Analysis of Functional Domains of Angiotensin II Type 2 Receptor Involved in Apoptosis
Jukka Y. A. Lehtonen1,
Laurent Daviet1,
Clara Nahmias,
Masatsugu Horiuchi and
Victor J. Dzau
Division of Cardiovascular Medicine (J.Y.A.L., L.D., M.H.,
V.J.D.) Harvard Medical School Brigham and Womens
Hospital Boston, Massachusetts 02115
Institut
Cochin de Génétique Moléculaire (C.N.) Centre
Nationale de Recherche Scientifique UPR 0415 75014 Paris,
France
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ABSTRACT
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We previously demonstrated that the intracellular
third loop (i3 loop) of angiotensin II type 2 receptor
(AT2) plays a key role in mediating the
biological functions of this receptor. To determine which residues are
important for AT2 signaling, mutated receptors
with serial deletions within the i3 loop were stably expressed in PC12
cells. Deletion of residues 240244 within the intermediate portion of
the i3 loop resulted in a complete loss of
AT2-mediated apoptosis, inhibition of
extracellular signal-regulated kinases (ERK), and SHP-1 activation. In
contrast to well characterized heptahelical receptors, the
AT2 functions were not affected by deletions of
the amino- or carboxyl-terminal portions of the i3 loop. Alanine
substitutions further demonstrated that lysine 240, asparagine 242, and
serine 243 are key residues for AT2-induced
apoptosis, ERK inhibition, and SHP-1 activation. To examine whether a
functional link exists between activation of SHP-1 and apoptosis, we
used a catalytically inactive SHP-1 mutant and demonstrated that
preventing SHP-1 activation strongly attenuates
AT2-induced ERK inhibition and apoptosis. Our
data demonstrate that the intermediate portion of the i3 loop is
important for AT2 function and that SHP-1 is a
proximal effector of the AT2 receptor that is
implicated in the inhibition of ERKs and in the apoptotic effect of
this receptor.
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INTRODUCTION
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Angiotensin II (AngII) is a potent vasoactive peptide and a growth
factor. Two subtypes of high-affinity AngII receptors (designated
AT1 and AT2 receptors) have been cloned, both
of which belong to the superfamily of G protein-coupled receptors
(GPCRs) (1, 2, 3, 4). The high levels and transient expression of
AT2 receptors in fetal tissues (5, 6) and in some
pathological states (7, 8) have led to the hypothesis that this
receptor has a role in cardiovascular development and remodeling.
The AT2 receptor exerts growth-inhibitory effects in
cultured cells and in vivo, one of which has been proposed
to be programmed cell death (7, 9, 10, 11, 12, 13, 14, 15, 16). Despite growing interest in
AT2 receptor-mediated apoptosis, relatively little is known
about the molecular basis of this process. Growth-inhibitory effects of
the AT2 receptor have been reported to be mediated by the
activation of protein tyrosine phosphatases (PTPs). Serine/threonine
phosphatase 2A activation and consequent extracellular signal-regulated
kinase (ERK) inhibition via the AT2 receptor have been
reported in some rat neuronal cells (17). In rat pheochromocytoma PC12W
cells, mitogen-activated protein kinase phosphatase-1 is
involved in AT2 receptor-mediated ERK inhibition and
apoptosis (16), whereas in murine neuroblastoma N1E-115 cells,
AT2 receptor stimulation is associated with a rapid
activation of SHP-1 (18). However, at present, no functional role has
been demonstrated for SHP-1 activation by the AT2 receptor.
It is interesting to note that SHP-1 has been shown to function as a
negative regulator of tyrosine kinase receptor signaling (19, 20). The
potential biological significance of AT2 receptor-induced
programmed cell death led us to investigate whether SHP-1 activation
could be involved in this process.
Functional characterization of i3 loop-mutated GPCRs has demonstrated a
key role for this domain in G protein-coupling specificity (21, 22, 23, 24, 25, 26).
