Pertussis Toxin-Sensitive and -Insensitive Thrombin Stimulation of Shc Phosphorylation and Mitogenesis Are Mediated through Distinct Pathways
William A. Ricketts,
Joan Heller Brown and
Jerrold M. Olefsky
Program in Biomedical Sciences (W.A.R., J.H.B., J.M.O.)
University of California San Diego, La Jolla, California
92037-0673
Department of Pharmacology (J.H.B.) University
of California San Diego La Jolla, California 92037-0645
Veterans Administration Research Service (J.M.O.), San
Diego, California 92161
Whittier Diabetes Program
(J.M.O.) La Jolla, California 92093
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ABSTRACT
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Activation of both receptor tyrosine kinases
(RTKs) and G protein-coupled receptors (GPCRs) result in
phosphorylation of the adaptor protein Shc, providing sites of
interaction for proteins in downstream signal transduction cascades.
The mechanism of Shc phosphorylation and its function in G protein
signaling pathways is still unclear. By examining Shc phosphorylation
in response to thrombin in two cell lines, we have defined distinct
pertussis toxin (PTX)-sensitive and -insensitive mechanisms by which
GPCRs can stimulate tyrosine phosphorylation of Shc. By mutating the
tyrosines in Shc, we show that the three sites of tyrosine
phosphorylation, Y239, Y240, and Y317, are necessary for thrombin
signaling in both systems. The SH2 (src homology 2) domain of Shc is
also critical for signaling, but not required for phosphorylation of
Shc. In both cell types, inhibition of src family member kinases by
chemical inhibitors or microinjection block Shc phosphorylation and
bromodeoxyuridine (BrdU) incorporation in response to thrombin.
However, in the PTX-sensitive thrombin pathway, both ß
function
and the epidermal growth factor receptor (EGFR) are necessary for Shc
phosphorylation and BrdU incorporation. In contrast, signaling in
the PTX-insensitive pathway is not mediated through ß
or the
EGFR. Thus, while phosphorylation and function of Shc appear to be the
same in both thrombin pathways, the mechanism of tyrosine kinase
activation proximal to Shc is different. The differences in signaling
between the two thrombin pathways may be representative of mechanisms
used by other PTX-sensitive and -insensitive GPCRs to mediate
specific responses. In addition, transactivation of RTKs may be a
manner by which GPCRs can amplify their signal.
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INTRODUCTION
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Signaling by receptor tyrosine kinases (RTKs) and G
protein-coupled receptors (GPCRs) has been viewed as occurring through
distinct mechanisms. RTKs possess an intrinsic kinase activity that
allows them to activate intracellular signaling components by direct
phosphorylation (1). Phosphorylation of the other partner in the
receptor dimer can also activate cytoplasmic proteins by providing
sites of interaction that localize the proteins near to their
activators or effectors (2, 3). GPCR signaling is initially mediated by
the heterotrimeric G proteins (4). GPCRs do not have tyrosine kinase
activity but, instead, increase the exchange of GDP for GTP on the
heterotrimeric G proteins, resulting in the separation of the
- and
ß
-subunits (5). However, the finding that both pathways can
activate a similar set of signal transducers, including src, Ras, and
mitogen-activated protein kinase (MAPK), indicated more
parallels than originally thought (6, 7, 8, 9, 10, 11).
One of the proteins recently established as being required for
signaling by both RTKs and GPCRs is the adaptor protein Shc (12, 13, 14, 15, 16, 17, 18).
Shc contains no catalytic domain but does encode an amino-terminal
phosphotyrosine binding (PTB) domain and a carboxy-terminal src
homology 2 (SH2) domain (19, 20). The PTB and SH2 both interact with
phosphotyrosine-containing sequences, but these regions are used
differentially to mediate signals in response to specific growth
factors (21). Between these two domains is a region termed the collagen
homology (CH) domain, which contains three tyrosines, Y239, Y240, and
Y317 (20, 22). The best studied of the three tyrosines, Y317, is
involved in Ras activation by targeting the Grb2-SOS complex to the
membrane (23, 24). Less understood, Y239 and Y240 appear necessary for
the induction of c-myc expression (22, 25).
Shc phosphorylation has been well documented as an early
signaling event leading to MAPK activation (11, 14). There appear to be
multiple pathways to achieve Shc phosphorylation in response to stimuli
(16). The classical pathway for Shc activation by a RTK was via
interaction of one of the phosphotyrosine interaction domains with the
activated RTK and its subsequent phosphorylation on tyrosine 317 (18, 23). It is now clear that Shc is phosphorylated and necessary in GPCR
pathways, but the precise mechanism by which GPCR stimulation induces
Shc phosphorylation remains unclear (12, 13, 15, 26). Expression of
ß
-subunits of the heterotrimeric G proteins can lead to Shc
phosphorylation by members of the src family of kinases (SFKs), but
expression of a wild-type or a constitutively activated form of the
-subunit of G12 can also induce Shc phosphorylation (12, 15). In
addition, several lines of evidence support cross-talk and
transactivation between RTKs and GPCRs (5, 27). In particular, several
groups have implicated the epidermal growth factor (EGF) receptor
(EGFR) as a necessary signaling component in response to GPCR
activation (28, 29, 30, 31, 32, 33, 34).
We examined Shc phosphorylation by thrombin as a means to further
elucidate the mechanisms of tyrosine kinase activation by GPCRs. The
experiments were conducted in cell lines that are distinguished by
whether thrombin induces Shc phosphorylation and DNA synthesis in a
pertussis toxin (PTX)-sensitive or -insensitive manner. Our results
demonstrate that there are at least two distinct mechanisms of Shc
phosphorylation by G proteins: ligand activation of PTX-insensitive G
proteins induces Shc phosphorylation through the
-subunit and SFK
activation, but PTX-sensitive G proteins function through
ß
-subunits and transactivation of the EGFR. Therefore, we conclude
that G protein activation of tyrosine kinases occurs differently,
depending on the role of
- and ß
-subunits in the pathway.
