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(Received for publication, July 2, 1996)
From the Departments of Pharmacology and ABSTRACT A variety of receptors coupled to
GTP-binding regulatory proteins (G proteins) initiate signals that
culminate in activation of the mitogen-activated protein kinases ERK1
and ERK2. We demonstrate here that the human 5-HT1A
receptor expressed in Chinese hamster ovary cells similarly promotes
activation of ERK1 and ERK2, but that the pathway used does not conform
entirely to those proposed previously for G protein-coupled receptors.
Activation of ERK2 by the 5-HT1A receptor-selective agonist
8-hydroxy-N,N-dipropyl-2-aminotetralin hydrobromide
(8-OH-DPAT) was inhibited completely by pertussis toxin and
substantially by prolonged treatment of cells with phorbol 12-myristate
13-acetate. The implied requirement for protein kinase C, however, was
negated in studies with bisindolylmaleimide and Ro-31-8220, which,
although completely inhibiting activation of ERK2 by phorbol ester, had
no impact on activation by 8-OH-DPAT. The anticipated inhibition by the
tyrosine kinase inhibitors genistein and herbimycin A, moreover, was
marginal at best. As expected for a Gi-coupled receptor,
the inhibitors of phosphatidylinositol 3-kinase wortmannin and LY294002
inhibited activation of ERK2, albeit only partly (70%). Of
significance, an inhibitor of a phosphatidylcholine-specific
phospholipase C, tricyclodecan-9-yl-xanthogenate (D609), caused a
similar degree of inhibition. When the two types of inhibitors were
combined, an almost complete inhibition was achieved. Our data suggest
that phosphatidylinositol 3-kinase and phosphatidylcholine-specific
phospholipase C represent components of different, but partly
overlapping pathways that can account almost entirely for the
activation of ERK2 by the 5-HT1A receptor.
INTRODUCTION Perhaps the most detailed models of activation of
mitogen-activated protein kinases (MAP
kinases)1 such as ERK1 and ERK2 by
receptors coupled to GTP-binding regulatory proteins (G proteins) are
those provided by Lefkowitz and colleagues, in which the scheme of
events depends on the G protein utilized. For agonists proceeding
through Gi, release of Despite the compelling nature of these models, a greater degree of
complexity clearly exists. In many instances, the pharmacological
interventions used to identify the requisite steps do not completely
attenuate activation of the MAP kinases. Conflicts also exist as to
whether inhibitors of tyrosine kinases necessarily inhibit activation
of Ras and/or MAP kinases achieved through Gi (5), an issue
that may ultimately devolve to the nature of the cells and agonists
employed. The extent to which PKC is involved in the activation of MAP
kinases, too, is elusive. Phorbol ester-promoted down-regulation of
PKC, the most often used means of assay, is selective for only certain
forms of PKC (6, 7) and can additionally affect processes in which PKC
need not be an upstream component (8, 9). Additional permutations in
pathways leading to activation of MAP kinases, moreover, may exist.
Daub et al. (10), for example, provide evidence for a scheme
wherein the stimulation of MAP kinases in fibroblasts by endothelin,
lysophosphatidic acid, and thrombin occurs through G protein-coupled
receptors, but with epidermal growth factor (EGF) receptors used as an
intermediate in the activation cascade.
In the studies described here, the human
5-hydroxytryptophan1A (5-HT1A) receptor was
employed to investigate further the pathways leading to activation of
MAP kinases by G protein-coupled receptors. We report that the
5-HT1A receptor is coupled through Gi to the
activation of ERK1 and ERK2 in CHO cells, but that the pathway(s) used
does not conform entirely to those proposed previously for G proteins.
While the activation is sensitive to inhibitors of PI 3-kinase, as
expected for a Gi-coupled process, a similar degree of
inhibition can also be achieved with tricyclodecan-9-yl-xanthogenate
(D609), an inhibitor of phosphatidylcholine-specific phospholipase C
(PC-PLC). Our data suggest that PI 3-kinase and PC-PLC represent
components of different but interacting pathways that can account
almost entirely for agonist-induced activation of MAP kinase.
