Contrasting Signaling Pathways of α_1A- and α_1B-Adrenergic Receptor Subtype Activation of Phosphatidylinositol 3-Kinase and Ras in Transfected NIH3T3 Cells

Abstract

Activation of protein kinases is an important intermediate step in signaling pathways of many G protein-coupled receptors including α₁-adrenergic receptors. The present study was designed to investigate the capacity of the three cloned subtypes of human α₁-receptors, namely, α_1A, α_1B and α_1D, to activate phosphatidylinositol 3-kinase (PI 3-kinase) and p21^ras in transfected NIH3T3 cells. Norepinephrine activated PI 3-kinase in cells expressing human α_1A andα _1B via pertussis toxin-insensitive G proteins; α_1D-receptors did not detectably activate this kinase. Transient transfection of NIH 3T3 cells with theα -subunit of the G protein transducin (α_t) a scavenger of βγ-subunits released from activated G proteins, inhibited α_1B-receptor but notα _1A-receptor-stimulated PI 3-kinase activity. Stimulation of both α_1A- andα _1B-receptors activated p21^ras and stimulated guanine nucleotide exchange on Ras protein. Overexpression of a dominant negative mutant of p21^ras attenuatedα _1B-receptor but notα _1A-receptor activation of PI 3-kinase. Overexpression of a dominant negative mutant of PI 3-kinase attenuatedα _1A- but notα _1B-receptor-stimulated mitogen-activated protein kinase activity. These results demonstrate the capacity for heterologous signaling of theα ₁-adrenergic receptor subtypes in promoting cellular responses in NIH3T3 cells.

INTRODUCTION

α₁-Adrenergic receptors, members of the class of G protein-coupled receptors, mediate many of the important physiological effects of catecholamines such as norepinephrine or epinephrine (1, 2).α ₁-Adrenergic receptors may play a role in many human diseases, such as atherosclerosis and hypertension (3, 4), restenosis after coronary dilation (5), myocardial hypertrophy, and cardiac arrhythmia (6, 7). There are at least three subtypes ofα ₁-receptors expressed in human vascular smooth muscle cells (VSMCs) and many other cells, namely, α_1A-,α _1B-, and α_1D-receptors (8, 9). It is generally accepted that activation of all three subtypes ofα ₁-receptors increases hydrolysis of phosphatidylinositol (PI) 4,5-bisphosphate to inositol (1, 4, 5)-triphosphate and diacylglycerol via the α-subunit of Gq, a family of pertussis toxin (PTx)-insensitive G proteins (10, 11). Production of inositol triphosphate and diacylglycerol can also occur via activation of phospholipase Cβ mediated by βγ-subunits released from G proteins such as Go and Gi (12). It is becoming increasingly clear thatα ₁-receptors activate other signaling pathways; for example, α₁-receptors activate phospholipase D in brain and promote the release of arachidonic acid via activation of phospholipase A2 via PTx-sensitive G proteins (13, 14). Recent studies indicate that α₁-adrenergic receptors may share tyrosine protein kinase/Ras/mitogen-activated protein (MAP) kinases signaling pathways with peptide growth factors to stimulate growth responses in several types of cells including myocytes and VSMCs (Ref. 15 and Z.-W. Hu, X. Y. Shi, R. Z. Lin, and B. B. Hoffman, submitted for publication).

Phosphatidylinositol 3-kinase (PI 3-kinase) is a kinase that plays an important role in mediation of mitogenic actions of many peptide growth factors and G protein-coupled receptors in various types of cells (for reviews see Refs. 17, 18). PI-3 kinase has been implicated in the regulation of cell growth by receptor tyrosine kinases (19, 20), cytokine receptors (21), and G protein-coupled receptors (22, 23). PI 3-kinase mediates a number of intracellular events including the PKC-independent serine phosphorylation and activation of a ribosomal S6 kinase family designated p70/p90^S6K (24). PI 3-kinases and ribosomal S6 kinases play a major part in mitogen-stimulated increase in protein synthesis and changes in cytosolic structure of growing and dividing cells. Mitogens such as platelet-derived growth factor (PDGF), insulin, and insulin-like growth factor (IGF) activate PI 3-kinase leading to rapid phosphorylation of phosphatidylinositol (4, 5)-diphosphate at the D-3 position of the inositol ring to form phosphatidylinositol (3–5)-triphosphate (25). Increasing evidence suggests that this product is involved in the regulation of the actin cytoskeleton that plays a critical role in a number of cellular processes including motility, chemotaxis, and cell division (26, 27).