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RESULTS
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The Mitogenic Response to Thrombin Can Be Mediated through a
PTX-Sensitive or Insensitive Pathway
Thrombin stimulates DNA synthesis and Shc phosphorylation in both
HIRcB and 1321N1. To assess the PTX sensitivity of these responses, we
treated cells with 100 ng/ml PTX for 6 h before assaying the
effects on subsequent thrombin signaling (Fig. 1
). Thrombin-stimulated bromodeoxyuridine
(BrdU) incorporation in HIRcB cells was inhibited by 84%
(P < 0.01), whereas PTX treatment had no effect in
1321N1 cells (P > 0.05) (Fig. 1A
). Longer treatments
with PTX produced similar results (35). PTX treatment was also an
effective inhibitor of thrombin-stimulated Shc phosphorylation in HIRcB
cells but not in 1321N1 cells. EGF-stimulated Shc phosphorylation was
unaffected by PTX treatment in either cell type (Fig. 1B
). These data
demonstrate that thrombin can stimulate Shc phosphorylation and DNA
synthesis through both PTX-sensitive or -insensitive pathways.

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Figure 1. Differential PTX Sensitivity of the Thrombin
Pathways
A, HIRcB and 1321N1 cells were treated with 100 ng/ml PTX for 6 h
before stimulation with 0.5 U/ml thrombin. Cells were assayed for BrdU
incorporation by immunofluorescence. B, HIRcB and 1321N1 cells were
either treated as above except cells were stimulated for 5 min with 0.5
U/ml thrombin and lysed, and Shc proteins were immunoprecipitated.
Phosphorylation of Shc was analyzed by Western blotting with a
monoclonal antiphosphotyrosine antibody.
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The Function of Shc Is Similar in Both Pathways
We previously reported that Shc was necessary for thrombin-induced
DNA synthesis in 1321N1 cells (12). To determine whether Shc functioned
differently in PTX-sensitive and -insensitive thrombin signaling, the
different structural elements of Shc were mutated. We constructed
mammalian expression vectors encoding full-length FLAG-tagged Shc with
point mutations that abolished PTB function (S154P), SH2 function
(R401L), or tyrosine phosphorylation at tyrosines 239 and 240
(Y239/240F), tyrosine 317 (Y317F), or all three tyrosines (3YF). These
constructs were transfected into either HIRcB cells or 1321N1 cells.
After transfection and serum deprivation, cells were treated with 0.5
U/ml thrombin or vehicle. Incorporation of BrdU in cells expressing the
FLAG-tagged Shc proteins were then assayed by immunofluorescence
18 h later (Fig. 2
).

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Figure 2. The Shc SH2, Y239/240, and Y317 Are Necessary for
Thrombin Signaling
A, HIRcB cells were transfected with Shc proteins containing the
indicated point mutations. Cells were stimulated with 0.5 U/ml
thrombin, and cells that were expressing the FLAG Shc proteins were
assayed by immunofluorescence for BrdU incorporation. B, 1321N1 cells
were transfected with the same constructs and assayed for BrdU
incorporation. Levels of incorporation are expressed as percent of mock
transfected cells stimulated with thrombin. Each bar
represents three to five experiments and error bars
represent SD.
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Expression of wild-type p52 Shc did not significantly alter
thrombin- stimulated BrdU incorporation (Fig. 2
, A and B). In cells
expressing Shc lacking a functional PTB domain (S154P),
thrombin-stimulated BrdU incorporation remained at control (mock
transfected) levels. In contrast, expression of Shc with a
nonfunctional SH2 domain (R401L) completely blocked thrombin-stimulated
BrdU incorporation in HIRcB cells and inhibited BrdU incorporation by
71% in 1321N1 cells. Results from these experiments suggest that the
Shc SH2 mutant is unable to signal but interferes with the function of
endogenous Shc. Thus, the Shc SH2 and not the Shc PTB participates in
mediating both PTX-sensitive and -insensitive thrombin signaling.
Consistent with previous work, mutation at Y317 of Shc, the site
implicated in Grb2-SOS association and activation of Ras, blocked
mitogenesis in HIRcB and 1321N1 cells by 98% and 64%, respectively.
Y239/240 were critical for thrombin signaling since expression of
Y239/240F also blocked the mitogenic response to thrombin by 92% in
HIRcB cells and 58% in 1321N1 cells. Finally, mutation of all three
tyrosines (3YF) markedly attenuated thrombin-induced DNA synthesis in
both cell types. The novel finding that Y239/240 is essential for cell
cycle events mediated by thrombin in conjunction with Y317 and 3YF data
suggests all three tyrosines are necessary for mitogenic signaling by
thrombin. In conclusion, all of these elements, the SH2, Y239, Y240,
and Y317, are important for both PTX-sensitive and -insensitive
thrombin signaling pathways.
The Shc SH2 Does Not Mediate Tyrosine Phosphorylation of Shc
To determine whether the SH2 of Shc is required for its
phosphorylation in response to thrombin, we assayed phosphorylation of
Shc constructs both in vivo (Fig. 3
) and in vitro (data not
shown). Wild-type or R401L FLAG-tagged Shc was expressed in HIRcB cells
and stimulated with thrombin for 5 min, and the amount of tyrosine
phosphorylation was determined by Western blotting. There was no
detectable difference in thrombin-induced phosphorylation of R401L
compared with wild-type Shc. In vitro phosphorylation data
of mutant Shc proteins resembled the in vivo situation with
phosphorylation of wild-type Shc and R401L Shc being similar (data not
shown). Since mutation of the SH2 does not affect phosphorylation, we
conclude that the Shc SH2 is not used in the process by which
activation of the thrombin receptor leads to Shc phosphorylation. The
Shc SH2 does appear to participate in thrombin siganling by mediating
complex formation with other phosphotyrosine-containing signaling
proteins. In both HIRcB cells and in 1321N1 cells, the GST-SH2
interacted with a number of phosphorylated proteins, several of whose
phosphorylation state was altered by stimulating the cells with
thrombin (data not shown).

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Figure 3. Phosphorylation of Shc Mutants
FLAG-tagged wild-type and R401L Shc were immunoprecipitated with
anti-FLAG polyclonal antibodies from transfected HIRcB cells either
unstimulated or stimulated with 0.5 U/ml thrombin. Western blotting was
done as above.