Using synthetic peptide and AT2/AT1 chimeric
receptors, previous studies demonstrated that the i3 loop is necessary
and sufficient for the cellular effects of the AT2 receptor
(10, 27, 28). However, little is known about the structural
determinants within this domain that functionally couple to downstream
signal transduction pathways. In the present study, we first examined
which specific sequence and amino acids of the i3 loop are essential
for AT2 receptor function. Toward this goal, mutated
receptors with serial deletions or amino acid substitutions within the
i3 loop were stably expressed in PC12 cells, and their functional
properties were characterized, including AngII-induced apoptosis, ERK
inhibition, and SHP-1 activation. Our data demonstrate that
AT2 receptor function is critically dependent on three
amino acids (K240, N242, and S243) located in the midportion of the i3
loop and suggest a functional link among SHP-1 activation, ERK
dephosphorylation, and apoptosis. On the basis of this observation, we
hypothesized that SHP-1 is an effector in the signal transduction
pathway leading to programmed cell death. To test this hypothesis, we
coexpressed a catalytically inactive SHP-1 mutant together with the
wild-type AT2 receptor and found that the inactive SHP-1
mutant significantly attenuated AT2 receptor-evoked ERK
dephosphorylation as well as apoptosis.
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RESULTS
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Effect of Serial Deletions of AT2
Receptor i3 Loop on Receptor Function
Wild-type or mutated AT2 receptors were stably
expressed in PC12 cells, and binding characteristics of the different
receptors were determined. As shown in Tables 1
and 2
,
[Sar1,Ile8]AngII binding affinity of all the
mutants was in the range described for the wild-type AT2
receptor. We did not observe any specific AT1 receptor
binding in the transfected cells.
Recently, Bedecs et al. (18) reported that SHP-1 is rapidly
activated by the AT2 receptor in N1E-115 cells. We first
examined the expression level of SHP-1 in PC12 cells and, in agreement
with previous reports (29), found that SHP-1 is expressed at a
relatively high level in this cell line. Although no functional role
has yet been described for AT2-mediated SHP-1 activation,
AT2 receptor-induced ERK inhibition and apoptosis are both
orthovanadate-sensitive, which suggests that PTPs act upstream of these
two events (16). We observed AT2 receptor-mediated SHP-1
activation in PC12 cells stably transfected with a wild-type
AT2 receptor cDNA construct (Fig. 1A
). To
explore the structural basis underlying SHP-1 activation, the effects
of serial i3 loop deletions on SHP-1 activation were investigated.
Mutants lacking either the last five amino- or carboxyl-terminal amino
acids of the i3 loop (
235239 and
250254, respectively) were
fully functional in terms of SHP-1 activation (Fig. 1A
). In assays
performed with the
240244 mutant, no AngII-mediated
stimulation of SHP-1 was observed. Deletion of residues 245249
(
245249) attenuated by 70% the AngII-induced SHP-1 activation
(Fig. 1A
). On the basis of these results, a stretch of five amino acids
located in the middle part of the i3 loop appears to be critically
involved in AT2 receptor-mediated stimulation of SHP-1.

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Figure 1. Effects of i3 Loop Deletions on AT2
Receptor Function
A, AngII-stimulated SHP-1 activity in PC12 cells stably transfected
with the deleted AT2 receptors. Cells were maintained
overnight in serum-free medium and then stimulated with AngII
(10-7 M) for 2 min at 37 C. Cell lysates were
assayed for PTP activity as described in Materials and
Methods. Results are expressed as percentage of PTP activity
obtained from nonstimulated cells. B, Effect of i3 loop deletions on
AT2 receptor-mediated inhibition of NGF-stimulated ERK.
Cells were maintained overnight in serum-free medium and then
stimulated with NGF (10 ng/ml) in the presence or absence of AngII.
