Gα12/13 Mediates α1-Adrenergic Receptor–Induced Cardiac Hypertrophy
In neonatal cardiomyocytes, activation of the Gq-coupled α1-adrenergic receptor (α1AR) induces hypertrophy by activating mitogen-activated protein kinases, including c-Jun NH2-terminal kinase (JNK). Here, we show that JNK activation is essential for α1AR-induced hypertrophy, in that α1AR-induced hypertrophic responses, such as reorganization of the actin cytoskeleton and increased protein synthesis, could be blocked by expressing the JNK-binding domain of JNK-interacting protein-1, a specific inhibitor of JNK. We also identified the classes and subunits of G proteins that mediate α1AR-induced JNK activation and hypertrophic responses by generating several recombinant adenoviruses that express polypeptides capable of inhibiting the function of specific G-protein subunits. α1AR-induced JNK activation was inhibited by the expression of carboxyl terminal regions of Gαq, Gα12, and Gα13. JNK activation was also inhibited by the Gαq/11- or Gα12/13-specific regulator of G-protein signaling (RGS) domains and by C3 toxin but was not affected by treatment with pertussis toxin or by expression of the carboxyl terminal region of G protein–coupled receptor kinase 2, a polypeptide that sequesters Gβγ. α1AR-induced hypertrophic responses were inhibited by Gαq/11- and Gα12/13-specific RGS domains, C3 toxin, and the carboxyl terminal region of G protein–coupled receptor kinase 2 but not by pertussis toxin. Activation of Rho was inhibited by carboxyl terminal regions of Gα12 and Gα13 but not by Gαq. Our findings suggest that α1AR-induced hypertrophic responses are mediated in part by a Gα12/13-Rho-JNK pathway, in part by a Gq/11-JNK pathway that is Rho independent, and in part by a Gβγ pathway that is JNK independent.
In cardiomyocytes, stimulation of G-protein–coupled receptors activates mitogen-activated protein kinases, including c-Jun NH2-terminal kinase (JNK), which can subsequently trigger hypertrophic responses.1 α1-Adrenergic receptors (α1ARs) are Gq-coupled receptors expressed in the heart, and they are thought to play a role in cardiac hypertrophy. Significant evidence points to an important role for Gαq in receptor-induced hypertrophic responses. For example, α1AR-induced hypertrophy can be inhibited by neutralizing antibodies that recognize Gαq. In addition, a mutated M1 muscarinic acetylcholine receptor with impaired coupling to Gq, in contrast to the wild-type receptor, is unable to induce hypertrophy.2,3⇓ It has also been reported that cardiac hypertrophy can be induced by expressing a constitutively active mutant of Gαq.4 In total, these results suggest that Gαq plays an essential role in Gq-coupled receptor–mediated hypertrophy, a conclusion strengthened by evidence that overexpression of protein kinase Cβ, which is activated by diacylglycerol, can induce hypertrophy in a transgenic animal model.5 Recent reports have demonstrated that various proteins acting downstream from Gq-coupled receptors, including JNK and RhoA, are also involved in α1AR-induced hypertrophy.6,7⇓ α1AR-induced JNK activation and hypertrophic responses can be inhibited by the expression of dominant-negative RhoA and Clostridium botulinum C3 toxin,6 whereas transgenic overexpression of wild-type Gαq in the heart has been shown to induce hypertrophy.7 Consequently, it is generally thought that α1AR-induced hypertrophy is mediated, in part, by Gαq, RhoA, and JNK. The mechanism through which Gαq activates Rho has yet to be fully established. In COS-7 cells, expression of Gαq leads to a direct interaction between Gαq and the Rho guanine nucleotide exchange factor (RhoGEF) but does not result in an associated increase in activated Rho.8 Much about the signal transduction mechanisms mediating Gαq-dependent activation of JNK and Rho remain unresolved in cardiomyocytes. Also yet to be described is whether pertussis toxin (PTX)-insensitive G proteins other than Gq, such as G12 and G13, play a role in α1AR-induced JNK activation and hypertrophy.