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Functions of the EGFR and SFKs Differ between PTX-Sensitive and
-Insensitive Thrombin Pathways
Earlier studies have implicated the activation of SFKs and/or the
EGFR in GPCR signaling (6, 7, 8, 36, 37). To determine whether SFKs or the
EGFR were involved in the thrombin signaling in HIRcB or 1321N1 cells,
we assessed the effects of pharmacological inhibitors of these kinases
on BrdU incorporation (Fig. 4
).
Pretreatment of HIRcB cells with the EGFR inhibitor AG1478 or the src
inhibitor PP1 blocked thrombin-induced BrdU incorporation to basal
levels (Fig. 4A
). In contrast, thrombin-stimulated BrdU incorporation
in 1321N1 was inhibited by PP1 (P < 0.01) but was
unaffected by AG1478 (95% of stimulated, P > 0.05)
(Fig. 4B
). The EGFR inhibitors A48 and Compound 56 and src inhibitor
PP2 gave similar results, but inhibitors of the HER/neu,
platelet-derived growth factor, fibroblast growth factor, or insulin
receptors had no effect in either cell type (data not shown).

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Figure 4. Inhibition of the EGFR and SFKs Have Different
Effects on Thrombin- Stimulated BrdU Incorporation in the Two Cell
Lines
A, HIRcB cells were treated with DMSO, the EGFR inhibitor AG1478, or
the src inhibitor PP1 for 30 min before stimulation. Cells were
stimulated with 0.5 U/ml thrombin and DNA synthesis was measured by
BrdU incorporation. BrdU was detected by immunofluorescence with an
anti-BrdU antibody and FRITC-conjugated antirat antibody. B, 1321N1
cells were treated and assayed in the same manner as the HIRcB cells.
Bars represent the average of three experiments with
SD.
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To determine whether similar differences were exhibited with respect to
Shc phosphorylation, cells were incubated with dimethylsulfoxide (DMSO;
vehicle), A63 (inactive tyrphostin control), AG1478, or PP1 for 30 min
before stimulation with thrombin. After stimulation for 5 min with
thrombin, the cells were lysed, Shc proteins were immunoprecipitated
and Western blotted with a monoclonal antiphosphotyrosine antibody
(4G10). As observed for BrdU inhibition, both the EGFR inhibitor and
SFK inhibitor blocked thrombin-stimulated Shc phosphorylation in HIRcB
cells (Fig. 5A
). In contrast, Shc
phosphorylation in 1321N1 cells was unaffected by the presence of
AG1478 but sensitive to PP1 (Fig. 5B
). Neither inhibitor had an effect
on insulin-stimulated Shc phosphorylation in HIRcB cells, but the EGFR
inhibitor blocked EGF-stimulated Shc phosphorylation as expected (data
not shown), demonstrating the specificity of these compounds. These
results indicate that PTX-sensitive and -insensitive thrombin-signaling
pathways may differentially utilize the EGFR to mediate signaling, and
the role of the EGFR in the two pathways may represent a major
mechanistic difference in the signaling properties of PTX-sensitive and
-insensitive pathways.

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Figure 5. Shc Phosphorylation in HIRcB and 1321N1 Cells Is
Differentially Affected by the EGFR and src Inhibitors
A, HIRcB cells were treated with DMSO (vehicle), A63 (inactive
tyrphostin), AG1478 (EGFR inhibitor), or PP1 (src inhibitor) for
30 min before stimulation. Cells were stimulated with 0.5 U/ml thrombin
for 5 min and lysed, and Shc was immunoprecipitated with polyclonal
anti-Shc antibodies. Proteins were separated on a 10% gel by SDS-PAGE
and transferred to Immobilon, and phosphorylation of Shc was
determined by Western blotting with a monoclonal antiphosphotyrosine
antibody (4G10). Membranes were stripped and reprobed with a monoclonal
anti-Shc antibody to verify equal loading (data not shown). B, 1321N1
cells were treated and analyzed as above. Bar graphs
represent densitometry analysis of three to six experiments.
Bars are expressed as percent of stimulated
control in the presence of DMSO and include SE.
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The Kinase Activity of the EGFR Is Necessary for Mediating
PTX-Sensitive Thrombin Signaling
To further evaluate the role of the EGFR in response to thrombin,
we transfected cells with wild-type EGFR, kinase-inactive EGFR
(
kinase), or HIS-tagged LacZ. Transfected cells were stimulated with
EGF or thrombin, fixed after 18 h, and stained to detect BrdU
incorporation. In LacZ-expressing HIRcB cells, BrdU increased from 16%
to 44% after stimulation with EGF. Overexpression of wild-type EGFR
had no effect on basal levels of BrdU incorporation (16% in the basal
cells) but produced a slight augmentation in BrdU incorporation when
stimulated with EGF (52% compared with 44% in stimulated cells).
Overexpression of the
kinase EGFR inhibited EGF-stimulated BrdU
incorporation by 75%.
The effect of the EGFR constructs on thrombin signaling in the HIRcB
cells strongly resembled that observed in cells stimulated with EGF
(Fig. 6B
). An augmentation of thrombin
signaling was seen in cells overexpressing the wild-type EGFR (an
increase of 12%), and expression of the
kinase EGFR inhibited
thrombin-stimulated BrdU incorporation almost to basal levels (24%
compared with basal levels of 16%). In the 1321N1 cells (Fig. 6C
),
expression of wild type EGFR did not enhance BrdU incorporation (94%
of control stimulation), and expression of the
kinase EGFR had no
effect (93% of control stimulated). These results support the
aforementioned hypothesis, based on inhibitor data, that the EGFR is
necessary for PTX-sensitive thrombin signaling but does not function in
PTX-insensitive thrombin signaling.