Upper panel, Phosphorylated or total ERK was detected by
immunoblotting with phospho-ERK (upper blot) or ERK
(lower blot) antibodies, respectively. Lower
panel, Quantification of phospho-ERK by densitometric scanning of
the autoradiograms (n = 3). C, Proapoptotic effect of deleted and
wild-type AT2 receptors. Upper panel,
Representative autoradiogram of a DNA fragmentation assay. Cells
maintained in serum-free medium containing NGF (10 ng/ml) were
stimulated by AngII (10-7 M) for 48 h.
After genomic DNA extraction, oligosomal DNA fragmentation was assessed
as described in Materials and Methods. Lower
panel, Quantification of the DNA fragmentation assay described
above. The amount of radiolabeled dideoxy-ATP incorporated into low
molecular weight (<20 kb) fractions were quantified as described in
Materials and Methods (mean ± SE, n =
3 for each construct). *, P < 0.05; and **,
P < 0.01 vs. control. Wt AT2,
Wild-type AT2 receptor.
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In accordance with previously published results (16), activation of the
AT2 receptor elicited an approximatively 60% decrease in
nerve growth factor (NGF)-stimulated ERK phosphorylation (Fig. 1B
). In
the absence of AngII treatment, the wild-type and mutant
receptor-transfected cells showed similar levels of NGF-stimulated ERK
phosphorylation (data not shown). Deletion of the last five amino acids
of either the amino- or the carboxyl-terminal part of the i3 loop had
no effect on AngII-evoked ERK inhibition. In contrast, the
240244
mutant completely lost its ability to mediate agonist-dependent
inhibition of ERK. Deletion of residues 245249 (
245249) yielded
a partially functional receptor, causing a modest 15% reduction in ERK
phosphorylation (Fig. 1B
). Taken together, these results suggest that
the central part of the i3 loop is important for both SHP-1 activation
and ERK inhibition.
We then examined the effects of i3 loop deletions on AngII-induced
apoptosis. In control cells maintained under NGF, DNA fragmentation was
barely detectable. DNA fragmentation was undetectable in PC12 cells
expressing the wild-type or the deleted AT2 receptors in
the absence of AngII treatment (data not shown). Incubation of PC12
cells expressing wild-type AT2 receptor with AngII for
48 h resulted in the appearance of typical oligonucleosomal DNA
fragmentation that increased by a maximum of 3-fold (Fig. 1C
).
Consistent with SHP-1 activation and ERK inhibition, deletion of the
last five amino- or carboxyl-terminal amino acids of the i3 loop did
not affect AngII-induced DNA fragmentation (Fig. 1C
). Deletion of
residues 240244 (
240244) completely abolished AngII-induced
apoptosis, whereas the
245249 mutant showed an intermediate
phenotype (1.8-fold increase in DNA fragmentation). Taken together,
these results indicate that the central part of the i3 loop (residues
240 to 244) is pivotal for AT2 receptor-mediated apoptosis
as well as SHP-1 activation and ERK inhibition.
Alanine Scanning Mutagenesis of Residues 240244 in the i3 Loop of
the AT2 Receptor
In an attempt to identify the critical residues for
AT2 receptor function, we systematically substituted K240,
T241, N242, S243, and Y244 by alanine. In the absence of AngII
treatment, the wild-type and mutant receptor-transfected cells showed
similar levels of ERK phosphorylation and undetectable DNA
fragmentation (data not shown). As shown in Fig. 2
, the K240/A, N242/A, and S243/A mutants completely lost their ability to
activate SHP-1, inhibit ERK, and induce apoptosis, whereas the T241/A
mutant showed a wild-type phenotype. The Y244/A mutant showed a
substantial loss in AngII-mediated SHP-1 activation, ERK inhibition,
and apoptosis. As shown in Table 2
, the mutated receptors displayed
similar AngII binding characteristics, thus excluding the possibility
that the observed phenotypes were a consequence of lower membrane
expression levels or failure to bind ligand. It should also be noted
that the expression levels of AT2 receptors reported in the
present study (Tables 1
and 2
) are similar to those reported in cell
lines that endogenously express AT2 receptors such as PC12W
(30) or N1E-115 (31), thus excluding the possibility that the observed
phenotypes are a consequence of receptor expression levels. As outlined
above, we found a strong correlation between SHP-1 activation and
apoptosis. However, it is conceivable that SHP-1 activation coincides
with, rather than causes, cell death. To test the hypothesis that SHP-1
is involved in AT2 receptor-induced apoptosis, we used a
catalytically inactive mutant of this PTP.

