(Received for publication, April 5, 1995; and in revised form, June 13, 1995)
From the
The anaphylatoxin C5a receptor activates the
Ras/Raf/mitogen-activated protein (MAP) kinase pathway in human
neutrophils. The signal pathways involved in Ras/Raf/MAP kinase
activation in response to C5a and other chemoattractant receptors is
poorly understood. Stimulation of the C5a receptor expressed in HEK293
cells results in modest MAP kinase activation, which is inhibited by
pertussis toxin-catalyzed ADP-ribosylation of G.
Coexpression of the C5a receptor and the G
subunit
(
) results in the G
-mediated activation
of phospholipase C
and a robust MAP kinase activation. Pertussis
toxin treatment of C5a receptor/
-cotransfected cells
inhibits C5a stimulation of MAP kinase activity approximately 60%
relative to the control response. Similarly, the protein kinase C
inhibitor, GF109203X inhibits activation of MAP kinase activation in
C5a receptor/
-cotransfected cells by 60%; the protein
kinase C inhibitor does not affect the modest C5a receptor response in
the absence of
expression. These results demonstrate
that two independent signals are required for the maximal activation of
MAP kinase by G protein-coupled receptors.
Several groups have demonstrated that Raf-1 binds to
RasGTP (1, 2, 3, 4, 5) .
Functional Ras
GTP interaction with Raf-1 appears to be required
for activation of Raf-1 kinase activity. Activated Raf-1 phosphorylates
and activates MEK-1, (
)the MAP kinase/Erk
kinase(6, 7, 8) , which in turn
phosphorylates and activates MAP kinase(9) . Even though the
expression of v-Ras activates the Raf-1/MAP kinase pathway in
cells(10) , the interaction of Raf-1 with Ras
GTP in
vitro is insufficient to substantially activate Raf-1 kinase
activity(11, 12) . One predicted role of Ras
GTP
is to bring Raf-1 into proximity at the cytoplasmic surface of the
plasma membrane for a second regulatory event required for Raf-1
activation(13, 14) . This second regulatory event has
been proposed to involve protein kinase C in some cell
types(15) , and different protein kinase C isotypes have been
shown to directly phosphorylate Raf-1(16, 17) .
Protein kinase C-catalyzed phosphorylation of Raf-1 increased Raf-1
autophosphorylation but alone did not increase activity toward
MEK-1(11) . Thus, it is probable that both Ras-dependent and
Ras-independent regulatory events are required for Raf-1 activation
leading to increased MAP kinase activity. The Ras-independent events
leading to Raf-1 activation are not unequivocally defined but may
involve protein kinase C.
Seven transmembrane receptors coupled to
G and G
are capable of activating the
Ras/Raf/MAP kinase pathway in different cell
types(18, 19) .
subunits can activate the
MAP kinase pathway in a Ras dependent manner(20) . In most cell
types activated G
or G
subunits do
not activate the MAP kinase pathway by themselves. An exception is
Rat1a cells, where GTPase-deficient
(
Q205L) is capable of activating MAP
kinase(21) . In this report we have used the C5a receptor
(C5aR) expressed in HEK293 cells. The C5aR couples to G
and
G
, a homolog of G
expressed in
hematopoietic cells, but not to G
or
G
(22, 23) . Using this receptor system
and the ability to selectively signal through G
,
G
, or G
and G
, we demonstrate
that two signal inputs are required for maximal MAP kinase activation.
Figure 1:
Regulation of
MAP kinase and PLC activities by the transfected C5aR. A,
cells transfected with pCMV5 without a cDNA insert (Control),
pCMV5
alone, pCMV5C5aR, or the combination of
pCMV5
+ pCMV5C5aR were challenged with 50 nM C5a for 5 min. Cells were lysed and the lysates fractionated by
Mono Q FPLC and assayed for MAP kinase activity. B, cells
transfected with the same combinations of expression plasmid for
and the C5aR as described in A were
incubated for 16 h in the presence or absence of 100 ng/ml pertussis
toxin (Ptx). Cells were stimulated with 50 nM C5a for
5 min and then lysed and assayed for MAP kinase activity as in A. C, cells transfected with the indicated
combinations of expression plasmids for
and the C5aR
were incubated for 16 h with 1 µCi/ml myo-[2-
H]inositol in the absence or
presence of pertussis toxin (Ptx). Cells were then challenged
with 50 nM C5a for 5 min and assayed for phospholipase C
activity as described
previously(23) .