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Figure 6. PTX-Sensitive Thrombin Signaling Depends upon the
EGFR Kinase Activity
A, HIRcB cells were transfected with LacZ (expression marker),
wild-type EGFR, and kinase-inactive EGFR ( kinase). Cells were
stimulated with 100 ng/ml EGF and assayed for BrdU incorporation by
immunofluorescence. B, HIRcB cells were transfected and assayed as
above but were stimulated with 0.5 U/ml thrombin. C, 1321N1 cells were
transfected and assayed as above but were stimulated with 0.5 U/ml
thrombin. Control cells were stimulated with 1 µg/ml EGF.
Bars are the average of three experiments with
SD.
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The SH3 Domain of SFKs Is Necessary for Thrombin Signaling
To further define the functional role of SFKs in thrombin
signaling, we microinjected fusion proteins of the src SH3, fyn SH3, a
fyn SH3 mutant (W119K, labeled SH3*), which cannot bind target
sequences, or the fyn SH2 and measured the effects on BrdU
incorporation (Fig. 7
). The src SH2
inhibited thrombin-stimulated BrdU incorporation in HIRcB cells (32%,
P < 0.05) but had no significant effect in 1321N1
cells. In HIRcB cells, the src SH3 and fyn SH3 domains inhibited
thrombin-stimulated BrdU incorporation by 54% and 60%, respectively
(Fig. 7A
). Comparable results were observed in the 1321N1 cells (Fig. 7B
). As controls, we demonstrated that the src SH3 had no significant
effect on insulin-stimulated BrdU incorporation, and the fyn SH3* had
no effect on thrombin signaling. Although the SH2 domain may be of
varied importance between pathways, the involvement of SFK SH3 domains
in thrombin signaling is crucial in both PTX-sensitive and -insensitive
thrombin signaling.

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Figure 7. The SH3 Domain of src or fyn Is Necessary for Both
Thrombin-Signaling Pathways
A, HIRcB cells were microinjected with the fyn SH2, fyn SH3, fyn SH3
mutant (W119K), and src SH3 fusion proteins at concentrations of 10
mg/ml. After recovering from injection, cells were stimulated with 0.5
U/ml thrombin or 100 ng/ml insulin (control injection). The effect of
injection was measured by an immunofluorescence assay for BrdU
incorporation. B, Microinjections were performed in 1321N1 cells, and
the effects of injections on BrdU incorporation were determined.
Bars represent three to five experiments expressed as
percent of control stimulated BrdU incorporation with SD.
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Thrombin-Activated SFKs Phosphorylate Shc in An SH3-Dependent
Manner
SFK activation is a necessary event for thrombin-induced BrdU
incorporation and Shc phosphorylation, suggesting that an SFK is the
Shc kinase. Since the fyn SH3 has been reported to interact with Shc
and recombinant src or fyn were able to directly phosphorylate Shc
(Fig. 8A
), a possible mechanism for the
inhibition of BrdU incorporation by the SH3 could be the disruption
of Shc phosphorylation (6, 36, 38). Using 1321N1 cell lysates, we
addressed this hypothesis by performing kinase assays on FLAG-tagged
Shc preincubated with varying amounts of fyn GST-SH3 (Fig. 8B
). The
W119K mutant GST-SH3* was used as a control for nonspecific inhibition.
The fyn GST-SH3 inhibited the ability of lysates from
thrombin-stimulated cells to phosphorylate Shc, while the W119K mutant
had no effect.

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Figure 8. Fyn Phosphorylates Shc through an SH3-Dependent
Mechanism
A, Wild-type FLAG tagged Shc proteins were expressed in COS7 cells and
immunoprecipitated with polyclonal anti-FLAG. This pellet was washed in
kinase buffer before addition of 5 µg recombinant src or fyn. Kinase
reactions were allowed to proceed for 1 h at 4 C. Proteins were
separated on a 10% gel by SDS-PAGE, transferred to Immobilon, and
blotted with a monoclonal antiphosphotyrosine antibody (4G10). Controls
were 1321N1 lysates from cells stimulated with 0.5 U/ml thrombin for 2
min. B, After immunoprecipitation, FLAG-tagged Shc was incubated with
varying concentrations of either GST-SH3 of fyn or GST-W119K SH3 of
fyn. Lysates from 1321N1 cells that had been stimulated for 2 min with
0.5 U/ml thrombin were added to the FLAG-tagged Shc. The kinase
reaction was incubated for 1 h at 4 C. Samples were run on a 10%
gel with SDS-PAGE, transferred to Immobilon, and blotted for
phosphotyrosine with a monoclonal antiphosphotyrosine antibody (4G10).
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PTX-Sensitive Thrombin Signaling Is Dependent on ß
-Subunits
but PTX-Insensitive Thrombin Signaling Is Not
An early event in G protein signaling is the dissociation of
-
and ß
-subunits after GTP binding (4). Either
or ß
can
propagate specific signaling events (5, 39). To determine whether
PTX-sensitive and -insensitive pathways differed in their dependence on
ß
-subunits, we microinjected a fusion protein of the
ß-adrenergic receptor kinase carboxy terminus (ßARK CT), which
binds to ß
-subunits (40). Injection of the ßARK CT into HIRcB
cells had no effect on EGF-stimulated BrdU incorporation but blocked
PTX-sensitive L-
-lysophosphatidic acid (LPA) and
thrombin signaling by 75% and 76% (Fig. 9A
). In contrast, injection of the ßARK
CT into 1321N1 cells had no significant effect on thrombin signaling
(Fig. 9B
). This shows that a proximal difference in signaling by
PTX-sensitive and -insensitive pathways involves the function of
ß
-subunits.

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Figure 9. Inhibition of ß -Subunits Suppresses
PTX-Sensitive Thrombin Signaling but Not the PTX-Insensitive Pathway
A, HIRcB cells were microinjected with 10 mg/ml of a GST fusion with
the ßARK CT. After microinjection, cells were stimulated with 100
ng/ml EGF, 1 mM LPA, or 0.5 U/ml thrombin and the effects
of injection the GST- ßARK CT on BrdU incorporation were
determined. B, 1321N1 cells were treated as above.
Bars represent the average of three experiments with the
SDs.