There are multiple forms of PI 3-kinase with distinct mechanisms of regulation and different substrate specificities in mammalian cells; these enzymes are activated both by peptide growth factors and G protein-coupled receptor agonists. The first identified PI 3-kinase is a heterodimer consisting of a p85-regulatory subunit with SRC homology 2 (SH2) domains and a p110 catalytic subunit (28). Another PI 3-kinase has been described; this isoform consisted of a p110 monomeric subunit that is activated independently of a p85-regulatory subunit (29). A major mode of activation of the heterodimeric PI 3-kinase by growth factors likely involves docking of the kinase through SH2 domains on the p85 subunit to phosphorylated tyrosine residues(s) on receptor tyrosine kinases (30). This PI-3 kinase isoform is also directly activated by βγ-subunits released from activated G proteins in platelets (31) and by Gi in cell lines expressingα _2A-adrenergic receptors (22). Activation of the heterodimeric PI 3-kinase has a complex relationship with p21^ras activation. Activation of PI 3-kinase may occur via Ras-dependent (32) or independent (33) pathways; PI 3-kinase may function as a target of P21^ras (34) in some cells or function upstream of p21^ras in others (22, 35). We recently demonstrated in human VSMCs, likely expressing the three subtypes ofα ₁-receptors, that α₁ receptor-stimulated mitogenesis is associated with activation of PI 3-kinase (36). In those cells, α₁-receptor activation of PI 3-kinase is mediated by a PTx-sensitive G protein(s), and PI 3-kinase activity is associated with activation of p21^Ras and increased tyrosine protein kinase activity. However, there is little specific information about signaling pathways that mediate α₁-receptor subtype stimulation of PI 3-kinase, particularly in a model system where potential differences in the signaling pathways used by the three cloned α₁-receptor subtypes could be discerned.

Increasing evidence suggests that each of the subtypes ofα ₁-receptors may activate overlapping or potentially distinct signaling pathways (2, 13). However, little is known about the potential biological significance of the various specificα ₁-receptor subtypes being expressed in same cells. Availability of expression vectors containing cDNAs encoding each of the α₁-receptor subtypes provides an approach to the question of which subtype(s) of α₁-receptors mediate activation of PI-3 kinase. NIH3T3 cells stably transfected with each of three subtypes of α₁-receptors provides an interesting model system as these cells have been extensively characterized in terms of classical growth factor-stimulated mitogenesis. In the present study, we have found that stimulation both α_1A- andα _1B-, but not α_1D-receptor subtypes of human α₁-receptors, stably expressed in these cells, activated PI 3-kinase. The results demonstrate that α_1A- and α_1B-receptor subtypes activated PI 3-kinase via different subunits of G proteins; and there were differentiable patterns of activation of p21^Ras protein and MAP kinase cascades by α₁-receptor subtypes in these cells.

RESULTS

NIH3T3 cells were used for transfection of control vectors or expression vectors containing three subtypes (α_1A,α _1B, and α_1D) of humanα ₁-receptors. To confirm functional expression of each of the three subtypes of α₁-receptors in NIH3T3 cells,α ₁ receptor-stimulated expression of c-fos mRNA was measured in NIH3T3 cells stably expressing α_1A,α _1B, and α_1D receptors. Induction of c-fos mRNA was selected as a functional index of biologically significant expression of α₁-receptors (37). Stimulation of the cells with norepinephrine caused similar increases in expression of the c-fos gene in the cells transfected with each of the three subtypes of α₁-receptors, suggesting that these cells expressed functional receptors for each of the three subtypes (Fig. 1A). Norepinephrine did not stimulate c-fos expression in the wild-type cells (data not shown). Also, norepinephrine-stimulated increased expression of c-fos mRNA was attenuated by theα ₁-receptor antagonist prazosin (Fig. 1B).

Figure 1.

Characterization of Functional Expression ofα ₁-Receptor Subtypes A, NIH3T3 cells, which individually stably expressed each of the three subtypes of α₁-receptors, were grown to confluence in a series of 100-cm² dishes in DMEM containing 10% FBS in the presence of geneticin (100 ng/ml) for 24 h. The near-confluent 3T3 cells were then incubated with serum-free DMEM for 24 h. After norepinephrine or vehicle treatment for 60 min, cells were harvested for isolation of total RNA, and Northern blots were performed as described in Materials and Methods. The data are representative of three experiments. Panel B demonstrates that theα ₁-receptor antagonist prazosin blocked the induction of norepinephrine-stimulated c-fos mRNA expression (mean ± sem of four experiments).

Norepinephrine did not activate PI-3 kinase activity in wild-type NIH3T3 cells or in cells transfected with control vectors, confirming that these cells do not express endogenous α₁-receptors. As illustrated in Fig. 2, norepinephrine (10 μm) stimulated activation of PI 3-kinase in NIH3T3 cells transfected with α_1A- orα _1B-receptors. However, although cells transfected withα _1D-receptors reproducibly had an increased basal activity of PI 3-kinase compared with cells transfected with control vectors, norepinephrine did not significantly increase PI 3-kinase activity in cells transfected with the α_1D-receptors. Interestingly, the increased basal activity of PI 3-kinase in cells transfected α_1D-receptors was inhibited by theα ₁-receptor antagonists, doxazosin and prazosin (data not shown). This result suggests that overexpression ofα _1D-receptors may spontaneously (in the absence of added agonist), albeit weakly, activate down-stream signaling mechanisms and that these antagonists are acting as inverse agonists. Norepinephrine stimulated a concentration-dependent increase in PI 3-kinase activity in cells expressing α_1A- or α_1B-receptors, and theses responses were blocked by α₁-receptor antagonists, doxazosin and prazosin (data not shown), as we reported previously for VSMCs (36).