Figure 2:
Immunoblotting of expressed C5aR,
, and
subunit. Sixty-five h after
transfection with pCMV5C5aR, pCMV5
or
pCMV5
+ pCMV5
cells were lysed
and 200 µg of each lysate separated by SDS-polyacrylamide gel
electrophoresis. Rabbit sera for C5aR,
, and
proteins were used for immunoblotting as described
under ``Experimental
Procedures.''
The modest activation of MAP
kinase in response to stimulation of the C5aR was pertussis
toxin-sensitive, indicating this response was coupled to G proteins in 293 cells (Fig. 1B). Interestingly,
the MAP kinase activation observed with C5aR stimulation in the
presence of
was inhibited approximately 60% by
pertussis toxin. Although pertussis toxin treatment of cells markedly
inhibited the MAP kinase response to C5aR stimulation in the presence
of
, it had no significant effect on the C5aR
activation of phospholipase C
activity (Fig. 1C).
These findings indicate that both a functional G
and
G
protein were required for maximal MAP kinase activation
in response to C5aR stimulation, but only G
was required
for phospholipase C activation.
Figure 3:
Activation of MAP kinase activity by G
protein subunits. A, cells were cotransfected with
pCMV5 expression plasmids encoding
and
, or
and
and
, or
. Sixty-five h after
transfection, cells were lysed and the lysates fractionated by Mono
Q-FPLC and assayed for MAP kinase activity. B, cells were
transfected with pCMV5PLC
, pCMV5
+ pCMV5
, or the combination of all three
expression plasmids. Forty-nine h after transfection, cells were
labeled with 1 µCi/ml myo-[2-
H]inositol and 16 h later assayed
for phospholipase C activity. C, cells transfected as in B were assayed for MAP kinase activity as described in A.
The
C5aR coexpressed with the subunit polypeptide gives
a strong acute activation of MAP kinase. Since
activates phospholipase C
(PLC
)(22, 23) , this suggests that products of
PLC
-catalyzed reactions involving either diacylglycerol and/or
inositol phosphates are critical for mediating maximal MAP kinase
activation in response to C5aR stimulation. Coexpression of
G protein subunits with PLC
2
resulted in a significant PLC
activation in HEK293 cells (Fig. 3B). However, persistent activation of PLC
2
in the presence of
overexpression
did not significantly stimulate MAP kinase activity over that observed
with
alone (Fig. 3C). Thus, persistent PLC
stimulation in the presence of
overexpression cannot
sustain a maximal MAP kinase activity. This contrasts with activated
Ras which is able to sustain a high MAP kinase activity (Table 1).
Figure 4:
Effect of the protein kinase C inhibitor
GF109203X (GF) on C5aR stimulation of MAP kinase activity.
Cells were transfected with the indicated pCMV5 expression plasmids
encoding the C5aR, , or having no cDNA insert (Control). Sixty-four h after transfection cells were
incubated with 3.5 µM GF109203X in 0.1% dimethyl sulfoxide
or 0.1% dimethyl sulfoxide alone for 60 min, and then stimulated with
50 nM C5a for 5 min. The cells were then lysed, and the lysate
fractionated by Mono Q FPLC and assayed for MAP kinase
activity.
Figure 5:
Inhibitory effects of sG226A
expression on C5aR/
16 regulation of MAP kinase activation. A, cells were transfected with pCMV5C5aR or
pCMV5C5aR+pCMV5
16 with or without
sG226A (1 µg/ml).
Sixty-five h post-transfection, MAP kinase activity was measured
following a 5-min stimulation with C5a. B, cells were
transfected with the various cDNAs as in A. Forty-five h
post-transfection, the cells were incubated with 1 µCi/ml myo-[2-
H]inositol and 20 h later assayed
for phospholipase C activity.