EXPERIMENTAL PROCEDURES (±)8-Hydroxy-N,N-dipropyl-2-aminotetralin
hydrobromide (8-OH-DPAT) was purchased from Research Biochemicals
(Natick, MA). Rabbit polyclonal IgGs recognizing ERK1 (C-16) and ERK2
(C-14) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
The 9-amino acid MAP kinase substrate peptide, containing residues
95-98 of myelin basic protein, was purchased from Upstate
Biotechnology Inc. (Lake Placid, NY). Wortmannin, phorbol 12-myristate
13-acetate (PMA), herbimycin A, Ro-31-8220, LY294002,
bisindolylmaleimide, and PTX were purchased from Calbiochem (San Diego,
CA). Tricyclodecan-9-yl-xanthogenate, D609, was purchased from
Calbiochem or Biomol (Plymouth Meeting, PA).
4-(2 CHO cells expressing the human
5-HT1A receptor (by means of a
XbaI-BamHI restriction fragment of G21 (11) in
pcDNA1/neo) were provided as a gift from Dr. Perry B. Molinoff. The
cells were maintained in monolayer culture in Ham's F-12 nutrient
mixture with L-glutamine, 10% charcoal-treated fetal
bovine serum, 400 µg/ml Geneticin, and 1% penicillin-streptomycin at
37 °C in a humidified atmosphere (5% CO2). The
Bmax for [125I]MPPI was 2.5 pmol/mg of membrane protein.
MAP kinase activity was
measured following immunoprecipitation of ERK1 or ERK2 essentially as
described by Flordellis et al. (12). Cells were first
cultured overnight in serum- and Geneticin-free medium, then treated
with agonists and/or other reagents as described in the text.
Incorporation of 32P into substrate peptide was measured by
scintillation spectrometry after extensive washing of filters with
0.75% phosphoric acid and acetone.
Inositol phosphate accumulation was measured
as [3H]IP2 and
[3H]IP3 production following labeling of
cells with myo-[3H]inositol (13).
Water-soluble metabolites of phosphatidylcholine were measured 2-3
days following labeling of cells with
[methyl-3H]choline as release of radioactivity
into extracellular medium (14); the period of treatment with
agonists was 40 min. Immunoprecipitation of G protein subunits labeled
in CHO cells with [35S]methionine (150 µCi/ml) was
achieved with the peptide-directed antibodies 8730 (recognizing
RESULTS AND DISCUSSION Activation of the human 5-HT1A receptor by the
selective agonist 8-OH-DPAT promotes activation of ERK1 (data not
shown) and ERK2 (Fig. 1) in CHO cells. As shown for
ERK2, the activation is dose-dependent, with an
EC50 of 4 nM, and transient. The activity
achieved with 8-OH-DPAT is almost identical to that observed with
serotonin (data not shown). The activation is blocked by the
5-HT1A receptor-selective antagonist MPPI, and does not
occur for CHO cells that were not transfected with the plasmid encoding
the 5-HT1A receptor.
In most types of cells, the 5-HT1A receptor utilizes G
proteins sensitive to PTX, i.e. Gi and/or
Go (16, 17). Gz, which is not PTX-sensitive,
may also be used (15). When the CHO cells were treated overnight with
PTX, activation of ERK2 by 8-OH-DPAT was completely suppressed (Fig.
2). Thus, the receptor uses Gi or
Go. CHO cells contain Gi, but data for the
existence of Go are inconsistent (4, 18, 19). We therefore
investigated the complement of potentially relevant G proteins using
immunoprecipitation of
8-OH-DPAT causes a greater than 80% inhibition of forskolin-stimulated
cAMP accumulation in CHO cells expressing the 5-HT1A
receptor (data not shown). However, inhibition of adenylyl cyclase
cannot account for the activation of MAP kinase. Treatment of the CHO
cells with the cAMP analogs 8-bromo-adenosine-3 Like other investigators, we found that exposure of cells to PMA for a
short period of time results in activation of MAP kinase (Fig.
3). This activation can be completely prevented by
pretreatment of the cells overnight with PMA. Consistent with studies
on platelet-activating factor signaling in CHO cells (4), pretreatment
with PMA also inhibits activation of MAP kinase by 8-OH-DPAT. However,
the inhibition was incomplete, and the fact that 8-OH-DPAT might rely
on PKC to any extent was surprising since we had found that this
agonist does not stimulate PI hydrolysis in CHO cells (data not shown).