Figure 2.

α₁-Receptor Subtype-Mediated Activation of PI-3 Kinase NIH3T3 cells, stably transfected with control vectors or expression vectors containing each of the three subtypes of human α₁ receptors, were incubated for 24 h in serum-free DMEM and then pretreated with β-receptor antagonist timolol (10 μm) and α₂-receptor antagonist idazoxan (10 μm) for 2 h. Cells were then treated with vehicle or 10μ m of norepinephrine for 5 min. Cell lysates (2 mg of protein) were immunoprecipitated with anti-P85α subunit of PI-3 kinase and subjected to determination of PI-3 kinase activity as described in Materials and Methods. The autoradiogram of TLC of PI-3 kinase was exposed for 20 h. A, Data from a representative experiment. B, Data are average ± sem of three experiments. **, P < 0.01.

To determine whether stimulation of PI 3-kinase in cells expressing either α_1A- or α_1B-receptor subtypes required PTx-sensitive G proteins, cells were incubated with PTx (50 ng/ml) for 16 h. PTx did not block norepinephrine-stimulated PI 3-kinase in cells transfected with either α_1A-receptors (Fig. 3A, lane 4) orα _1B-receptors (Fig. 3B, lane 4), suggesting that both of these subtypes of α₁-receptors activate PI 3-kinase via PTx-insensitive G proteins in NIH3T3 cells.

Figure 3.

Effects of PTx and Expression of α-Subunit of Transducin (α-Transducin) on α₁-Receptor Subtype-Stimulated Activation of PI-3 Kinase Cells expressing α_1A (panel A) or α_1B (panel B) receptors were grown to 70–80% of confluence and transiently transfected with α-subunit of transducin orβ ₁-adrenergic receptor kinase (β-ARK) as described in Materials and Methods. Cells were incubated with serum-free DMEM for 24 h and were pretreated with PTx (100 ng/ml) or not for 16 h. Cells were then stimulated with vehicle or 10μ m norepinephrine for 5 min. Cell lysates (2 mg of protein) were prepared, immunoprecipitated with anti-p85 of PI-3 kinase antibody, and subjected to determination of PI-3 kinase as described above. The autoradiogram of TLC of PI-3 kinase was exposure for 24 h. Experiments were repeated 3 times with similar results. C, Transfection of 3T3 cells with the β-ARK and α_t genes demonstrated expression of the GRK2 protein. Cells were transiently transfected with β-ARK, α_t (10 μg), or control vector (10 μg) as described above. Cell lysates were prepared, and 100 μg of protein/sample were separated by PAGE and subjected by Western blotting as described in Materials and Methods. β-ARK or α_t was detected by an anti-GRK2 antibody or by anti-α_t antibody. D, Cotransfection of 3T3 cells with the α_2A-receptor gene plus either α_t or GRK2. Cells were then treated with or without norepinephrine, and MAP kinase activity was measured as described in Materials and Methods. β-ARK andα _t inhibited norepinephrine-stimulated MAP kinase activity in cells epxressing α_2A-receptors. Data are a representative of four experiments.

To investigate potential roles of α- and βγ-subunits of G proteins in activation of PI 3-kinase, cells transfected stably withα _1A- or α_1B-receptors were transiently transfected with the α_t-construct as α_t functions as a scavenger of free βγ (38). As illustrated in Fig. 3, overexpression of α_t did not inhibit norepinephrine-stimulated activation of PI 3-kinase in cells transfected with α_1A-receptors. This result suggests that either an α-subunit or a βγ-subunit not recognized byα _t, released from a PTx-insensitive G protein, mediates this response (Fig. 3A, lane 5). However, overexpression ofα _t blocked norepinephrine-stimulated activation of PI 3-kinase in cells transfected with α_1B-receptors (Fig. 3B, lane 5), suggesting that α_1B-receptor-mediated activation of PI 3-kinase may be due to direct effects ofβγ -subunits released from a PTx-insensitive G protein. However, overexpression of an β-adrenergic receptor kinase peptide (β-ARK)[ also called G-protein receptor kinase-2 (GRK2)], which is a different scavenger of free βγ-subunits (22), did not inhibit either α_1A- or α_1B-receptor-stimulated PI 3-kinase activity. Western blotting (Fig. 3C) demonstrated successful expression of GRK2 as well α_t in these cells. To demonstrate functional expression of GRK2 in these cells, cells were cotransfected with the α_2A-adrenergic receptor gene and either α_t or GRK2. GRK2 has been shown to blockα _2A-receptor-stimulated activation of MAP kinase (43). These cells were then treated with or without norepinephrine, and MAP kinase activity was measured. Results indicate that bothα _t and GRK2 attentuated theα _2A-receptor-stimulated increase in activity of MAP kinase, suggesting that these two molecules functioned effectively as scavengers of the βγ-subunits released by these receptors (Fig. 3D).