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Figure 2. Effect of Systematic Alanine Substitutions of Amino
Acids 240244 on AT2 Receptor Functions
A, AngII-stimulated SHP-1 activity in PC12 cells stably transfected
with the mutated AT2 receptors. Cells were maintained
overnight in serum-free medium and then stimulated with AngII
(10-7 M) for 2 min at 37 C. Cell lysates were
assessed for PTP activity as described in Materials and
Methods. Results are expressed as percentage of PTP activity
obtained from nonstimulated cells. B, Effect of alanine substitutions
on AT2 receptor-mediated inhibition of NGF-stimulated ERK.
Cells were maintained overnight in serum-free medium and then
stimulated with NGF (10 ng/ml) in the presence or absence of AngII.
Upper panel, Phosphorylated or total ERK was detected by
immunoblotting using phospho-ERK (upper blot) or ERK
(lower blot) antibodies, respectively. Lower
panel, Quantification of phospho-ERK was performed by
densitometric scanning of the autoradiograms (n = 3). C,
Proapoptotic effect of mutated and wild-type AT2 receptors.
Upper panel, Representative autoradiogram of a DNA
fragmentation assay. Cells maintained in serum-free medium containing
10 ng/ml NGF were stimulated by AngII (10-7 M)
for 48 h. After genomic DNA extraction, oligosomal DNA
fragmentation was assessed as described in Materials and
Methods. Lower panel, Quantification of the DNA
fragmentation assay described above. The amount of radiolabeled
dideoxy-ATP incorporated into low molecular weight (< 20 kb) fractions
was quantified as described in Materials and Methods
(mean ± SE, n = 3 for each construct). *,
P < 0.05; and **, P < 0.01
vs. control.
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Role of SHP-1 in AT2 Receptor Signal
Transduction
To examine the role of SHP-1 in AT2 receptor
signaling, we stably cotransfected PC12 cells with AT2
receptor and a dominant negative SHP-1 mutant in which the active site
cysteine 453 was mutated to serine (C453/S) (32). Ligand binding
experiments using membranes from the cotransfected cells yielded
Kd and Bmax values similar to those for the
cell line expressing only the AT2 receptor (data not
shown). Overexpression of the SHP-1 mutant (C453/S) was confirmed by
immunoblot showing a 5-fold increase in SHP-1 immunoreactivity compared
with the parental cell line (Fig. 3A
).
Overexpression of SHP-1 (C453/S) abolished AT2
receptor-induced SHP-1 activation (Fig. 3B
). As shown in Fig. 4
, expression of SHP-1 (C453/S) strongly
attenuated AngII-induced ERK dephosphorylation, thus suggesting that
SHP-1 is involved in ERK dephosphorylation by the AT2
receptor. This is also indirectly supported by the chronology of these
two events. Indeed, the onset of SHP-1 activation (23 min) clearly
precedes the onset of ERK inhibition (Fig. 4B
). Since AT2
receptor-mediated ERK inhibition may be part of the signal transduction
cascade leading to apoptosis (16), we hypothesized that
AT2-generated apoptotic signals are interrupted by SHP-1
(C453/S). As shown in Fig. 5
, the
overexpression of SHP-1 (C453/S) markedly reduced the proapoptotic
effect of AngII. To exclude the possibility that SHP-1 (C453/S) acts as
a nonspecific inhibitor of apoptosis, we treated the cotransfected cell
line with a cell-permeable ceramide analog and found that
overexpression of SHP-1 (C453/S) did not affect ceramide-induced
apoptosis when compared with the parental cells that only express
AT2 receptors (Fig. 6
).