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DISCUSSION
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The mechanism by which tyrosine kinases are activated by GPCRs is
an area of extreme interest. Although lacking the intrinsic kinase
activity and sites for protein interaction that the RTKs possess, GPCRs
are still able to induce tyrosine phosphorylation of intracellular
proteins upon stimulation. PTX-sensitive and -insensitive thrombin
signaling pathways both lead to Shc phosphorylation and DNA synthesis.
Our results indicate that the function of Shc within these two pathways
is similar, but variations in signaling proximal to Shc phosphorylation
exist between the two G protein-signaling pathways. Both pathways
require a src family member, but PTX-sensitive thrombin signaling
proceeds through ß
-subunits and the transactivation of the EGFR
while PTX-insensitive thrombin signaling occurs independently of ß
and the EGFR. The differential role of ß
-subunits and the EGFR may
represent a fundamental difference between signaling by PTX-sensitive
and -insensitive GPCRs.
Shc Performs Multiple Functions in Thrombin Signaling
From the results obtained with the mutant Shc proteins, we
conclude that Shc participates in at least three separate facets of
thrombin mitogenic signaling through its tyrosine phosphorylation sites
and through its SH2 domain.
Mutations at either Y239/240, Y317, or all three tyrosine residues in
Shc were able to dramatically reduce thrombin-stimulated BrdU
incorporation. Since Y239/240F and Y317F inhibited equally well, we
reason that two parallel pathways originate from Shc, and both are
necessary for later signaling events, such as DNA synthesis. Y317 has
been shown to be necessary for MAPK activation in fibroblasts
challenged with thrombin (13). Studies of Y239/240 function
implicate this tyrosine phosphorylation site in growth
factor-induced expression of c-myc (22, 25). However, the
importance of Y239/240 in GPCR signaling has not been previously
established. Based on the requirement for both sites of tyrosine
phosphorylation, it seems likely that thrombin acts through Shc not
only to regulate Ras but also via another pathway such as myc
expression, which ultimately work in a concerted manner to promote cell
cycle progression.
The Shc SH2 is also necessary for thrombin signaling, but it does
not function to mediate phosphorylation of Shc since Shc proteins with
a nonfunctional SH2 domain were phosphorylated to the same levels as
wild-type Shc in vivo and in vitro. The role of
the Shc SH2 in thrombin signaling therefore appears to be in the
formation of signaling complexes containing other phosphoproteins.
Interactions with the Shc SH2 may be necessary to target proteins to
subcellular compartments or to an activating enzyme or substrate (21, 41, 42). We observed similar results with the Shc SH2 in EGF
signaling, and others have proposed similar mechanisms for Shc SH2
function (21, 43). Complex formation mediated by the Shc SH2, along
with Shc phosphorylation, are required for transducing the thrombin
mitogenic signaling.
Activation of Tyrosine Kinases by Thrombin
Tyrosine phosphorylation of cytoplasmic proteins is a necessary
signaling event for thrombin-stimulated mitogenesis, but the
mechanism(s) by which this occurs remain unclear. Using Shc
phosphorylation as a marker for tyrosine kinase activation by thrombin,
we found that Shc phosphorylation occurs through both PTX-sensitive and
-insensitive pathways, but each pathway employed a different mechanism
to obtain this goal. PTX-sensitive thrombin-stimulated Shc
phosphorylation and BrdU incorporation were dependent on both the EGFR
and an SFK, while PTX-insensitive Shc phosphorylation and BrdU
incorporation were only dependent upon a src family member. These
results suggest that the transactivation of the EGFR may be a more
important component of PTX-sensitive G protein signaling.
Interestingly, when wild-type EGFR was overexpressed in HIRcB cells, a
slight increase in both EGF- and thrombin-induced BrdU incorporation
was detected as compared with mock transfected cells. This increase did
not occur in thrombin-stimulated 1321N1 cells, suggesting that these
cells do not transactivate the EGFR even at high EGFR expression
levels. This may reflect the different mechanism used for
PTX-insensitive thrombin signaling, which is incompatible with
PTX-sensitive thrombin signaling. Although transactivation of the
platelet-derived growth factor receptor has also been reported as a
pathway used by GPCRs (44), inhibitors of the HER/neu,
platelet-derived growth factor, fibroblast growth factor, or
insulin receptors had no effect on BrdU incorporation in either cell
type (data not shown). Thus, transactivation by thrombin occurs either
through an EGFR-specific mechanism or independently of the EGFR and
other known growth factors.
Several reports of EGFR involvement in GPCR signaling have recently
been published, but the mechanism of this transactivation is still
incompletely understood (29, 30, 31, 32, 33, 34, 45). Tice et al. (46) have
reported that G protein receptor agonists can stimulate src to
phosphorylate the EGFR on a novel phosphorylation site, Y845 (46). This
site was necessary for LPA-induced BrdU incorporation, and
phosphorylation at Y845 can be induced by thrombin (Ref. 47 and S.
Parsons, personal communication). Since the EGFR can function as a
tyrosine kinase and/or a scaffold protein, transactivation by a GPCR
would provide two functions the GPCR cannot perform on its own. In the
PTX-sensitive thrombin pathway, the kinase activity of the EGFR was
necessary for Shc phosphorylation and DNA synthesis, as determined by
both chemical inhibitors and the expression of a kinase-inactive EGFR.
Therefore, the kinase activity of the EGFR is necessary for
PTX-sensitive thrombin signaling, but an additional role as a scaffold
protein cannot be ruled out.
SFK activation and function are necessary for Shc phosphorylation and
DNA synthesis in both thrombin-signaling pathways. SFK activation in
response to thrombin differs between the PTX-sensitive and -insensitive
pathways may differ since microinjection of the src SH2 inhibited BrdU
incorporation in HIRcB cells but not in 1321N1 cells. It is possible
that microinjecting the src SH2 blocks association with and activation
of the EGFR by a src family member in HIRcB cells and, since 1321N1
cells do not transactivate the EGFR, signaling would be unaffected in
1321N1 cells (47). Once activated, the SFKs perform similar functions
within the two thrombin mitogenic pathways. The fact that Shc
phosphorylation by SFKs has been documented in several cell types, and
that the SH3 domain of fyn can disrupt Shc phosphorylation, leads us to
conclude that src or fyn is the Shc kinase activated by thrombin in
both cell types, and the interaction is mediated by SH3 domain
interacting within the proline-rich CH domain of Shc (36, 48, 49, 50).