We have previously found thatα ₁-receptor-activated PI 3-kinase activity can be detected in anti-Ras immunocomplexes, and stimulation ofα ₁-receptors directly increases active Ras-GTP in human VSMCs (36). Also, our previous study suggested that activation of Ras-GTP is a downstream target of PI 3-kinase after stimulation ofα ₁-receptors in those cells. NIH3T3 cells provide an excellent model system by which to pursue the role of the individualα ₁-receptor subtypes in activating Ras-GTP and PI 3-kinase. We first tested which subtypes of α₁-receptors activated p21^Ras protein. The results indicate that norepinephrine stimulates an increase in active Ras-GTP in cells expressing either α_1A- or α_1B-receptors, but no measurable change was detected in cells expressingα _1D-receptors (Fig. 4A and B). Both α_1A- and α_1B-receptor activation of p21^Ras protein was insensitive to PTx (Fig. 4C). Overexpression of α-transducin did not inhibitα _1A-receptor-activation of Ras-GTP. On the other hand,α _t markedly inhibitedα _1B-receptor-stimulated activation of Ras-GTP, suggesting that βγ-subunits released from Gq were used byα _1A-receptors in signaling to activate p21^Ras.

Figure 4.

Activation of PI 3-Kinase and p21^Ras-GTP by α₁-Receptors A, Near-confluent cells stably expressing each of the three subtypes ofα ₁-receptors were metabolically labeled with[ ³²P]Pi in phosphate-free DMEM for 12 h. The cells were treated with vehicle or norepinephrine (10 μm) for 5 min. Cell lysates (1 mg of protein) were subjected to immunoprecipitation with an rat polyclonal antibody against H-Ras. Ras-bound GTP was determined as described in Materials and Methods. The autoradiogram after TLC was exposed for 16 h. Panel A is representative of three experiments. Panel B illustrates the mean of three experiments. Panel C illustrates norepinephrine-stimulated increases in active Ras-GTP. Cells expressingα _1A- or α_1B-receptors were grown for 24 h, and then the cells were transfected with α-subunit of transducin as described in Materials and Methods. Cells were metabolically labeled with [³²P]Pi for 12 h. Cells were treated with vehicle or norepinephrine (10 μm) for 10 min. Cell lysates were prepared and immunoprecipitated with anti-H-Ras antibody and subjected to TLC as described above. PTx (pretreated for 16 h) had no effect on activation byα _1A- or α_1B-receptors, whereas expression of the α-subunit of transducin (α-transducin) inhibited theα _1B but not the α_1A response. Data are average ± sem of three experiments. *, P < 0.05; **, P < 0.01.

To obtain additional information about the interactions between p21^Ras protein and PI 3-kinase, overexpression of a dominant negative mutant of p21^Ras (P21^Ala15ras) was used. Transfection of cells with this dominant negative mutant of p21^Ras inhibits agonist-induced increases in active Ras-GTP (39). If activation of PI 3-kinase were upstream of p21^Ras activation, then transfection of cells with this mutated gene should not inhibit α₁-receptor activation of PI 3-kinase. On the other hand, if activation of PI 3-kinase by stimulation of α₁-receptors were a downstream target of Ras-GTP, the construct would be expected to inhibit PI 3-kinase activation. As illustrated in Fig. 5, overexpression of the dominant negative mutant of p21^Ras blocked norepinephrine-stimulated increases in active Ras-GTP in cells expressing α_1A- andα _1B-receptors as expected (Fig. 5A). Expression of this negative mutant of p21^Ras did not inhibit norepinephrine-stimulated activation of PI 3-kinases in the cells expressing α_1A receptors (Fig. 5B). However, expression of the mutant p21^ras attenuated activation of PI 3-kinase in cells expressing α_1B-receptors (Fig. 5C), suggesting that p21^Ras protein functions downstream or independently of activation of PI 3-kinase after stimulation ofα _1A-receptors but upstream of PI 3-kinase after stimulation of α_1B-receptors.

Figure 5.

Effects of Expression of Dominant Negative Mutant of p21^Ras Gene on Norepinephrine-Stimulated Increase in Ras-GTP and PI 3-Kinase A, Cells expressing α_1A- or α_1B-receptors were grown for 24 h, after which the cells were transfected with the p21^Ala15ras dominant negative mutant (Ras15) as described in Materials and Methods. Forty eight hours after transfection, cells were metabolically labeled with[ ³²P]Pi in Pi-free DMEM for 12 h. Cells were treated with norepinephrine (10 μm) for 10 min. Cell lysates were prepared and immunoprecipitated with anti-H-Ras antibody and subjected to TLC. The autoradiogram of the TLC plate was exposed for 16 h. Experiments were repeated twice with essentially identical results. The results indicate that both α_1A- andα _1B-receptor-mediated increases in Ras-GTP were blocked by expression of the negative mutation of p21^Ras gene. B and C, Cells expressing α_1A- orα _1B-receptors were grown for 24 h, after which the cells were transfected with the p21^Ala15ras dominant negative mutant as described in panel A. After 24 h incubation with serum-free DMEM, cells were treated with vehicle or norepinephrine (10 μm) for 5 min. PDGF-BB (1 nm for 5 min) was used as positive control. Cell lysates were prepared, immunoprecipitated with anti-p85 of PI 3-kinase, and kinase activity was measured. α_1B- but notα _1A receptor-stimulated increase in PI 3-kinases was blocked by expression of dominant negative mutation of the p21^Ras gene. The autoradiogram was exposed for 16 h. Experiments were repeated twice with essentially identical results.