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Figure 3. Characterization of the PC12 Cell Line Stably
Expressing Wild-Type AT2 Receptor and Catalytically
Inactive SHP-1 Mutant (C453/S)
A, Immunoblot analysis of SHP-1 expression in the parental and SHP-1
(C453/S)-transfected PC12 cells with polyclonal antibodies to SHP-1. B,
Overexpression of the inactive SHP-1 mutant (C453/S) inhibits
AT2 receptor-induced SHP-1 activation. Serum-starved cells
were stimulated with AngII (10-7 M) for 2 min
at 37 C and lysed. Cell lysates were subjected to immunoprecipitation
with antibodies to SHP-1, and immunocomplexes were assessed for PTP
activity with 33P-labeled myelin basic protein as a
substrate. Results are expressed as a percentage of PTP activity
obtained from nonstimulated cells (mean ± SE, n
= 3). **, P < 0.01 vs. control
cells.
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Figure 4. The Involvement of SHP-1 in ERK Dephosphorylation
by the AT2 Receptor
A, The catalytically inactive SHP-1 mutant (C453/S) prevents
AngII-evoked ERK dephosphorylation. NGF-stimulated (10 ng/ml) PC12
cells were treated with AngII (10-7 M) for 10
min and lysed. The level of ERK phosphorylation was assessed by
immunoblotting (upper panel) and densitometric scanning
of the autoradiograms (lower panel) as described in Fig. 1 (mean ± SE, n = 3). B, Time courses of SHP-1
activation and ERK inhibition after AT2 receptor
stimulation. SHP-1 activity and ERK phosphorylation levels were
assessed after the indicated period of AngII stimulation as described
in Materials and Methods.
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Figure 5. Overexpression of the Dominant-Negative SHP-1
Mutant (C453/S) Inhibits AT2 Receptor-Induced DNA
Fragmentation
SHP-1 (C453/S) stably transfected PC12 cells were maintained in
serum-free medium supplemented with NGF (10 ng/ml) and then stimulated
with AngII (10-7 M) for 48 h. After
genomic DNA extraction, oligosomal DNA fragmentation was assessed as
described in Materials and Methods. Upper
panel, Representative autoradiogram of a DNA fragmentation
assay. Lower panel, Quantification of the DNA
fragmentation assay described above. The amount of radiolabeled
dideoxy-ATP incorporated into low (<20 kb) molecular weight fractions
was quantified as described in Materials and Methods
(mean ± SE, n = 3). *, P <
0.05; and **, P < 0.01 vs. control.
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Figure 6. Overexpression of the Dominant-Negative SHP-1
Mutant Does Not Affect Ceramide-Induced Apoptosis
PC12 cells stably expressing the AT2 receptor alone (Wt) or together
with the inactive SHP-1 mutant [SHP-1 (C453/S)] were maintained in
serum-free medium supplemented with NGF (10 ng/ml) and then treated for
24 h with C2-ceramide (100 µM) or the vehicle.
Upper panel, Representative autoradiogram of a DNA
fragmentation assay. Lower panel, Quantification of the
DNA fragmentation assay described above. After genomic DNA extraction,
oligosomal DNA fragmentation was assessed as described in
Materials and Methods (mean ± SE,
n = 3). *, P < 0.05; and **,
P < 0.01 vs. control.
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DISCUSSION
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The peptidic hormone AngII exerts positive or negative effects on
cell growth and survival depending on which subtype of receptor
(AT1 or AT2) is activated. Recent data suggest
that the AT2 receptor exerts growth-inhibitory and
proapoptotic effects (7, 9, 10, 11, 12, 15, 16). Despite growing interest in
AT2 receptor-mediated apoptosis, little is known about
the intracellular signaling pathways regulating this process.