The Role of
- and ß
-Subunits in Thrombin Signaling
To determine whether differences in
- and ß
-subunit
function play a role in the variations we observed between
PTX-sensitive and -insensitive thrombin signaling, we microinjected a
fusion protein of the ßARK carboxy terminus (CT). Microinjection of
the ßARK CT inhibited thrombin-stimulated BrdU incorporation in HIRcB
cells through the PTX-sensitive pathway, presumably by sequestering
ß
-subunits away from their normal effectors (40). In contrast, the
PTX-insensitive cascade in 1321N1 cells was unaffected, indicating that
the ß
-subunits do not function within this pathway.
ß
-Subunits may thus be involved in the transactivation of the
EGFR, giving rise to the differential tyrosine kinase activation in
response to thrombin in the cell types studied.
Activation of tyrosine kinases by ß
-subunits in the PTX-sensitive
thrombin pathway has been recently suggested to occur as a multiple
step process. ß
-Subunits function in localizing the G protein
receptor kinases (GRKs) to the activated G protein receptor, the GRKs
phosphorylate the GPCR and create sites of interaction for a family of
proteins known as the arrestins (51). The arrestins function to
down-regulate the GPCRs and also appear to function in forming a
signaling scaffold, activating src kinases and leading to the
phosphorylation of the EGFR (52). Hence, the role of the
ß
-subunits in PTX-sensitive thrombin signaling may be to recruit
the foundation for the signaling scaffold to the receptor.
Since the ß
-subunits are not necessary for PTX-insensitive
thrombin signaling, this pathway must activate tyrosine kinases by a
different mechanism. We have previously shown that expression of a
constitutively activated G
12 in 1321N1 cells can mimic several
thrombin-stimulated responses, including Shc phosphorylation and AP-1
reporter gene activation (12). Our hypothesis is that only the G
q or
G
12 subunits function to transmit PTX-insensitive thrombin signals.
Activation of tyrosine kinases could be mediated by direct interactions
of thrombin-activated G
subunits in a manner similar to stimulation
of phospholipase C (PLC) and Brutons tyrosine kinase (53, 54, 55, 56). In
addition, second messengers produced by the activation of such enzymes
could increase tyrosine kinase activity. For example, Src activation by
lipid second messengers has been reported (50).
In conclusion, we have found that Shc tyrosine phosphorylation and its
SH2 work in conjunction to mediate both PTX-sensitive and -insensitive
thrombin mitogenic signaling. Shc phosphorylation is accomplished by
two different mechanisms, depending on the PTX sensitivity of the cell
type studied. The difference in PTX-sensitive and -insensitive
phosphorylation of Shc was characterized by differential EGFR
involvement. We also found that ß
- subunits were necessary for
PTX-sensitive thrombin signaling in the EGFR- dependent HIRcB pathway
but did not participate in PTX-insensitive thrombin signaling in 1321N1
cells. An interesting question posed by our data is whether or not the
differences we observed are representative of differences between
PTX-sensitive and -insensitive GPCRs and signaling across a broad range
of cell types and ligands. Our model suggests PTX-sensitive G protein
signaling is more dependent on ß
-subunits than
-subunits. The
role of the G
-subunits in PTX-sensitive G protein signaling requires
additional investigation. Recently, however, Neptune et al.
(57) reported that G
i is not required for chemotaxis in response to
stimulation of receptors coupled to G
i, suggesting that ß
function is more important than G
function in their system. These
results are consistent with a broad and prominent role for
ß
-subunits in PTX-sensitive pathways.
In PTX-insensitive G protein signaling, our model assumes that ß
-
subunits perform little or no function, while the G
-subunits are
responsible for signal transduction. We hypothesize that ß
function is minimal, and that EGFRs are not recruited and not
involved in downstream signaling. In PTX-insensitive pathways, G
subunits, such as G
q, can directly activate enzymes, such as PLC,
and the production of second messengers can initiate subsequent
downstream events. As mentioned earlier, expression of G
q or G
12
subunits can mimic responses of PTX-insensitive pathways, supporting a
dominant role for
-subunits in these pathways.
 |
MATERIALS AND METHODS
|
---|
Cell Lines and Culture Conditions
Rat1 cells overexpressing the human insulin receptor, HIRcBs,
were established in our laboratory (58). HIRcB cells were grown
in DME/F12 supplemented with 10% FBS, 100 µM
methotrexate, 1 mM glutamine, 100 U penicillin, and 100
µg/ml streptomycin. To increase expression of transfected constructs
(see below), transfected HIRcB cells were grown in the same media as
above but lacking methotrexate. 1321N1 cells were grown in DMEM regular
glucose supplemented with 5% FBS, 1 mM glutamine, 100 U
penicillin, and 100 µg/ml streptomycin. COS7 cells were grown in DMEM
regular glucose supplemented with 10% FBS, 1 mM glutamine,
100 U penicillin, and 100 µg/ml streptomycin. All cell lines were
grown at 37 C in a humidified chamber with 5% CO2.
Expression Vectors
The FLAG-tagged Shc expression vector, pRK5 Shc, was a generous
gift from Dr. Edward Y. Skolnik (Skirball Institute, New York,
NY). Point mutations were introduced into Shc by using the QuikChange
site-directed mutagenesis kit (Stratagene, La Jolla, CA).