Since activation of PI 3-kinase and p21^Ras protein induces activation of a series of growth or differentiation-related protein kinase cascades in various cells, we determined whether activation of specific protein kinase cascades was associated with the different subtypes of α₁-receptors. As illustrated in Fig. 6, norepinephrine activated MAP kinase in NIH 3T3 cells expressing α_1A- andα _1B-receptors but not significantly in cells expressingα _1D-receptors or in wild-type NIH 3T3 cells. These results were consistent with the results demonstrating that norepinephrine stimulated activation of p21^Ras protein in cells expressing α_1A- and α_1B-receptors, but not detectably in cells expressing α_1D-receptors (Fig. 4A). Coexpression of the P21^A15ras dominant negative mutant in cells expressing α_1A- andα _1B-receptors significantly inhibited norepinephrine-stimulated activation of MAP kinase (Fig. 6C). These results demonstrate that stimulation of both α_1A- andα _1B-receptors activates MAP kinase at least in part via a p21^Ras-dependent signaling path-way.

Figure 6.

α₁-Receptor Subtype Activation of MAP Kinase via p21^Ras Signaling Pathways A, The near-confluent cells stably expressing each of the three subtypes of α₁-receptors were incubated with serum-free DMEM for 24 h. The cells were treated with vehicle or norepinephrine (10 μm) for 10 min. Cell lysates (400 μg of protein) were subjected to immunoprecipitation with an anti-p44^ERK1 antibody. Washed immunocomplexes were subjected to in vitro assay of MAP kinase activity as described in Materials and Methods using MBP as substrate. Reaction mixtures were loaded and separated on 14% SDS-PAGE, and the dried gels were exposed to Kodak XAR-5 film at −70 C with an intensifying screen for 16 h. B, The data are mean ± sem of three assays of MAP kinase activity. C, Cells expressing α_1A- or α_1B-receptors were grown for 24 h and then were transfected with the p21^Ala15ras dominant negative mutant as described in Materials and Methods. Two days after transfection, cells were incubated in serum-free DMEM for 18 h. Cells were treated with vehicle or norepinephrine (10 μm) for 10 min. Cell lysates were prepared and immunoprecipitated with anti-p44^ERK1 antibody and subjected to in vitro assay of MAP kinase activity as described above. The data are average ± sem of three experiments. *, P < 0.05; **, P < 0.01.

We were interested in determining the potential role of PI 3-kinase in MAP kinase signaling pathways used by α₁-receptor subtypes expressed in NIH3T3 cells. Figure 7 illustrates the effects of negative dominant mutant of PI 3-kinase P85 subunit on MAP kinase activity in cells expressing α_1A- and α_1B-receptors. Coexpression of this mutant of PI 3-kinase p85 significantly attenuated norepinephrine-stimulated increases in kinase activities in cells expressing α_1A-, but not in cells expressingα _1B-receptors (Fig. 7), suggesting that PI 3-kinase functions as upstream component in theα _1A-receptor-stimulated MAP kinase cascade. On the other hand, the MAP kinase cascade may not be a target of PI 3-kinase inα _1B receptor-signaling pathways.

Figure 7.

α₁-Receptor Subtype Activation of MAP Kinases via a PI 3-Kinase-Dependent Signaling Pathway Cells expressing α_1A- or α_1B-receptors were grown for 24 h and then the cells were transfected with a dominant negative mutant of PI 3-kinase p85 (Δp85) as described in Materials and Methods. Forty eight hours after transfection, cells were incubated in serum-free DMEM for 18 h. Cells were treated with vehicle or norepinephrine (10 μm) for 10 min. Cell lysates were prepared and immunoprecipitated with anti-p44^ERK1 antibody. They were subjected to in vitro assay of MAP kinase activity using MBP as substrate. The data are mean ± sem of three assays of MAP kinase activity. *, P < 0.01.

DISCUSSION

Stimulation of transfected α_1A- orα _1B-adrenergic receptors in NIH-3T3 cells led to activation of PI 3-kinase. Cells expressing α_1D receptors did not stimulate PI3-kinase activity above basal values in response to norepinephrine in these experiments. In parallel experiments we found that transfection of the α_1D-receptors led to norepinephrine-activation of c-fos in NIH-3T3 cells demonstrating that the α_1D-receptors were being functionally expressed in the cells. The major result of these studies is that α_1A- and α_1B-adrenergic receptors activated downstream effectors, particularly p21^ras and PI 3-kinase, by different signaling mechanisms.

Increased abundance of c-fos mRNA could be readily measured after stimulation with norepinephrine in the cells expressing inα _1D-receptors, but we did not detect changes in PI 3-kinase activity in those cells. While these results clearly demonstrate functional expression of the α_1D-receptors, they do not exclude the possibility that these receptors might activate this kinase in different cells or under other conditions. Using[ ³H]prazosin, the level of expression of each of the three α₁-receptor subtypes was below the sensitivity of detection with this ligand (data not shown). Consequently, we can exclude the possibility of promiscuous coupling due to overexpression of these receptors. It has been shown previously that a low level of expression of m1 muscarinic receptors (very difficult to measure directly with a radioligand) is sufficient to activate MAP kinase cascades (40).