We therefore further tested the involvement of PKC using the PKC
inhibitor bisindolylmaleimide at a concentration of 1 µM
(Fig. 3). As anticipated, activation of MAP kinase by PMA was inhibited
by this reagent. However, bisindolylmaleimide had no affect on
activation of MAP kinase by 8-OH-DPAT. Similar results were obtained
with the PKC inhibitor Ro-31-8220, also used at 1 µM. We
conclude that while prolonged treatment with PMA inhibits
activation of MAP kinase by 8-OH-DPAT, possibly indirectly through some
form of cellular accommodation (8, 9), 8-OH-DPAT utilizes a pathway
distinct from traditional forms of PKC.
Given previous work with Gi coupling to Shc phosphorylation
(2) and receptor coupling to MAP kinase (20, 21), a possibly key
component in the activation of MAP kinase induced by 8-OH-DPAT is PI
3-kinase. Indeed, we find the activation of MAP kinase to be inhibited
by wortmannin (Fig. 4). The inhibition is notable at 10 nM and achieves a maximum of 70% by 30 nM. The
concentrations causing inhibition are consistent with those required
for inhibition of PI 3-kinase (22). However, because the specificity of
wortmannin for PI 3-kinase alone has been questioned (23), we also
examined the actions of the structurally different inhibitor LY294002
(24). The inhibition of MAP kinase achieved with LY294002 was
similar to that achieved by wortmannin, again at concentrations
consistent with inhibition of PI 3-kinase. These observations suggest
that PI 3-kinase forms an important link between the 5-HT1A
receptor and the activation of MAP kinase. However, PI 3-kinase does
not form the only link, as a significant level of activation occurs
even in the presence of saturating concentrations of wortmannin or
LY294002.
A critical question, therefore, is what comprises the
wortmannin/LY294002-insensitive pathway of activation. We turned our
attention to the phosphatidylcholine-specific phospholipase C (PC-PLC).
While PC has long been viewed as a source of diacylglycerol for
sustained activation of PKC (25), products of its hydrolysis (perhaps
diacylglycerol itself) are also critical to the activation of Raf by
Ras in response to receptor tyrosine kinase activation (26). We
therefore tested the possibility that PC hydrolysis is required for
activation of MAP kinase by a G protein-coupled receptor, and that it
forms a pathway in part distinct from that sensitive to wortmannin and
LY294002. Tricyclodecan-9-yl-xanthogenate (D609) is a specific
inhibitor of PC-PLC (27, 28). Remarkably, D609 inhibits
8-OH-DPAT-stimulated MAP kinase activity to an extent comparable to
that of wortmannin or LY294002 (Fig. 4, bottom panel). The
concentration used was 50 µg/ml, a concentration comparable to those
used previously to inhibit PC-PLC (26, 27, 28, 29, 30, 31). Increasing the
concentration of D609 to 100 µg/ml caused no further inhibition (data
not shown). When CHO cells were treated simultaneously with both D609
and a PI 3-kinase inhibitor (either wortmannin or LY294002),
8-OH-DPAT stimulated activity was almost completely inhibited. Thus,
the pathways leading to activation of MAP kinase can be accounted for
almost entirely by those inhibited by wortmannin and D609.
Identification of PC-PLC using D609, of course, is subject to issues of
selectivity. As noted above, D609 inhibits PC-PLC in a variety of
cells. In separate experiments here, 8-OH-DPAT and PMA elicited small
but significant increases PC metabolism as measured by release of
radioactivity from cells labeled with [3H]choline
(60 ± 18% and 88 ± 12%, respectively (n = 4)). The increases were reversed by D609. The effects of D609 on the
actions of PMA were anticipated from previous studies examining
PMA-stimulated PC-PLC or sequellae (26, 27). As reported elsewhere,
D609 does not have an effect on phospholipase A2,
phospholipase D, PI-PLC, or sphingomyelinase activities in U937 cells
(28), nor on PI-PLC or PI 3-kinase activities in NIH 3T3 cells (26).
These data notwithstanding, the conclusive demonstration of a
role for PC-PLC will require other inhibitors and/or means to eliminate
enzymic activity.