The mutation of serine 154 to proline was introduced with the
oligonucleotides 5'-ATC TCT TTC GCG CCC GGT GGG GAT CCG GAC-3' and
5'-GTC CGG ATC CCC ACC GGG CGC GAA AGA GAT-3'. Arginine 401 was mutated
to leucine with the oligonucleotides 5'-GAC TTC TTG GTG CTA GAG AGC ACG
ACC ACG-3' and 5'-CGT GGT CGT GCT CTC TAG CAC CAA GAC GTC-3'. Mutations
of tyrosines 239 and 240 to phenylalanines were accomplished through
using oligonucleotides 5'- CCT GAC CAT CAG TTC TTT AAT GAC TTC CCG-3'
and 5'-CGG GAA GTC ATT AAA GAA CTG ATG GTC AGG-3'. Finally, the change
of tyrosine 317 to phenylalanine was created with the oligonucleotides
5'-GAT GAC CCC TCC TTT GTC AAC ATC CAG AAT-3' and 5'-ATT CTG GAT GTT
GAC AAA GGA GGG GTC ATC-3'. The presence of mutations was determined by
sequencing (CFAR Sequencing Facility, La Jolla, CA).
All EGFR constructs were obtained from Dr. Gordon N. Gill (University
of CA, San Diego). His tagged LacZ was purchased from
Invitrogen (La Jolla, CA). Vectors encoding fyn fusion
proteins were obtained from Dr. Hamid Band (Harvard University, Boston,
MA). The src SH3 fusion protein was obtained from Dr. David D.
Schlaepfer (Scripps Research Institute, La Jolla, CA). The
ßARK CT fusion protein construct was obtained from Dr. Robert J.
Lefkowitz (Duke University, Durham, NC).
Kinase Inhibitors
The inhibitors A63, AG1478, and PP1 were all purchased
from Calbiochem (La Jolla, CA). Compounds were resuspended
in DMSO and used at concentrations as follows: A63 was used at 2
mM, AG1478 was used at 50 nM, and PP1 was used
at 100 nM. Control cells were treated with DMSO and DMSO
concentrations never exceeded 0.2% of media volume. Cells were treated
for 30 min before stimulation and lysis as described below. PTX was
from Sigma (St. Louis, MO). Cells were treated with 100
ng/ml for 6 h before stimulation.
Transfection of Cell Lines
Transfections of all three cell lines were performed with
SuperFECT (Qiagen, Valencia, CA) in accordance with
manufacturers instructions. In brief, cells were plated 1 day before
transfection. DNA purified over a CsCl gradient was mixed with the
SuperFECT reagent, incubated at room temperature to allow complex
formation, and added to cells. Three hours later, the transfection mix
was removed by aspiration, cells were washed once with warm PBS
Life Technologies, Inc., Gaithersburg, MD), and fresh
growth media were added to the cells. Cells were allowed to grow in
this media for at least 20 h before being serum deprived for
24 h before experiments.
Cell Stimulation and Cell Lysates
HIRcB cells and 1321N1 cells were stimulated with 0.5 U/ml
thrombin (Calbiochem) in the presence of 0.1% fatty
acid-free BSA (Sigma) at 37 C. Times of stimulation are
indicated in the figure legends. Control cells were exposed to only
0.1% fatty acid-free BSA. In experiments in which other growth factors
were used, cells were stimulated with 1 mM LPA
(Sigma), 100 ng/ml insulin, or 1 µg/ml EGF (Life Technologies, Inc.). Recombinant human insulin used was a
generous gift from Dr. Bruce Frank (Eli Lilly & Co.,
Indianapolis, IN). After stimulation, cells were washed once in 4 C PBS
and lysed in fibroblast solubilization buffer [(FSB) 25 mM
HEPES, 8 mM EDTA, 120 mM NaCl, 5 mM
KCl, 1 mM MgCl2, 1 mM
CaCl2, 150 mM NaF, 10 mM Na
pyrophosphate, 2 mM Na2VO4, 1
mM phenylmethyl sulfonylfluoride (PMSF), 10% glycerol, and
1% Triton X-100 (pH 7.5)]. Insoluble material was removed by
centrifugation and the supernatant used for subsequent
experiments.
Immunoprecipitation and Western Blotting
Endogenous Shc proteins were immunoprecipitated to determine the
effect of kinase inhibitors on Shc tyrosine phosphorylation. Five
micrograms of anti-Shc polyclonal antibodies (Transduction Laboratories, Inc., Lexington, KY) and 50 µl Protein A agarose
(Upstate Biotechnology, Inc., Lake Placid, NY) were added
to lysates to immunoprecipitate Shc. The pellets were washed twice in
FSB and solubilized in sample buffer containing 10%
ß-mercaptoethanol (BME).
Samples were separated by SDS-PAGE on a 7.5% acrylamide gel and
transferred to Immobilon (Millipore Corp., Bedford,
MA). Membranes were blocked in either 5% BSA in TBST [0.5
M Tris, 1.5 M NaCl, 0.1% Tween 20 (pH 7.5)]
or 5% nonfat dried milk in TBST. Phosphorylation on tyrosines
was detected by Western blotting with a monoclonal antibody to
phosphotyrosine, 4G10 (Upstate Biotechnology, Inc.) at 0.5
µg/ml. In the GST-SH2 pull-down experiments (see below), polyclonal
antiphosphotyrosine antibodies at 2 µg/ml were used instead. In data
not shown, an anti-Shc monoclonal antibody (Transduction Laboratories, Inc.) was used to determine equal protein
loading. Detection of FLAG-tagged Shc proteins was done by
Western blotting with the monoclonal antibody M2 (Sigma)
at 10 µg/ml. Goat polyclonal antimouse and antirabbit IgG antibodies
conjugated to horseradish peroxidase (Amersham Pharmacia Biotech, Arlington Heights, IL) at a dilution of 1:1000 were
incubated with the membrane and visualized with the SuperSignal
chemiluminescence detection reagents (Pierce Chemical Co.,
Rockford, IL) upon exposure to X-omat AR scientific imaging film
(Kodak, Rochester, NY).
Membranes that needed to be probed again to determine levels of a
specific protein were treated as follows. Membranes were washed in
Tris-buffered saline (TBS) after the initial exposure to
enhanced chemiluminescence reagents. Two 10-min washes with stripping
buffer (0.5 M acetic acid and 0.5 M NaCl in
water) were used to remove antibodies bound to the membrane. After
stripping, the membrane was washed three times in TBS to remove excess
NaCl and neutralize the acetic acid. Western blotting was
then performed as above.