It is generally accepted that PTx-insensitive G proteins, particularly members of the Gq family, mediate increases in hydrolysis of PI 4,5-bisphosphate to inositol triphosphate and diacylglycerol by the three subtypes of α₁-receptors (11, 41). Recent evidence suggests that signal transduction mechanisms ofα ₁-receptors are much more complex than previously realized. For example, several studies have suggested that PTx-sensitive G proteins may also be involved inα ₁-receptor signaling pathways in several types of cells. Llahi and Fain (42) found that α₁-receptors activate phospholipase D in the brain and promote the release of arachidonic acid via activation of phospholipase A2 via PTx-sensitive G protein (13). Stimulation of α₁-receptors causes phosphorylation of c-Jun kinase mediated by a PTx-sensitive G protein (14). We recently demonstrated thatα ₁-receptor-stimulated mitogenic effects are associated with activation of PI 3-kinase in human VSMCs and that activation of PI 3-kinase is mediated by a PTx-sensitive G protein in those cells (36). In the present study, the finding that activation of PI 3-kinase by theα _1A- and α_1B-receptor subtypes is PTx insensitive in NIH3T3 cells emphasizes the importance of host cell factors in modulating receptor function. Our previous study was performed in human VSMCs that express mainly α_1B andα _1D mRNA with very low α_1A receptor subtype mRNA. A recent study in COS-7 cells demonstrated thatα _1B-receptor stimulation of MAP kinase involved a PTx-insensitive Gqα using a p21^ras-independent mechanism, as it was found that overexpression of the P21^N17ras dominant negative mutant did not inhibit α_1B-receptor stimulation of MAP kinase (43).

Direct interactions between Gβγ-subunits and protein kinases have been implicated in various cells. For example, Gβγ-subunits stimulate PI 3-kinase in platelets and neutrophils (44, 45). Moreover, transfection studies with COS-7 cells revealed that Gβγ-subunits activate MAP kinase after stimulation of Gs, Gi, and Gq-coupled receptors (46); these effects of the βγ-subunits are due to activation of p21^ras (46). Gβγ-subunits mediate tyrosine kinase receptor, IGF-I receptor activation of MAP kinase (47). van Biesen et al. (48) reported thatα ₂-adrenergic receptors activate MAP kinase via stimulation of G protein βγ-subunits, leading to interactions of adapter proteins Grb2/SOS1/Shc, indicating that an intact signaling pathway machinery used by classical receptor tyrosine kinases is shared by G protein-coupled receptors. Based on these findings, Gβγ-subunits, in G protein-coupled receptor signaling, have been suggested to be analogous to phosphorylated receptor tyrosine kinases (49).

In the present study, we obtained evidence that expression ofα -transducin attenuated the norepinephrine-induced increase of PI-3 kinase activity in cells expressing α_1B-receptors but not in cells expressing α_1A-receptors, suggesting thatβγ -subunits mediate α_1B-receptor activation of PI-3 kinase. However, we found that overexpression of GRK2,β -adrenergic receptor kinase peptide, used successfully to sequester βγ-subunits in other studies (47), did not inhibitα _1B receptor-stimulated PI-3 kinase activity in our experiments (Fig. 3, A and B). This result raises the possibility that the βγ-subunits involved in α_1B receptor-stimulated PI-3 kinase in NIH 3T3 cells have differing affinities for α_tvs. GRK2. Since there are multiple forms ofβγ -subunits, this possibility is tantalizing but requires further experimental testing.

A number of studies have suggested that there is a complex interaction between activation of PI 3-kinase and activation of the P21^ras/MAP kinase cascade (17). For instance, several studies have demonstrated that activation of PI 3-kinase by growth factors or by G protein-coupled receptor agonists is associated with activation of p21^ras. In PC12 cells, Downward et al. (50) demonstrated that PDGF-stimulated p21^ras targets PI3-kinase. On the other hand, activation of PI 3-kinase may also function upstream of p21^ras/MAP kinase cascade. A recent study suggests that the sequential activation of PI 3-kinase, Ras protein, and MAP kinase is involved in the insulin-signaling pathways during differentiation of adipocytes by hormones and phosphodiesterase inhibitors; inhibition of PI 3-kinase by wortmannin inactivated the Ras/MAP kinase pathway, leading to suppression of adipocyte differentiation (51). Other studies have demonstrated that PI 3-kinase activity is an important intermediate step in G protein-coupled receptor agonist activation of the p21^ras-signaling cascade (22, 36). Hawes et al. (22) reported that stimulation of α_2A-ARs or lysophosphatidic acid receptors activates p21^ras and MAP kinase cascade via Gβγ-subunits. Treatment of cells with specific inhibitors of PI 3-kinase, wortmannin or LY294002, or overexpression of a dominant negative mutant of the p85 subunit of PI 3-kinase, attenuated activation of the Ras/MAP kinase cascade. Moreover, a Gβγ-specific PI 3-kinase γ has recently been identified to mediate G protein-coupled receptor activation of MAP kinase cascades (52). Given the fact that α_1B-receptor mediates MAP kinase activation via Gβγ-subunits, it will be important to determine which subtype of PI 3-kinases (p85/p110 or p110γ ) plays a signaling role in α_1B-receptor signal transduction.