Many Gq-coupled receptors, including those for thrombin, thromboxane A2, and lysophosphatidic acid, also couple to and activate G12 and G13.9–11⇓⇓ These are members of a distinct subfamily of G-protein α subunits; they are insensitive to PTX12 and have previously been shown to regulate stress fiber formation, cell transformation, apoptosis, and activation of the Na+-H+ exchanger.13 GTPase-deficient mutants of Gα12 and Gα13 are capable of activating JNK in many cell types.14,15⇓ In rat neonatal cardiomyocytes, expression of a constitutively active mutant of Gα13 induces hypertrophic responses, including increased gene expression of atrial natriuretic factor and β-myosin heavy chain.16 A recent report establishing a link between Gα12/13 and Rho, a small G protein, in an in vitro system showed that Gα13 can directly stimulate p115-RhoGEF, thereby triggering Rho activation,17,18⇓ and that although p115-RhoGEF does not appear to be involved in activation of Rho by Gα12, the regulator of the G-protein signaling (RGS) domain of p115-RhoGEF (p115-RGS) can bind Gα12 as well.
There are a few available tools to probe the functions of PTX-insensitive G proteins, such as Gq and G12/13. Among the more promising approaches are the RGS domains, which are polypeptides of ≈125 amino acids that interact with and inhibit activated Gα subunits.19 For example, the RGS domain of G-protein–coupled receptor kinase 2 (GRK2) interacts with Gαq and blocks its activity.20 Likewise, the RGS domain of p115-RhoGEF interacts with both Gα12 and Gα13 but does not bind Gαq or Gαi.18 Another promising tool is the carboxyl terminal portion of Gα subunits. Several groups have reported that carboxyl terminal fragments of Gαq and Gα13 can inhibit the interaction of receptors with Gq and G13, respectively.21–23⇓⇓ Therefore, we have used RGS domains and carboxyl terminal fragments of Gα subunits as inhibitors for analyzing Gαq-, Gα12-, and Gα13-mediated signaling pathways. We have also constructed a carboxyl terminal region of GRK2 (GRK2-ct) that can bind to and block the function of Gβγ.
In the present study, we demonstrate that α1AR stimulation activates JNK in both a G12/13-Rho–dependent and Gq-Rho–independent manner. We also demonstrate that Gα12/13 and Gβγ, in addition to Gαq, are involved in α1AR-induced hypertrophy.
Materials and Methods
DMEM, penicillin/streptomycin, PTX, and Thermoscript were purchased from Invitrogen. A DNA thermal cycler sequencing kit was purchased from Pharmacia Biotech. l-Phenylephrine hydrochloride (PE) and (±)-propranolol hydrochloride were from Sigma-Aldrich, and collagenase A was from Roche Molecular Biochemicals. Anti-JNK1 (SC-474 for immunoprecipitation), anti-JNK1 (SC-571 for Western blotting), anti-RhoA (SC-418), anti-Gαq (SC-393), anti-Gα12 (SC-409), anti-Gα13 (SC-410), and horseradish peroxidase–conjugated anti-rabbit and anti-mouse IgG antibodies were purchased from Santa Cruz Biotechnology. Anti-Gαq, anti-Gα12, and anti-Gα13 antibodies show the expected specificity. [γ-32P]ATP and [3H]leucine were from Perkin-Elmer Life Sciences, the RNeasy kit was from Qiagen, and Alexa Fluor 594-phalloidin was from Molecular Probes.
Cloning of cDNA and Production of Recombinant Adenoviruses
Details of the primers used to amplify regions of Gαq (Gαq-ct, amino acids 305 to 359), Gα12 (Gα12-ct, amino acids 325 to 379), Gα13 (Gα13-ct, amino acids 322 to 378), p115-RhoGEF (p115-RGS, amino acids 1 to 252), RGS4 (entire coding region), and the RGS domain of GRK2 (GRK2-RGS, amino acids 1 to 188) have been described previously.24 The entire coding region of C3 toxin was amplified, and its sequence was confirmed by a thermal cycler sequencing kit. We identified a sequence error at position 325 (G to A transition) in the original plasmid, which resulted in an amino acid change (Thr to Ala) at position 109. The carboxyl end of the p115-RGS construct was supplemented with a geranylgeranylation signal (Cys-Val-Leu-Leu) to facilitate translocation to the membrane.24 Each polymerase chain reaction (PCR) product was inserted into the appropriate sites of pAdTrack-CMV or pShuttle-CMV, and recombinant adenoviruses were prepared according to the method of He et al.25 Recombinant adenoviruses expressing GRK2-ct (amino acids 542 to 685) were produced as previously described.26 Recombinant adenovirus expressing LacZ was provided by RIKEN DNA Bank and was amplified as previously described.26 The JNK-binding domain of JNK-interacting protein-1 (JIP1-JBD, amino acids 127 to 281) was amplified by PCR with two primers (forward primer, 5′-GCGCGAATTCATCCTGGAGGACCTCAATATG-3′; reverse primer, 5′-CGCGCTCGAGTCAGCCTGTCTTCTCCAGCAC-CTGGGC-3′). After confirming the sequence, JIP1-JBD was inserted into KpnI and HindIII sites of pShuttle-CMV. To pull down activated Rho, a glutathione S-transferase (GST)-Rho–binding domain (RBD) fusion protein was constructed. The RBD of Rhotekin (amino acids 7 to 89) was PCR-amplified from cDNA freshly prepared from mouse brain RNA and confirmed by DNA sequencing. The resulting product was inserted into EcoRI and XhoI sites of pGEX-4T1, and the GST fusion protein (GST-RBD) was prepared by standard methods.