Increasing evidence suggests the coupling of the AT2
receptor to PTPs. In murine N1E-115 cells, AT2 activates
the amino-terminal Src homology 2 (SH2) domain-containing tyrosine
phosphatase SHP-1 (18). In rat PC12W cells, mitogen-activated protein
kinase phosphatase-1 has been shown to mediate AT2
receptor-induced ERK inhibition and apoptosis (16). However, the
structural determinants of the AT2 receptor implicated in
intracellular signaling remain to be identified. In the present report,
the functional consequences of serial deletions and alanine
substitutions in the i3 loop of the AT2 receptor on
apoptosis, ERK inhibition, and the recently described, AT2
receptor-induced SHP-1 activation were investigated. We also examined
the potential role of SHP-1 as an early effector in
AT2-receptor-mediated ERK inhibition and apoptosis.
The role of the i3 loop in G protein coupling specificity has been
extensively studied for many GPCRs, including AT1 and
adrenergic and muscarinic acetylcholine (21, 22, 23, 24, 25, 26). In these reports,
residues and specific sequences of the proximal and distal parts of the
i3 loop were shown to be involved in G protein interactions and
specificities. Accumulating data indicate that the functional
differences between AT1 and AT2 receptors
depend on sequence differences in the i3 loop (21, 26). In accordance
with these results, our group has recently shown, using a peptide
transfer approach (10) and AT1/AT2 chimeric
receptors (27), that the i3 loop is a key structural determinant in the
signal transduction process of the AT2 receptor. The
results of our present deletional analysis confirm the functional
importance of the i3 loop in AT2 signaling and further
demonstrate that amino acids 240244 and, to a lesser extent, 245249
within the i3 loop are required for AT2 receptor-mediated
SHP-1 activation, ERK inhibition, and apoptosis. In contrast to a
number of other GPCRs (see above), these functionally important
residues are located in the central part of the i3 loop. An alanine
scan performed within the 240244 sequence further confirms the
pivotal role of this region in AT2 signaling and narrows
down the critical domain to three residues: lysine 240, asparagine 242,
and serine 243. These results do not, however, exclude the possible
contribution of other residues in the adjacent 245249 segment or,
alternatively, in other intracellular domain(s) to the AT2
receptor-mediated signaling. Indeed, the currently available data agree
with a model in which the recognition site for the G protein is a
discontinuous structure composed of several segments of the receptor,
some of them being masked in the basal state and unmasked by agonist
binding (33).
SHP-1 is a soluble tyrosine phosphatase that participates in the
negative regulation of receptor tyrosine kinase pathways (19, 20). It
has been recently reported that stimulation of AT2
receptors rapidly activates SHP-1 in N1E-115 and
AT2-transfected Chinese hamster ovary (CHO) cells (18). In
the present study, we documented that AT2 receptors also
activate SHP-1 in transfected PC12 cells with a time course and
amplitude similar to those reported by Bedecs et al. (18).
Moreover, the onset of SHP-1 activation clearly precedes the onset of
ERK inhibition (Fig. 4B
) and apoptosis, thus suggesting that SHP-1 is
an upstream, proximal effector in AT2 signaling. Moreover,
the observation that SHP-1 activation, ERK inhibition, and apoptosis
are affected by identical point mutations in the intermediate portion
of the i3 loop further suggest that they constitute sequential events
in the same signaling pathway. This hypothesis is also supported
by the observation that AT2-mediated ERK
inhibition and apoptosis are both orthovanadate-sensitive, which
implies that PTPs act upstream of these two events (16). To establish a
functional link between AT2-mediated activation of SHP-1
and inactivation of ERK, we used a catalytically inactive, dominant
negative mutant of SHP-1 and demonstrated that preventing SHP-1
activation abrogates AT2-induced ERK inhibition. The lag
time between maximal SHP-1 activation and ERK inhibition suggests that
ERKs are not direct cellular targets for SHP-1, but rather that SHP-1
activation affects upstream intermediates of the NGF-induced ERK
pathway, possibly at the level of the NGF TrkA receptor. Indeed, it has
been shown that SHP-1 physically interacts with the TrkA receptor in
PC12 cells (29) and is possibly involved in termination of TrkA
signaling, as already observed for a number of membrane receptors
(the epidermal growth factor receptor, c-Kit, the
interleukin-3 receptor ß-subunit, the erythropoietin receptor) (34, 35). In accordance with this, AT2 receptor stimulation has
recently been shown to promote dephosphorylation of
tyrosine-phosphorylated insulin receptors further suggesting that
AT2 receptor may directly target the activity of growth
factor receptors (C. Nahmias, submitted for publication). In addition,
sst2 somatostatin receptor-mediated activation of SHP-1 has been shown
to promote dephosphorylation of the insulin receptor (36) and to act as
an initial transducer of the antiproliferative signaling mediated by
this heptahelical receptor (32).