The magnitude of inhibition achieved with wortmannin and D609 alone and
together suggests that the pathways presumably using PI 3-kinase and
PC-PLC are not entirely redundant nor completely independent. PC-PLC
may well be interposed between Ras and Raf. Cai et al. (26,
32) report that the activation of PC-PLC in NIH 3T3 cells by serum and
EGF is attenuated by the dominant negative form of Ras, RasN17, as is
cell replication, and that introduction of a bacterial form of PC-PLC
bypasses the block to cell replication. Introduction of the enzyme does
not bypass the block to replication achieved with dominant negative
forms of Raf. Moreover, PC-PLC activates Raf, and the activation of Raf
by serum or EGF is completely inhibited by D609 (26). Thus, one or more
products of PC hydrolysis likely support the activation of Raf either
following its recruitment to membrane by Ras or in an independent
fashion. With respect to 8-OH-DPAT-stimulated MAP kinase activity, we
hypothesize that the wortmannin-sensitive pathway is relevant to the
activation of Ras, e.g. through Grb2·Sos, and that
activation of Raf by Ras in turn is enhanced by PC hydrolysis. PC
hydrolysis itself can nevertheless represent an independent input to
the activation of Raf or downstream components of the MAP kinase
activation cascade. Our data imply two convergent pathways relevant to
the activation of MAP kinases by a single G protein-coupled receptor,
both of which are required for optimal activation.
FOOTNOTES Acknowledgments We thank Dr. Perry B. Molinoff for the kind
gift of CHO cells expressing the 5-HT1A receptor and Dr.
Paul Butkerait for performing the analysis of G proteins present in
these cells.
REFERENCES
Volume 271, Number 37,
Issue of September 13, 1996
pp. 22297-22300
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
COMMUNICATION:
,
Psychiatry
at the University of Pennsylvania School of Medicine,
Philadelphia, Pennsylvania 19104-6084
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
leads to phosphorylation of
Shc and consequent engagement of Grb2 and Sos through steps thought to
involve phosphatidylinositol 3-kinase (PI 3-kinase) and a Src-like
tyrosine kinase (1, 2). For agonists employing Gq, the
sequence of events appears to entail stimulation of phosphoinositide
metabolism by 
q and subsequent activation of protein
kinase C (PKC) and thus Raf (3). A similar pathway has been proposed
for an agonist using Go, wherein o is the
subunit implied to stimulate phosphoinositide metabolism (4).
-Methoxyphenyl)-1-[2-[N-(2"-pyridinyl)-p-iodobenzamidoethyl]piperazine
(MPPI) was kindly provided by Dr. H.F. Kung.
i1 and i2 equally, and
i3 less well), 9072 (directed toward o1,
presumably recognizing o2 also), and 2921 (z) as described previously (15).
Fig. 1.
Stimulation of MAP kinase activity by
8-OH-DPAT. CHO cells expressing the 5-HT1A receptor
were incubated with the indicated concentrations of 8-OH-DPAT for 5 min
(A), with 1 µM 8-OH-DPAT for the indicated
times (B), and in the absence (Vehicle) or
presence (DPAT) of 1 µM 8-OH DPAT ± 10 µM MPPI for 5 min (C). ERK2 was
immunoprecipitated, and its activity was measured as described under
``Experimental Procedures.'' The data shown in panels A
and B are single experiments that are representative of four
total each. The data shown in panel C are the means ± S.E. of four separate experiments, each performed in triplicate, with
activity expressed as a percentage of 8-OH-DPAT-stimulated
activity.
subunits labeled with
[35S]methionine. The CHO cells employed here contain one
or more forms of i. They do not contain detectable
levels of o or z.
Fig. 2.
Sensitivity of 8-OH-DPAT-stimulated MAP
kinase activity to pertussis toxin. CHO cells expressing the
5-HT1A receptor were treated overnight with 100 ng/ml PTX,
or not, then incubated for 5 min with vehicle or 1 µM
8-OH-DPAT. ERK2 was immunoprecipitated, and its activity was measured
as described under ``Experimental Procedures.'' The data shown are
the means ± S.E. of three separate experiments, each performed in
duplicate. The figure on the right is an autoradiogram (18-h
exposure) of immunoprecipitates obtained from cells incubated with
[35S]methionine using antibodies directed toward
i, o, and z. Bands other
than the one depicted by the arrow (pointing to
i) are nonspecific.
-5-monophosphate or
N6,2-O-dibutyryl-adenosine-3-5-monophosphate
(1 mM each) had no effect on agonist activation of ERK2,
nor did forskolin or 3-isobutyl-1-methylxanthine. Herbimycin A and
genistein, inhibitors of tyrosine kinases, had little impact.