Preparation of Rat Neonatal Cardiomyocytes, Adenoviral Infection, and JNK Kinase Assay
Ventricular cardiomyocytes were isolated from 1-day-old Sprague-Dawley rat hearts and cultured as described previously.24,26⇓ Cardiomyocytes were plated on 1% gelatin-coated plates and cultured in DMEM supplemented with 5% FBS. Myocytes were seeded at a density of 7×106 cells per 6-cm dish for JNK kinase assay, 3.5×106 cells per well of a 6-well plate for Rho activation assay and intracellular cAMP measurement, 5×105 cells per well of a 12-well plate for [3H]leucine incorporation, and 3×105 cells per well of a 4-well slide chamber for actin staining. After 24 hours, the cells were infected with recombinant adenoviruses at a multiplicity of infection of 100 or 300 for 2 hours at 37°C. Cells were then starved in serum-free DMEM containing 10 nmol/L insulin and 5 mmol/L taurine and cultured for an additional 48 hours before treatment. Under these conditions, infection with adenoviruses coding for LacZ or green fluorescent protein resulted in almost 100% of cells that were LacZ or green fluorescent protein positive. JNK kinase activity in an immunocomplex prepared from stimulated or unstimulated cells was determined as previously described.24
Rho Activation Assay
Rho activation was determined by a pull-down assay using GST-RBD.27 Forty-eight hours after adenovirus infection, cardiomyocytes were treated with 30 μmol/L PE (PE is always supplemented with 1.5 μmol/L propranolol to block the β-stimulating activity of PE) for 1 minute and harvested in lysis buffer (in mmol/L: NaCl 150, MgCl2 30, Tris-HCl [pH 7.5] 50, dithiothreitol 1, and phenylmethylsulfonyl fluoride 1) supplemented with 0.1% Triton X-100, 10% glycerol, 1 μg/mL leupeptin, and 1 μg/mL aprotinin. Cell lysates were homogenized and centrifuged at 25 000g for 10 minutes at 4°C. The supernatant was then incubated with 12 μg of GST-RBD and glutathione-Sepharose beads for 1 hour at 4°C. The beads were washed twice with lysis buffer, and proteins were eluted with SDS-sample buffer, boiled, and subjected to Western blot analysis as previously described.24,26⇓ Densitometry was analyzed using Image Gauge (FUJIFILM), an image analysis program . Aliquots of cell lysates from each sample were subjected to Western blot analysis to confirm that equal amounts of RhoA were present under each condition.
Protein synthesis and actin reorganization were monitored by [3H]leucine incorporation into trichloroacetic acid–precipitable materials and by staining with Alexa Fluor 594-phalloidin, respectively.
Intracellular cAMP Measurement
Twenty-four hours after infection, rat neonatal cardiomyocytes were labeled with [3H]adenine for 24 hours, washed with serum-free DMEM, and stimulated by each agonist for 15 minutes. Intracellular cAMP accumulation was determined as previously described.28
Statistical significance was evaluated by ANOVA, followed by the Dunnett test.
