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 Women’s Hospital Boston, Massachusetts 02115
Institut Cochin de Génétique Moléculaire (C.N.) Centre Nationale de Recherche Scientifique UPR 0415 75014 Paris, France


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 240–244 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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 1Go and 2Go, [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.


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Table 1. Binding Parameters of 125I-[Sar1, Ile8]AngII in Stably Transfected PC12 Cells

 

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Table 2. Binding Parameters of 125I-[Sar1, Ile8]AngII in Stably Transfected PC12 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. 1AGo). 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 ({Delta}235–239 and {Delta}250–254, respectively) were fully functional in terms of SHP-1 activation (Fig. 1AGo). In assays performed with the {Delta}240–244 mutant, no AngII-mediated stimulation of SHP-1 was observed. Deletion of residues 245–249 ({Delta}245–249) attenuated by 70% the AngII-induced SHP-1 activation (Fig. 1AGo). 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.

 
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. 1BGo). 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 {Delta}240–244 mutant completely lost its ability to mediate agonist-dependent inhibition of ERK. Deletion of residues 245–249 ({Delta}245–249) yielded a partially functional receptor, causing a modest 15% reduction in ERK phosphorylation (Fig. 1BGo). 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. 1CGo). 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. 1CGo). Deletion of residues 240–244 ({Delta}240–244) completely abolished AngII-induced apoptosis, whereas the {Delta}245–249 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 240–244 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. 2Go, 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 2Go, 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 1Go and 2Go) 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 240–244 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.

 
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. 3AGo). Overexpression of SHP-1 (C453/S) abolished AT2 receptor-induced SHP-1 activation (Fig. 3BGo). As shown in Fig. 4Go, 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 (2–3 min) clearly precedes the onset of ERK inhibition (Fig. 4BGo). 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. 5Go, 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. 6Go).



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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. 1Go (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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 240–244 and, to a lesser extent, 245–249 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 240–244 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 245–249 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. 4BGo) 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 manufacturer’s 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 [{alpha}-32P]dideoxy-ATP (NEN Life Science Products, Boston, MA) as previously described (40). The amount of [{alpha}-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
 
Address requests for reprints to: Victor Dzau, M.D., Cardiovascular Division, Department of Medicine, Brigham and Women’s 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. Back

Received for publication January 21, 1999. Revision received March 2, 1999. Accepted for publication March 19, 1999.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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