We recently found that α₁-receptor stimulation of PI 3-kinase activity is associated with activation of Ras protein, and Ras may function as a target of PI 3-kinase in smooth muscle cells (36). Our present study provides evidence demonstrating that activation of PI 3-kinase is greatly involved in α₁-receptor subtype-signaling pathways. Overexpression of the dominant negative mutant of p21^A15Ras markedly inhibited the capacity ofα _1A- and α_1B-receptor subtypes to activate p21^ras (Fig. 4). This construct attenuatedα _1B-receptor-stimulated, but did not attenuateα _1A receptor-stimulated, PI 3-kinase activity, suggesting that p21^Ras may act either as downstream component ofα _1A receptor-stimulated PI 3-kinase or upstream component of α_1B receptor-stimulated PI 3-kinase. However, overexpression of a dominant negative mutant of the PI 3-kinase p85 subunit, namely Δp85, blocked α_1A- but notα _1B-receptor subtype-stimulated MAP kinase activity. This result demonstrates that PI 3-kinase is critical for activation of MAP kinase by α_1A-receptors. However, the results suggest that α_1B-receptor subtype activates PI 3-kinase and MAP kinase in parallel. There is other evidence indicating thatα ₁- receptors may utilize other intracellular signal pathways such as protein kinase C/Raf-1 to activate MAP kinase independently of p21^Ras (43). These complex interactions among the α₁-receptors, subunits of G proteins, and downstream protein kinase cascades are presented schematically in Fig. 8.

Figure 8.

Proposed Scheme of G Protein, PI 3-Kinase, and MAP Kinase Signaling Pathways Transduced by α_1A- andα _1B-Adrenergic Receptors The data suggest that α_1A- andα _1B-receptors, coupled to PTx-insensitive G protein(s), have signaling mechanisms involving triggering by Gα- or Gβγ-subunits. The activated α- or βγ-subunits subsequently trigger PI 3-kinase, p21^Ras, and MAP kinase or p21^Ras and PI 3-kinase, respectively. The position of Ras, either upstream or downstream of PI 3-kinase, is different for these two α₁-receptor subtypes.

In summary, the results indicate that α_1A- andα _1B-receptors activate PI 3-kinase, likely via different subunits of PTx-insensitive G proteins. Moreover, theα _1A-receptor subtype may use Gqα and α_1B receptor subtype may use βγ-subunits of Gq to stimulate PI 3-kinase activity in NIH3T3 cells. We also provide evidence demonstrating that increases in PI 3-kinase activity by stimulation ofα _1A-receptor subtype turn on p21^ras activation, which in turn activates the MAP kinase cascade. However,α _1B-receptor subtype activation of MAP kinase cascade is likely independent of activation of PI 3-kinase. These experiments demonstrate the heterogeneity of the signaling pathways activated by the various receptor subtypes. Further experiments aimed at understanding structure-function relationships forα ₁-receptor-coupling mechanisms should provide new insights into the functions of the physiologically very important receptor systems.

MATERIALS AND METHODS

Materials

Norepinephrine and myelin basic protein (MBP) were purchased from Sigma Chemical Co. (St Louis, MA); [γ³²P]ATP (2000 Ci/mmol), [α³²P]dCTP, and enhanced chemiluminescence (ECL) Western detection system were purchased from Amersham Corp.(Arlington, IL); expression vectors of human α_1A-,α _1B-, and α_1D-adrenergic receptor and control vectors were a gift of Dr. Johnston and colleagues of Central Research (Pfizer Inc., Sandwich, England); expression vectors ofα -subunit of transducin (αt-pcDNA-I) and control vector (pcDNA-I) were kindly provided by Dr. Henry Bourne of University of California at San Francisco; expression vectors of a minigene (cDNA 495–689) of GRK2 (β-ARK1) was a gift from Dr. R. J. Lefkowitz’s laboratory of Duke University (Durham, NC); the p21^Ala15ras dominant negative mutant and control vectors were kindly provided by Joan H. Brown of University of California at San Diego; phosphatidylinositol was obtained from Avanti Polar Lipids Inc. (Alabaster, AL); human recombinant IGF-I, cell culture medium, and FBS were purchased from GIBCO/BRL (Grand Island, NY); wortmannin was from Worthington Biochemical Co. (Freehold, NJ); and antibodies against PI-3 kinase p85-α, p110, tyrosine protein kinases, H-ras, p44^ERK1/2, GRK2, and α_t, as well as protein A/G-agarose, were from Santa Cruz Biotechnology (Santa Cruz, CA). All other chemicals were reagent or molecular biology grade and were obtained from standard commercial sources.

