular loop of fMLP receptor may function
as an inhibitory domain, which constrains activation of the unliganded
wild-type receptor(19) ; (b) the first intracellular
loop of the C5a receptor may directly activate G
when
removed from the context of the wild-type receptor; or (c)
exchange of the first intracellular loop could disrupt the conformation
of the receptor and indirectly unmask a receptor domain, which
activates G
.
Mutagenesis of the first
intracellular domain of the fMLP receptor confirms the involvement of
this region of the receptor in signal transduction through
G. Each of these mutant receptors manifested dramatic
decreases in ligand binding, suggesting that alterations in the first
intracellular domain disrupted either the conformation of contiguous
ligand-binding regions of the receptor or the receptor-G-protein
interaction, which is necessary for high affinity binding. Previously,
small changes in sequence in the first and second extracellular domains
of the fMLP receptor were shown to disrupt ligand
binding(10, 20) . Replacement of residues 54-56
and 60-62 with alanines attenuated or abolished the signaling
properties of the receptors. These amino acids are predicted to sit at
the intracellular face of the receptor, a region that has been
implicated in G-protein activation through mutagenesis of several
classes of receptors(21) . The observation that replacement of
these residues did not lead to constitutive activation argues that the
first intracellular domain of the fMLP receptor is not a specific
inhibitory region that constrains receptor activation(19) . Our
results are most consistent with the hypothesis that the first
intracellular domain of both the fMLP and C5a receptors activates
G
and predict that mutagenesis of the homologous
region of the C5a receptor or the Ch 16 chimera will also decrease
activation of G
by these receptors.
Our findings
have several implications for the model of chemoattractant peptide
signaling. First, we confirm that chemoattractant peptide ligands can
activate PI-PLC through a G class signal transduction
pathway (G
). The physiological importance of this
pathway remains undetermined. Second, our results confirm that exchange
of certain domains of the fMLP and C5a receptor results in receptors
that are capable of binding ligand and activating a G-protein signal
transduction pathway. Third, we have demonstrated that the first
intracellular domains of the fMLP and C5a receptors are not
interchangeable and that the first intracellular domain of the fMLP
receptor is necessary for activation of G
. The
mechanism by which the chimeric receptor is constitutively activated
remains unclear but apparently does not entail the elimination of a
constraining or inhibitory region in the first intracellular domain of
the fMLPR. The constitutive activation of G
by this
chimeric receptor implies that these two receptors with apparently
similar physiological actions differ in the mechanism by which they
activate a G-protein pathway. This hypothesis receives indirect support
from phylogenetic analysis. The first intracellular domain of the fMLP
receptor is strongly conserved between mouse (FRMKHTVTT) and
human (FRMTHTVTT)(13) , and the human fMLP receptor is capable
of activating the murine homologue of G
,
G
15.
In contrast, the primary sequence of the C5a
receptor homologue from mouse (FEPDGPSNA) diverges from the
human receptor in this region (FEAKRTINA)(13) . Understanding
the physiological significance of these differences in receptor
properties will require further insight into the role of G
in the physiology of cells of hematopoietic lineage.