Abrogation of AT2 receptor-mediated apoptosis by
expression of the inactive SHP-1 mutant supports the concept that the
proapoptotic effect of AT2 is associated with the
inhibition of ERK via a signaling pathway involving the activation of
SHP-1. It is interesting that SHP-1-mediated protein dephosphorylation
has been reported to be required for the delivery of the Fas apoptosis
signal in lymphoid cells (37). Altogether, these observations suggest
that SHP-1 is a transducer of the antimitogenic and/or proapoptotic
signals mediated by different membrane receptors, including
AT2 and sst2 receptors, probably through negative
regulation of growth factor signaling.
In conclusion, this study demonstrates that the central part
of the i3 loop of the AT2 receptor plays an important role
in the activation of the signaling pathway that leads to SHP-1
activation, ERK inhibition, and programmed cell death. Moreover, our
results strongly suggest that SHP-1 constitutes one of the proximal
effectors of the AT2 receptor and is implicated in the
negative regulation of ERK and in the apoptotic effect of
AT2 receptors.
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MATERIALS AND METHODS
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Construction of Mutated AT2
Receptor cDNAs
To introduce amino acid deletions and substitutions in the
putative i3 loop of the human AT2 receptor, we have
exploited the fact that the cDNA fragment encoding this domain lies
between two unique restriction sites: KpnI (position 429)
and AlwNI (position 778). For each mutant, the i3 loop
coding region was amplified by PCR from a pUC19/hAT2
receptor vector with an antisense oligonucleotide bearing the desired
deletion/mutation and activated by an AlwNI restriction site
and a sense primer activated by a KpnI restriction site (the
sequences of the primers are available upon request). Each antisense
primer also contained a silent mutation removing the Tth111I
restriction site that naturally occurred at position 757 of the
AT2 receptor cDNA to facilitate the screening of colonies.
The resulting PCR fragments encoding the mutated i3 loop were first
subcloned into a KpnI-AlwNI-digested
pUC19/hAT2 receptor vector to reconstitute full-length
AT2 receptor cDNAs. Finally, the mutated cDNAs were
transferred between the NcoI and BamHI sites of
the pBC-SF mammalian expression vector (38), fusing the 5'-end of the
AT2 receptor cDNA in frame with the Flag (DYKDDDDK)
epitope. Mutations were confirmed by dideoxy sequencing.
Cell Culture
PC12 cells (which do not express AT1 or
AT2 receptor) were cultured as described previously (16, 39). In brief, PC12 cells were maintained in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 10% FCS, 5%
horse serum, 100 U/ml penicillin, and 1 mg/ml streptomycin in
humidified atmosphere of 95% air and 5% CO2.
Generation of Stably Transfected PC12 Cell Lines
Mutated AT2 receptor cDNAs were cotransfected with
pSV2-Neo into PC12 cells with the Lipofectamine reagent
(Life Technologies). The SHP-1 (C453/S) mutant-expressing
cell line was generated by cotransfecting the SHP-1 (C453/S) mutant in
a pcDNA3 vector (32) and the wild-type AT2 receptor in a
pBC-SF vector (using a 40:1 DNA ratio). Stably transfected cells were
selected in G418 (750 µg/ml; Life Technologies) for 3
weeks, and the cells expressing high levels of AT2
receptors were sorted by fluorescence-activated cell sorting
after immunolabeling with the anti-Flag M1 monoclonal antibody (Babco,
Richmond, VA). SHP-1 (C435/S) expression was assessed by immunoblot as
previously described (32). The immunoselected, stably transfected cells
were maintained in G418 (750 µg/ml) and used for up to four
passages.