G protein-regulated pathways leading to MAP kinase activation
have not been clearly delineated. As with tyrosine kinases, a
functional Ras protein is required for G protein-coupled seven
transmembrane receptor activation of MAP
kinase(18, 30) . In several studies the inhibitory
mutant Asn17Ras protein inhibits G protein-mediated MAP kinase
activation(20) , ()and it has also been postulated
that
subunits mediate G protein activation of Ras leading to
increased MAP kinase activity(20) . In one study in particular,
using COS cell transient transfections, it was postulated that G
protein
subunits were sufficient to activate MAP kinase and
that
subunits mimicked receptor stimulation of the MAP
kinase pathway(20) . A second study also using COS cell
transfections suggested the ability of G protein
subunits to
activate MAP kinase was modest at best(28) , suggesting that
overexpression of
subunits was insufficient to give a strong
MAP kinase activation.
Our studies presented here with the seven
transmembrane G protein-coupled C5a receptor and the work of others (11, 12) using tyrosine kinase-regulated MAP kinase
activation indicate that two regulatory responses are required for
maximal MAP kinase stimulation. We demonstrate that for the C5aR both a
G and a G
regulatory event are required for
maximal MAP kinase activation. The G
regulatory event is
inhibited by the protein kinase C inhibitor GF109203X. The role of
protein kinase C may be to directly regulate Raf-1 activity (16) or to regulate the activity of a second kinase that
regulates Raf-1 activity(15) . GF109203X treatment of cells has
no effect on C5aR stimulation of PLC
activity, so the action of
the inhibitor must be downstream of G protein activation. (
)The ability of pertussis toxin to block C5aR activation of
MAP kinase indicates that G
is also involved. Because
GTPase-deficient
does not activate MAP kinase in
HEK293 cells,
the apparent contribution from G
is its
subunit. This is consistent with the magnitude
of
-mediated MAP kinase activation being similar to the C5aR
response in the absence of G
. Coexpression of
subunits and PLC
was insufficient, however, to
persistently activate a strong MAP kinase activity. It is likely that
either the protein kinase C or the Ras GDP exchange factor regulatory
pathways become refractory to activation with persistent PLC
stimulation. Consistent with this idea is the finding that phorbol
ester down-regulation of protein kinase C partially inhibits
stimulation of MAP kinase activity by G
-coupled
receptors(32) .
From these observations and the necessity of
two signal inputs for maximal MAP kinase activation, G protein
subunits alone could maximally activate MAP kinase activity
only if the two signal inputs were both
-stimulated. For
example, significant expression of PLC
2 or PLC
3 activated by
subunits could provide the second signal input in addition
to Ras activation for strong MAP kinase stimulation. Neither HEK293 nor
COS (not shown) cells have a measurable PLC
response to the C5aR
in the absence of G
expression, indicating that there
is insufficient PLC
2 or PLC
3 expressed for a
G
-dependent,
-mediated C5aR response; the
PLC
response must be reconstituted by expression of
G
. The requirement for two signal inputs for G
protein regulation of MAP kinase activation obviously restricts the
ability of different G protein-coupled receptors to regulate MAP kinase
activity. Release of
subunits without an appropriate second
input will give only modest responses. If cAMP is also elevated, the
MAP kinase response may actually be
inhibited(33, 34, 35, 36, 37, 38) ,
preventing the modest
activation of the pathway. Similarly,
persistent G
subunit responses such as those observed with the
GTPase-deficient
Q205L and
Q209L
polypeptides is insufficient for MAP kinase
activation(32) .
The requirement for two signal
inputs for maximal MAP kinase activity allows a graded MAP kinase
response to be regulated by one or more G protein signals from one or
more receptor inputs. Thus, the MAP kinase pathway is another example
of ``coincidence detection'' of multiple signal inputs in
regulating cytoplasmic signaling in response to extracellular stimuli
for the control of cell phenotype(31) .