Herbimycin A at a concentration of 1 µg/ml had no effect on
8-OH-DPAT-stimulated MAP kinase activity. Genistein at 75 µM similarly had no effect, although at 100 µM caused an approximately 35% inhibition.
Concentrations beyond 100 µM appeared to be toxic. We
also tested whether EGF had any effect, given the report by Daub
et al. (10) that the EGF receptor was a necessary link to
activation of MAP kinases by G protein-coupled receptors. EGF did not
stimulate ERK2 in the CHO cells, implying the absence of EGF receptors
capable of serving as intermediates for 8-OH-DPAT.
Fig. 3.
Sensitivity of 8-OH-DPAT-stimulated MAP
kinase activity to inhibition of PKC. CHO cells expressing the
5-HT1A receptor were pretreated with or without 100 nM PMA for 16 h (A) or with or without 1 µM bisindolylmaleimide for 30 min (B), then
with 100 nM PMA or 1 µM 8-OH-DPAT for 5 min.
ERK2 was immunoprecipitated, and its activity was measured as described
under ``Experimental Procedures.'' Data are expressed as the
means ± S.E. of four separate experiments, each performed in
triplicate.
Fig. 4.
Sensitivity of 8-OH-DPAT-stimulated MAP
kinase activity to inhibitors of PI 3-kinase and PC-PLC. CHO cells
expressing the 5-HT1A receptor were incubated for 5 min
with 1 µM 8-OH-DPAT after preincubation for 30 min with
either vehicle (0.1% Me2SO) or wortmannin at the specified
concentrations (A), LY294002 at the specified concentrations
(B), or 30 nM wortmannin, 50 µg/ml D609, or a
combination of the two drugs (C). ERK2 was
immunoprecipitated, and its activity was measured as described under
``Experimental Procedures.'' Results are expressed as means ± S.E. of triplicate samples and are representative of three or more
experiments.
§
To whom correspondence should be addressed: Dept. of Pharmacology,
University of Pennsylvania School of Medicine, 3620 Hamilton
Walk, Philadelphia, PA 19104-6084. Tel.: 215-898-1775; Fax:
215-573-2236.
1
The abbreviations used are: MAP kinase,
mitogen-activated protein kinase; 8-OH-DPAT,
8-hydroxy-N,N-dipropyl-2-aminotetralin hydrobromide; 5-HT,
5-hydroxytryptamine(serotonin); CHO, Chinese hamster ovary; EGF,
epidermal growth factor; ERK, extracellular signal-regulated kinase,
also referred to as MAP kinase; MPPI,
4-(2-methoxyphenyl)-1-[2-[N-(2"-pyridinyl)-p-iodobenzamidoethyl]piperazine;
PC, phosphatidylcholine; PI, phosphatidylinositol; PKC, protein
kinase C; PLC, phospholipase C; PMA, phorbol 12-myristate
13-acetate; PI 3-kinase, phosphatidylinositol 3-kinase; PTX, pertussis
toxin.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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A. J. Barr and D. R. Manning Agonist-independent Activation of Gz by the 5-Hydroxytryptamine1A Receptor Co-expressed in Spodoptera frugiperda Cells. DISTINGUISHING INVERSE AGONISTS FROM NEUTRAL ANTAGONISTS J. Biol. Chem., December 26, 1997; 272(52): 32979 - 32987. [Abstract] [Full Text] [PDF]
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J. Cheng, J. D. Weber, J. J. Baldassare, and D. M. Raben Ablation of Go alpha -Subunit Results in a Transformed Phenotype and Constitutively Active Phosphatidylcholine-specific Phospholipase C J. Biol. Chem., July 11, 1997; 272(28): 17312 - 17319. [Abstract] [Full Text] [PDF]
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Y. Wan, K. Bence, A. Hata, T. Kurosaki, A. Veillette, and X.-Y. Huang Genetic Evidence for a Tyrosine Kinase Cascade Preceding the Mitogen-activated Protein Kinase Cascade in Vertebrate G Protein Signaling J. Biol. Chem., July 4, 1997; 272(27): 17209 - 17215. [Abstract] [Full Text] [PDF]
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C. A. Chen and D. R. Manning Regulation of Galpha i Palmitoylation by Activation of the 5-Hydroxytryptamine-1A Receptor J. Biol. Chem., July 28, 2000; 275(31): 23516 - 23522. [Abstract] [Full Text] [PDF]
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