JNK Activation and Hypertrophic Responses
To delineate the signal transduction cascades mediating α1AR-induced JNK activation in rat neonatal cardiomyocytes, it is necessary to introduce foreign cDNAs or cDNA fragments into the cells. However, rat neonatal cardiomyocytes resist typical transfection procedures, such as calcium phosphate precipitation and lipid-mediated transfection. As an alternative, we decided to produce various recombinant adenoviruses that express polypeptides that inhibit protein-protein interactions between different cellular signaling components. To determine whether JNK mediates α1AR-induced hypertrophic responses, we expressed JIP1-JBD, a specific inhibitor of JNK. α1AR stimulation by PE increased JNK activity by ≈5-fold (Figure 1A), which was blocked almost completely by the expression of JIP1-JBD. H2O2 also produced a marked increase in JNK activation, which was inhibited by the expression of JIP1-JBD. Prolonged α1AR-stimulation induced hypertrophic responses such as increased protein synthesis and reorganization of the actin cytoskeleton (Figures 1B and 1C). These responses were also inhibited by JIP1-JBD, suggesting that JNK activation is essential for the hypertrophic responses induced by the α1AR.
Regulation of α1AR-Induced JNK Activation by Gαq, Gα12, and Gα13
We have produced recombinant adenoviruses coding the carboxyl terminal regions of Gαq (Gαq-ct), Gα12 (Gα12-ct), and Gα13 (Gα13-ct). Previously, we demonstrated that these Gα-ct constructs can inhibit receptor–G-protein coupling with subunit-specific selectivity.24 In CHO-K1 cells, adenovirus-mediated expression of Gαq-ct, but not Gα12-ct and Gα13-ct, inhibited the ATP-induced increase in [Ca2+] by activation of phospholipase C. In contrast, ATP-induced actin polymerization, possibly mediated through Rho, was inhibited by Gα12-ct and Gα13-ct, but not Gαq-ct. The expression of Gα-ct constructs was previously confirmed by reverse transcription–PCR. Before further examining the selectivity of these constructs, we also confirmed the expression of Gα12, Gα13, and Gαq in membranes of neonatal cardiomyocytes by Western blot (Figure 2A. These results indicate that these constructs are capable of competing with endogenously expressed Gα12, Gα13, and Gαq. We next examined whether Gαq-ct, Gα12-ct, and Gα13-ct could affect Gs-mediated, Gi-mediated, or receptor-independent signaling pathways. Figure 2B shows that isoproterenol-stimulated (Gs-mediated) cAMP accumulation and carbachol-stimulated (Gi-mediated) inhibition of isoproterenol-induced cAMP accumulation were not affected by Gαq-ct, Gα12-ct, or Gα13-ct. H2O2 has previously been shown to strongly activate JNK in neonatal cardiomyocytes, although the precise mechanism through which this occurs remains unknown. Again, expression of Gαq-ct, Gα12-ct, and Gα13-ct did not affect H2O2-induced (receptor-independent) JNK activation (Figure 2C). These and previous results demonstrate that Gαq-ct, Gα12-ct, and Gα13-ct selectively inhibit receptor-Gq, -G12, and -G13 coupling, respectively.21,24⇓ As shown in Figure 3, rat neonatal cardiomyocytes expressing Gαq-ct, Gα12-ct, or Gα13-ct were stimulated with 30 μmol/L PE for 30 minutes, and JNK activity was determined. Overexpression of Gαq-ct strongly inhibited PE-induced JNK activation, as did overexpression of Gα12-ct and Gα13-ct, suggesting that JNK activation by α1AR stimulation is mediated by Gq, G12, and G13.
We next determined which G-protein subunits may be involved in PE-induced JNK activation. To examine the involvement of α subunits of Gq, G12, and G13, we produced adenoviruses coding RGS domains of GRK2 (GRK2-RGS) and p115-RhoGEF (p115-RGS), which function as specific inhibitors of Gαq and Gα12/Gα13, respectively. We also produced adenoviruses coding RGS4, which inhibits both Gαq and Gαi. JNK activation after PE stimulation was strongly inhibited by p115-RGS, RGS4, and GRK2-RGS (Figures 4A and 4B). Pretreatment of cells with 100 ng/mL PTX, which ADP-ribosylates Gαi and uncouples receptors from Gi, had no effect on PE-induced JNK activation (Figure 4C). We also examined the role of Gβγ using GRK2-ct, which binds Gβγ and blocks Gβγ-mediated signaling pathways. GRK2-ct did not affect PE-induced JNK activation (Figure 4D), suggesting that a Gβγ-mediated signal is not required for JNK activation by the α1AR. These results indicate that in cardiomyocytes JNK activation by the α1AR is mediated by the activation of Gαq, Gα12, and Gα13, but not Gβγ or Gαi.
