©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mitogen-activated Protein Kinase Activation Requires Two Signal Inputs from the Human Anaphylatoxin C5a Receptor (*)

(Received for publication, April 5, 1995; and in revised form, June 13, 1995)

Anne Mette Buhl (1) (2)(§) Shoji Osawa (3) Gary L. Johnson (2) (4)(¶)

From the  (1)Division of Biostructural Chemistry, Department of Chemistry, Aarhus University, DK-8000 Aarhus C, Denmark, the (2)Division of Basic Sciences, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206, the (3)Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill, North Carolina 27599, and the (4)Department of Pharmacology, University of Colorado Medical School, Denver, Colorado 80262

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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(i). Coexpression of the C5a receptor and the G alpha subunit (alpha) results in the G-mediated activation of phospholipase Cbeta and a robust MAP kinase activation. Pertussis toxin treatment of C5a receptor/alpha-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/alpha-cotransfected cells by 60%; the protein kinase C inhibitor does not affect the modest C5a receptor response in the absence of alpha expression. These results demonstrate that two independent signals are required for the maximal activation of MAP kinase by G protein-coupled receptors.


INTRODUCTION

Several groups have demonstrated that Raf-1 binds to RasbulletGTP (1, 2, 3, 4, 5) . Functional RasbulletGTP interaction with Raf-1 appears to be required for activation of Raf-1 kinase activity. Activated Raf-1 phosphorylates and activates MEK-1, (^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 RasbulletGTP in vitro is insufficient to substantially activate Raf-1 kinase activity(11, 12) . One predicted role of RasbulletGTP 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(i) and G(q) are capable of activating the Ras/Raf/MAP kinase pathway in different cell types(18, 19) . beta subunits can activate the MAP kinase pathway in a Ras dependent manner(20) . In most cell types activated Galpha(i) or Galpha(q) subunits do not activate the MAP kinase pathway by themselves. An exception is Rat1a cells, where GTPase-deficient alpha (alpha(i)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(i) and G, a homolog of G expressed in hematopoietic cells, but not to G(q) or G(22, 23) . Using this receptor system and the ability to selectively signal through G(i), G, or G(i) and G, we demonstrate that two signal inputs are required for maximal MAP kinase activation.


EXPERIMENTAL PROCEDURES

Materials

C5a was from Sigma. The protein kinase C inhibitor bisindoylmaleimide (GF109203X) was from Calbiochem.

Cell Culture and Transfection

HEK293 cells were grown in Dulbecco's modified Eagle's medium and 10% bovine calf serum. cDNAs were inserted in pCMV5 and used for transfection using the calcium phosphate precipitation procedure with 1 µg/ml of each expression plasmid(24) . Cells were incubated for 15 h and glycerol-shocked in 15% glycerol in Dulbecco's modified Eagle's medium for 1 min. After 65 h the cells were assayed in the various experiments.

Other Procedures

Phospholipase C activity was assayed as described(23) . MAP kinase activity was assayed following fractionation of cell lysates using Mono Q anion-exchange chromatography(21) . Detection of transfected proteins by immunoblot was performed as described previously(23) . All antisera were raised in rabbits against C-terminal peptides of the C5aR, alpha16, and transducin beta, respectively. All results are representative of at least three independent experiments giving similar results.


RESULTS

MAP Kinase Activation in Response to C5a Receptor Stimulation

The C5aR was expressed in HEK293 cells and characterized for its ability to regulate MAP kinase activity. C5a activation of the expressed C5aR modestly stimulates MAP kinase activity (Fig. 1A). When coexpressed with the G alpha subunit (alpha), activation of the C5aR gives a strong MAP kinase stimulation. Expression of alpha in the absence of the C5aR did not influence basal MAP kinase activity. Immunoblotting demonstrated expression of both the C5aR and alpha (Fig. 2).