Radioligand-Binding Assay
Ligand-binding assays were performed using membrane preparations
from the stably transfected cells as previously described (4).
Immunoprecipitation and Measurement of SHP-1 Tyrosine Phosphatase
Activity
Stably transfected PC12 cells were maintained overnight in
serum-free medium and then stimulated by AngII (10-7
M; Sigma Chemical Co., St. Louis, MO) for the
indicated time periods. The reaction was terminated by washing the
cells with ice-cold PBS, after which the cells were frozen in liquid
nitrogen and scraped. Cell lysis, SHP-1 immunoprecipitation, and
tyrosine phosphatase assay were performed as previously described (18)
with Abl-tyrosine-phosphorylated myelin basic protein as a substrate
according to the manufacturers instructions (New England Biolabs, Inc., Beverly, MA).
Phospho-ERK Immunoblot
Growth-arrested PC12 cells were stimulated with 10 ng/ml murine
NGF (Life Technologies) for 10 min with or without AngII
(10-7 M), washed twice with ice-cold PBS,
frozen in liquid nitrogen, and scraped. Cell lysates were subjected to
SDS-PAGE and electrotransferred onto Hybond-ECL nitrocellulose
membranes (Amersham, Arlington Heights, IL).
Phosphorylated or total ERK was detected with phospho-ERK (New England Biolabs, Inc.) or ERK (Upstate Biotechnology, Inc., Lake Placid, NY) antibodies, respectively, visualized by
enzyme-linked chemiluminescence (Amersham), and quantified
by scanning densitometry.
Apoptosis Assay
Oligosomal fragmentation of PC12 genomic DNA was measured using
cells seeded on six-well plates at a density of 5 x
105 cells per well. Equal amounts of DNA (500 ng) were used
for 3'-end labeling using terminal transferase (25 U/reaction;
Boehringer Mannheim, Indianapolis, IN) and
[
-32P]dideoxy-ATP (NEN Life Science Products, Boston,
MA) as previously described (40). The amount of
[
-32P]dideoxy-ATP incorporated into the low molecular
weight DNA fraction was quantified by scintillation counting of the
subchromosomal fraction of DNA cut from the dried gel.
Treatment of the Transfected PC12 Cells with a Membrane-Permeable
Ceramide Analog (C2-Ceramide)
PC12 cells stably expressing the AT2 receptor alone or together
with the SHP-1 (C453/S) mutant were seeded on six-well plates at a
density of 5 x 105 cells per well. The cells were
then treated for 24 h with 100 µM of C2-ceramide
(BIOMOL Research Laboratories, Inc., Plymouth Meeting, PA)
in serum-free medium containing 10 ng/ml NGF. After genomic DNA
extraction, oligosomal DNA fragmentation was performed as described
above.
Statistics
Results are expressed as mean ± SE.
Statistical significance was assessed by t test.
 |
FOOTNOTES
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---|
Address requests for reprints to: Victor Dzau, M.D., Cardiovascular Division, Department of Medicine, Brigham and Womens Hospital, 75 Francis Street, Boston, Massachusetts 02115.
This study was supported by NIH Grants HL-46631, HL-35252, HL-35610,
HL-48638, HL-07708, and HL-58616. Victor J. Dzau is the recipient of
NIH MERIT Award HL35610. Laurent Daviet is the recipient of a
postdoctoral fellowship from the American Heart Association,
Massachusetts Affiliate, and of an international research fellowship
from the Institut National de la Santé et de la Recherche
Médicale, France.
1 These authors contributed equally to this work. 
Received for publication January 21, 1999.
Revision received March 2, 1999.
Accepted for publication March 19, 1999.
 |
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