Role of Rho in α1AR-Induced JNK Activation
Present knowledge suggests that Rho, a small G protein, is activated downstream from G12 and G13. In the present study, we have examined the contribution of Rho to α1AR-induced JNK activation. C3 toxin is frequently used to demonstrate an involvement of Rho in cellular functions, inasmuch as it has the ability to ADP-ribosylate and inactivate Rho. In cardiomyocytes, the expression of C3 toxin inhibited JNK activation after stimulation by PE (Figure 5A) and completely inhibited PE-induced Rho activation (Figure 5B), suggesting that Rho is involved in α1AR-induced JNK activation. We next examined whether Gq, G12, or G13 contributes to the PE-induced activation of Rho. α1AR stimulation increased the amount of activated Rho that was pulled down by GST-RBD by ≈4-fold, suggesting that α1AR stimulation activates Rho in neonatal cardiomyocytes (Figure 6). This activation could be inhibited by Gα12-ct and Gα13-ct, but not Gαq-ct. These results suggest that JNK is activated through Gα12/13-Rho–dependent and Gαq-Rho–independent pathways in a concerted manner.
Involvement of Gα12/13 in Hypertrophic Response Induced by α1AR
We next investigated the roles of individual G-protein subunits in α1AR-induced hypertrophic responses. Measures of hypertrophy, including reorganization of the actin cytoskeleton and changes in protein synthesis, were evaluated by staining actin filaments with phalloidin and by measuring the incorporation of 3H into cellular proteins, respectively. Compared with no stimulation of cardiomyocytes, stimulation of the α1AR with 30 μmol/L PE for 48 hours induced drastic reorganization of actin filaments (Figures 7A and 7B). α1AR-induced reorganization of actin filaments was not inhibited by PTX treatment (Figure 7A), consistent with previous results showing that PTX does not affect α1AR-induced JNK activation. In contrast, α1AR-induced actin reorganization was inhibited by p115-RGS, GRK2-RGS, and C3 toxin (Figure 7B). Figure 7B also shows that GRK2-ct could inhibit α1AR-induced actin reorganization, indicating that Gβγ contributes to actin reorganization through a signaling pathway that does not involve JNK activation. Measurements of protein synthesis rate produced similar results. α1AR stimulation increased the rate of protein synthesis by ≈2-fold (Figures 8A and 8B). This increase in protein synthesis was not affected by PTX treatment (Figure 8A) but was inhibited by p115-RGS, GRK2-RGS, GRK2-ct, and C3 toxin (Figure 8B).
In the present study, we demonstrated that α1AR stimulation activates JNK via Gq- and G12/13-mediated pathways in rat neonatal cardiomyocytes. This finding is consistent with previous reports indicating that transient expression of wild-type or constitutively active mutants of Gαq and Gα13 can induce JNK activation.14–16,29⇓⇓⇓ To demonstrate an involvement of Gαq, Gα12, and Gα13 in α1AR-induced JNK activation in cardiomyocytes, we used unique reagents, such as RGS domains and carboxyl terminal fragments of Gα subunits. Expression of Gαq-ct, Gα12-ct, and Gα13-ct inhibited JNK activation, indicating that Gq, G12, and G13 are involved in α1AR-induced JNK activation. This conclusion is further supported by data showing that overexpression of RGS domains specific for Gαq, Gα12, or Gα13 also significantly or almost completely inhibited α1AR-induced JNK activation. The RGS domains inhibited JNK activation with a rank order of potency of p115-RGS>GRK2-RGS>RGS4. Whether these differences reflect the differential contributions of the various Gα subunits to α1AR-mediated JNK activation remains unclear, inasmuch as the expression levels and Ki values of the various inhibitory peptides could not be determined. Nonetheless, the present study clearly demonstrates that RGS domains and Gα-ct are useful tools for delineating the role of individual PTX-insensitive Gα subunits in intracellular signal transduction pathways.