Figure 1: Regulation of MAP kinase and PLCbeta activities by the transfected C5aR. A, cells transfected with pCMV5 without a cDNA insert (Control), pCMV5alpha alone, pCMV5C5aR, or the combination of pCMV5alpha + 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 alpha 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 alpha and the C5aR were incubated for 16 h with 1 µCi/ml myo-[2-^3H]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, alpha, and beta(1) subunit. Sixty-five h after transfection with pCMV5C5aR, pCMV5alpha or pCMV5beta(1) + pCMV5(2) cells were lysed and 200 µg of each lysate separated by SDS-polyacrylamide gel electrophoresis. Rabbit sera for C5aR, alpha, and beta(1) 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(i) proteins in 293 cells (Fig. 1B). Interestingly, the MAP kinase activation observed with C5aR stimulation in the presence of alpha 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 alpha, it had no significant effect on the C5aR activation of phospholipase Cbeta activity (Fig. 1C). These findings indicate that both a functional G(i) and G protein were required for maximal MAP kinase activation in response to C5aR stimulation, but only G was required for phospholipase C activation.

beta Subunit Complex Activation of MAP Kinase

It has been postulated that G protein beta subunits, not alpha subunits, are the major contributor to seven transmembrane receptor stimulation of MAP kinase activity(20) . Fig. 3A demonstrates that transient expression of different beta(1), beta(2), (2), and (3) combinations are capable of modestly activating MAP kinase. The magnitude of the MAP kinase activation with beta subunit expression was comparable to that observed with the pertussis toxin-sensitive acute C5aR stimulation in the absence of alpha (Fig. 1). It is important to note that activated Ras (Val12Ras), when transiently expressed in HEK293 gave a very strong MAP kinase activation (Table 1). Thus, the MAP kinase pathway can sustain a high activity for the 48-65 h after transfection with a constitutively activated upstream regulator. The fact that beta-stimulated MAP kinase activity is modest in this assay suggests that beta overexpression is unable to sustain maximal MAP kinase activation even though beta subunit expression can persistently activate phospholipase Cbeta activity (see below). beta-Subunit overexpression was unable to activate MAP kinase in a manner similar to Val12Ras, indicating that beta-subunits alone cannot sustain a significant Ras activation in HEK293 cells.


Figure 3: Activation of MAP kinase activity by G protein beta subunits. A, cells were cotransfected with pCMV5 expression plasmids encoding beta(1) and (2), or (3) and beta(2) and (2), or (3). 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 pCMV5PLCbeta(2), pCMV5beta(2) + pCMV5(2), or the combination of all three expression plasmids. Forty-nine h after transfection, cells were labeled with 1 µCi/ml myo-[2-^3H]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 alpha subunit polypeptide gives a strong acute activation of MAP kinase. Since alpha activates phospholipase Cbeta (PLCbeta)(22, 23) , this suggests that products of PLCbeta-catalyzed reactions involving either diacylglycerol and/or inositol phosphates are critical for mediating maximal MAP kinase activation in response to C5aR stimulation. Coexpression of beta(2)(2) G protein subunits with PLCbeta2 resulted in a significant PLCbeta activation in HEK293 cells (Fig. 3B). However, persistent activation of PLCbeta2 in the presence of beta(2)(2) overexpression did not significantly stimulate MAP kinase activity over that observed with beta(2)(2) alone (Fig. 3C). Thus, persistent PLCbeta(2) stimulation in the presence of beta 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).

Protein Kinase C Inhibition Inhibits C5aR/GalphaStimulation of MAP Kinase

The protein kinase C inhibitor GF109203X (25) strongly inhibits the robust MAP kinase activation observed with coexpression of C5aR and alpha (Fig. 4). The modest MAP kinase activation seen with the C5aR in the absence of alpha coexpression is unaffected by incubation of HEK293 cells with GF109203X. We have previously shown inhibition of cells with GF109203X does not inhibit MAP kinase activation in response to N-formylmethionyl-leucyl-phenylalanine (FMLP) peptide in human neutrophils(26) . These findings therefore indicate that a functional protein kinase C is required for maximal C5aR stimulation of MAP kinase activity, and that GF109203X is not inhibiting component kinases in the sequential MAP kinase pathway (Raf/MEK/MAP kinase).