Glutatione-S-transferase (GST) Fusion Proteins
GST fusion proteins were expressed in and recovered from DH5
or BL21 (GST-src SH3) strains under conditions suggested by the
manufacturer of the pGEX vectors (Pharmacia Biotech,
Alameda, CA). In brief, bacteria containing the fusion protein plasmids
were grown in 1-liter cultures and induced with 0.1 mM IPTG
for 4 h. Fusion proteins were purified from bacterial lysates by
affinity precipitation with glutathione (GSH)-conjugated agarose beads
(Pharmacia Biotech), washed in PBS containing 1
mM PMSF, and eluted from the beads with 10 mM
GSH in 100 mM Tris (pH 8.0).
Fusion proteins for microinjection were concentrated in
microinjection buffer [5 mM Na phosphate (pH 7.2), 100
mM KCl] by using a Ultrafree-15 centrifugal filter device
(Millipore Corp.). Protein concentration was determined by
spectrophotometric analysis at OD280 and confirmed by
Coomassie blue staining on a 10% acrylamide gel. Fusion proteins were
injected at 10 mg/ml into HIRcB or 1321N1 cells as previously described
(1, 45). Affinity precipitation experiments were performed using the
GST-SH2 of Shc prepared in this manner. For the precipitations, 10 µg
of fusion protein and 50 µl GSH-conjugated agarose beads were added
to HIRcB and 1321N1 lysates and incubated while rotating for 1 h
at 4 C. Beads and associated proteins were collected by centrifugation,
washed once in FSB, solubilized in sample buffer containing 10% BME,
and analyzed by Western blotting as stated above.
In Vitro Kinase Assays
FLAG-tagged wild-type or mutant Shc proteins were produced for
use as a substrate in kinase assays by transfection of COS7 cells with
the appropriate expression vector. Cells were serum deprived for
24 h and lysed in KB (25 mM HEPES, 120 mM
NaCl, 5 mM KCl, 1 mM MgCl2, 1
mM CaCl2, 2 mM
Na3VO4 and 1 mM PMSF, 10%
glycerol, and 0.1% Triton X-100). FLAG-tagged Shc proteins were
immunoprecipitating from COS7 cell lysates with 5 µg of polyclonal
antibodies against the FLAG epitope (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and 50 µl Protein A-conjugated agarose
(Upstate Biotechnology, Inc.) per reaction. Pellets were
washed in EBG (25 mM HEPES, 120 mM NaCl, 5
mM KCl, 1 mM MgCl2, 1
mM CaCl2, 10% glycerol, and 0.01% Triton
X-100) containing 2 mM Na3VO4 and 1
mM PMSF. To these pellets, we added 300 µl KB, 200 µl
HIRcB or 1321N1 cell lysate, 2 mM MgCl2, 2
mM CaCl2, 2 mM MnCl2, 2
mM ATP, and 2 mM
Na3VO4. Cell lysates used in the kinase assay
were from cells that were serum deprived, stimulated for 2 min, and
lysed in KB, and the detergent-insoluble fraction was removed by
centrifugation.
In samples in which purified src or fyn was used in Shc kinase
assays, the src or fyn was recombinantly produced (both from
Upstate Biotechnology, Inc.). The Shc used as substrate
was produced in COS7 cells and isolated as before, but the pellet was
resuspended in KB including 2 mM MgCl2, 2
mM CaCl2, 2 mM MnCl2,
and 2 mM ATP. Five micrograms of recombinant kinase were
added, and both the kinase assay and analysis were done as above.
Controls for Shc phosphorylation in this experiment were kinase assays
performed in either unstimulated or thrombin-stimulated 1321N1 cell
lysates. To assay the effect of the fyn SH3 on Shc phosphorylation,
immunoprecipitated FLAG-tagged Shc was incubated in 300 µl KB with
the GST-SH3 (fyn), at 5, 10, or 25 µg per reaction, for 30 min before
the kinase assay was performed. Two hundred microliters of cell lysate
were added, and the kinase assays were done as above. Kinase reactions
were allowed to proceed for 1 h at 4 C. The
antibody-agarose-FLAG-tagged Shc complex was spun down in a
microcentrifuge at 4 C. This pellet was washed once in FSB, solubilized
in sample buffer containing 10% BME, and boiled for 2 min. Samples
were analyzed as above by Western blotting.
Immunofluorescence
Incorporation of BrdU, a thymidine analog, was used as a
marker for cell cycle progression as previously described (1, 45). BrdU
(Amersham Pharmacia Biotech) was added to cells 12 h
after being stimulated after microinjection, transfection, or treatment
with kinase inhibitors. Incorporation of BrdU was allowed to occur for
6 h, whereupon cells were fixed in 3.7% formaldehyde in PBS for
20 min. Coverslips were washed three times in PBS and labeled with rat
anti-BrdU (Amersham Pharmacia Biotech) and either a mouse
monoclonal antibody to the anti-Xpress epitope
(Invitrogen) or the polyclonal anti-FLAG antibody
(Santa Cruz Biotechnology, Inc.). Coverslips were washed
again in PBS and labeled with donkey antirat antibodies conjugated to
rhodamine (tetramethyl rhodamine isothiocyanate) and donkey
antimouse or antirabbit antibodies conjugated to fluorescein
(fluorescein isothiocyanate) (Jackson ImmunoResearch Laboratories, Inc. West Grove, PA). Analysis was performed on an
Axiophot fluorescence microscope (Carl Zeiss, Thornwood,
NY).
 |
ACKNOWLEDGMENTS
|
---|
The authors of this paper would like to thank the investigators
mentioned in Materials and Methods for their generous gifts
or reagents. We also thank Dr. Salme Taagepera and Dr. Lila Collins for
insightful discussions.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Jerrold Olefsky, Department of Medicine, Division of Endocrinology and Metabolism, University of California at San Diego, 9500 Gilman Drive, La Jolla, California 92093-0673.
This work was supported by NIDDK, NIH Grant DK-33651 and GM-36927
(J.H.B.).
Received for publication June 4, 1999.
Revision received August 16, 1999.
Accepted for publication August 18, 1999.
 |
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