The inhibition of JNK activation by GRK2-RGS, RGS4, and p115-RGS suggests that Gαq or Gα12/13 alone is not sufficient for α1AR-induced JNK activation and that a concerted contribution by Gαq, Gα12, and Gα13 may instead be necessary. Activation of Rho by the α1AR was inhibited by Gα12-ct and Gα13-ct, but not Gαq-ct. Therefore, Gαq- and Gα12/Gα13-derived signals converge at a point after Rho activation but preceding JNK activation. In the future, it will be necessary to determine the specific target molecule(s) of Gαq to precisely delineate the JNK activation pathway. We also showed that Gβγ is not involved in α1AR-induced JNK activation in rat neonatal cardiomyocytes. The inability of GRK2-ct and PTX to affect JNK activation cannot be explained by either low expression of GRK2-ct or insufficient ADP-ribosylation of Gαi by PTX, because under the same conditions, we observed that GRK2-ct and PTX treatment can block endothelin-1–induced ERK activation.24 Two groups have previously reported a role for Gβγ in the activation of JNK by Gq-coupled receptors in HEK293 cells.29,30⇓ It is possible that various cells may express different signaling components at different levels and that the relative contributions of these signaling components to JNK activation may differ in both a cell-type–specific and receptor-dependent manner.
The present study also demonstrated an essential role for G12/13 in α1AR-induced hypertrophy. Finn et al16 observed in rat neonatal cardiomyocytes that the transient overexpression of a constitutively active mutant of Gα13 causes hypertrophic responses, such as increased gene expression of atrial natriuretic factor and β-myosin heavy chain, and an increase in cell size. Consistent with that report, we have shown that inhibition of Gα12/13 by p115-RGS dramatically decreases α1AR-induced hypertrophic responses. Many groups have also reported that Gαq is essential for α1AR-mediated hypertrophy.2–4,7⇓⇓⇓ Likewise, we have shown that inhibition of Gαq by GRK2-RGS blocks α1AR-induced hypertrophic responses. These results suggest an important role for Gα12 and Gα13, in addition to Gαq, in α1AR-induced hypertrophy. Furthermore, these results suggest that Gαq-, Gα12-, and Gα13-mediated signal transduction pathways are all necessary to fully induce hypertrophic responses by α1AR stimulation. However, inasmuch as Gα12-ct and Gα13-ct, but not Gαq-ct, inhibited Rho activation, whereas Gα12-ct, Gα13-ct, and Gαq-ct inhibited JNK activation, it appears that Gαq activates JNK in a Rho-independent manner.
We have also demonstrated that Gβγ inhibition by GRK2-ct can inhibit the PE-induced reorganization of the actin cytoskeleton and the increased protein synthesis, suggesting that Gβγ also plays a role in α1AR-induced cardiac hypertrophy. The inhibitory action of GRK2-ct may be due to an inhibition of ERK activation. Yue et al31 reported that inactivation of ERK by U0126 can inhibit the hypertrophic responses induced by PE. Conversely, Gβγ could target a protein involved in Rho activation. In HeLa but not Swiss 3T3 cells, expression of Gβγ induces stress fiber formation in a Rho-dependent manner.32 In rat neonatal cardiomyocytes, expression of a dominant-negative mutant of Rho inhibits the hypertrophic response induced by PE.6 Therefore, it is possible that Gβγ may induce hypertrophic responses through the activation of Rho.
Few reports have investigated the role of Gβγ in α1AR-induced hypertrophy. A recent study showed that in vivo pressure-overload hypertrophy activates phosphatidylinositol 3-kinase and Akt through Gq-coupled receptor stimulation and subsequent Gβγ dissociation in the heart.33 However, blockade of Gβγ function by GRK2-ct in a transgenic mouse heart fails to affect pressure overload–induced cardiac hypertrophy.33 However, this pressure overload–induced hypertrophy could be attenuated by cardiac-specific overexpression of Gαq-ct, which inhibits receptor-Gq coupling.21,33⇓ Further studies will be necessary to elucidate the role of Gβγ in the induction of hypertrophic responses and to define the relationship between Gβγ and Rho activation in cardiomyocytes.
In summary, we have demonstrated in cardiomyocytes that the α1AR activates Gq, G12, and G13 and that α1AR-induced JNK activation is mediated via the activation of Gαq, Gα12, and Gα13, but not Gi or Gβγ. We have also shown that Gα12/13 and Gαq are required for α1AR-induced hypertrophy.
This study was supported by a grant from the Ministry of Education, Science, Sports, and Culture of Japan (to T.N. and H.K.). We thank Dr Melvin I. Simon for mouse Gα12 and Gα13 cDNAs and Dr Robert J. Lefkowitz for rat GRK2 cDNA. We also thank the RIKEN DNA Bank for adenovirus expressing LacZ.
Original received March 28, 2002; resubmission received September 9, 2002; revised resubmission received October 14, 2002; accepted October 14, 2002.
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