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, alpha, 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.



Expression of alpha(s)G226A Inhibits C5aR-stimulated MAP Kinase and PLCbeta Activity

Mutation of Gly-226 Ala in Galpha(s) (alpha(s)G226A) prevents the mutant alpha(s) polypeptide from dissociating from beta subunits even in the presence of bound GTP(27) . The alpha(s)G226A polypeptide can therefore function as a ``sink'' for beta subunits similar to the use of Galpha(t) by other investigators(20, 28) . Expression of alpha(s)G226A inhibited C5aR/alpha stimulated MAP kinase and PLCbeta activity (Fig. 5). The approximate 50% inhibition of both MAP kinase and PLCbeta activity by alpha(s)G226A, when alpha and the C5aR are coexpressed, indicates that alpha(s)G226A probably competes with alpha for beta subunits in transfected HEK293 cells. The concomitant loss of MAP kinase and PLCbeta stimulation argues that functional Galphabeta heterotrimers in addition to Galpha(i)beta are required for maximal C5aR activation of MAP kinase. This is similar to the requirement of Galphabeta complexes for PLCbeta stimulation by the C5aR(29) .


Figure 5: Inhibitory effects of alphasG226A expression on C5aR/alpha16 regulation of MAP kinase activation. A, cells were transfected with pCMV5C5aR or pCMV5C5aR+pCMV5alpha16 with or without alphasG226A (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-^3H]inositol and 20 h later assayed for phospholipase C activity.




DISCUSSION

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) , (^2)and it has also been postulated that beta 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 beta subunits were sufficient to activate MAP kinase and that beta subunits mimicked receptor stimulation of the MAP kinase pathway(20) . A second study also using COS cell transfections suggested the ability of G protein beta subunits to activate MAP kinase was modest at best(28) , suggesting that overexpression of beta 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(i) 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 PLCbeta activity, so the action of the inhibitor must be downstream of G protein activation. (^3)The ability of pertussis toxin to block C5aR activation of MAP kinase indicates that G(i) is also involved. Because GTPase-deficient alpha does not activate MAP kinase in HEK293 cells,^3 the apparent contribution from G(i) is its beta subunit. This is consistent with the magnitude of beta-mediated MAP kinase activation being similar to the C5aR response in the absence of Galpha. Coexpression of beta subunits and PLCbeta 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 PLCbeta 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(q)-coupled receptors(32) .

From these observations and the necessity of two signal inputs for maximal MAP kinase activation, G protein beta subunits alone could maximally activate MAP kinase activity only if the two signal inputs were both beta-stimulated. For example, significant expression of PLCbeta2 or PLCbeta3 activated by beta 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 PLCbeta response to the C5aR in the absence of Galpha expression, indicating that there is insufficient PLCbeta2 or PLCbeta3 expressed for a G(i)-dependent, beta-mediated C5aR response; the PLCbeta response must be reconstituted by expression of Galpha. 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 beta 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 beta activation of the pathway. Similarly, persistent Galpha subunit responses such as those observed with the GTPase-deficient alphaQ205L and alpha(q)Q209L polypeptides is insufficient for MAP kinase activation(32) .^3 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) .


FOOTNOTES

*
This work was supported by National Institutes of Health Grants GM30324 and DK37871. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a grant from the Danish Natural Science Research Council.

To whom correspondence should be addressed: Division of Basic Sciences, National Jewish Center for Immunology, and Respiratory Medicine, 1400 Jackson St., Denver, CO 80206. Tel.: 303-398-1504; Fax: 303-398-1225.

(^1)
The abbreviations used are: MEK, mitogen-activated protein kinase/Erk kinase; MAP, mitogen-activated protein kinase; C5aR, C5a receptor; PLC, phospholipase C.

(^2)
N.-X. Qian and G. L. Johnson, manuscript in preparation.

(^3)
A. M. Buhl and G. L. Johnson, unpublished results.


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