Cell Culture

NIH3T3 mouse fibroblasts were purchased from the American Type Culture Collection (Manassas, VA). Cells were grown in DMEM with 5% FBS at 37 C in a humidified atmosphere of 5% CO₂-95% air. The cells were harvested for passaging at confluence with trypsin-EDTA and plated in 100-mm dishes at a density about 5 × 10⁵, with a 80–90% confluence being reached about 5 days later. The medium was replaced every 2 days. To examine effects of agonist-stimulated changes, cells were incubated with DMEM without serum for indicated times after achieving confluence. The cells were treated with agonists or vehicle solution (as control) starting from the longest time point, and the cells were harvested at the same time.

Stable Transfection of NIH3T3 Cells withα ₁-Adrenergic Receptors

NIH3T3 cells (3 × 10⁵) were seeded in 100-mm² culture dishes with DMEM with 10% FBS. The following day, each adrenergic receptor subtype expression vector (α_1A and α_1B, 10 μg; α_1D, 50 μg) was transfected using 50 μg of lipofectamine (GIBCO-BRL) in serum-free medium after the instruction of manufacture. Five hours later, an equal volume of DMEM with 20% FBS was added. Twenty four hours after the start of transfection, the cells were washed and placed in fresh medium; 48 h later, the transfected cells were reseeded into ten 100-mm² dishes, and culture medium was changed to DMEM/10% FBS with 500 μg/ml of Geneticin (GIBCO-BRL). Ten to 14 days later, the surviving cell colony was isolated and grown in medium containing Geneticin. Cells expressing a similar number of receptors were used for the further experiments. For various assays, the cells were made quiescent by serum starving for 24 h before treatment and harvest.

Transient Transfection of NIH3T3 Cells with Expression Vectors Containing α-Transducin, β₁-Adrenergic Receptor Kinase, p21^Ala15ras, or p85α^PI3K

Transfection of NIH3T3 cells with control or expression vectors containing α-transducin, β₁-adrenergic receptor kinase (now termed GRK2), the p21^Ala15ras dominant negative mutant, or a dominant negative mutant of PI 3-kinase p85α (ΔP85) was performed as described above. NIH3T3 cells that stably expressed each of the three subtypes of α₁-adrenergic receptors were cultured in DMEM with 10% FBS in the presence of 500 μg/ml of geneticin as described above. The cells were seeded into 100-mm² dishes and transfected at ∼80% confluence. Transfection was performed in 3.0 ml of Optim-MEM (GIBCO-BRL) containing 50 μg of lipofectamine and 10 μg of control vectors or expression vectors containing α-transducin,β ₁-adrenergic receptor kinase, p21^Ala15ras, or ΔP85. Five hours later, 3 ml of DMEM with 20% FBS were added. Twenty four hours from the start of transfection, the cells were washed, and fresh DMEM with 10% FBS was replaced. Next day, the cells were made quiescent by serum starving for 18 h before treatment and harvest. α₁-Adrenergic receptor subtype-stimulated activation of PI 3-kinase, Ras-GTP, and MAP kinase was then determined as described below.

RNA Preparation and Northern Blotting Analysis

A single-step method of RNA isolation using acid guanidinium thiocyanate-phenol-chloroform extraction as described previously (53) was used to isolate total RNA from NIH3T3 cells. Briefly, cells were rinsed with cold calcium-magnesium-free PBS, and then the cells were homogenized with a Polytron in 10 vol of denaturing buffer containing 4 m guanidinium thiocyanate, 25 mm sodium citrate (pH 7), 0.5% sarcosyl, 0.1 m 2-mercaptoethanol. One volume of 2 m sodium acetate (pH 4.0), 10 vol of water-saturated phenol, and 2 vol of chloroform-isoamyl alcohol (49:1) were sequentially added to the homogenate with thorough mixing after addition of each reagent. The homogenate was incubated on ice for 20 min and then centrifuged at 12,000 × g for 20 min. The aqueous phase was taken and RNA was precipitated with isopropanol (1:1 volume). The resulting RNA pellet was dissolved in the denaturing buffer and again precipitated with isopropanol by cooling and centrifugation. The RNA pellet was washed with 75% ethanol, sedimented, vacuum dried, and dissolved in Tris-EDTA buffer. For Northern blotting analysis of c-fos, 10 μg of total RNA were heated at 65 C for 10 min, cooled rapidly on ice, and denatured with 6% formaldehyde. The RNA was fractionated by 1% agarose gel electrophoresis and transferred to a nylon filter by capillary blotting. The blot was prehybridized in 50% formamide, 5 × SSPE buffer (1 × SSPE = 0.18 m NaCl, 10 mm sodium phosphate, pH 7.7, and 1 mm EDTA), 5 × Denhardt’s solution, 0.5% SDS, at 42 C for 4 h, and hybridized at 42 C for 12–16 h to the rat c-fos orβ -actin cDNA probes that were labeled by [³²P]dCTP using Amersham’s random priming labeling kit. After hybridization the filter was washed twice in 2 × SSPE, 0.1% SDS at 65 C for 15 min and once in 0.1 × SSPE, 0.1% SDS at 56 C for 30 min. The filter was exposed to Kodak XAR-5 film at −70 C with an intensifying screen for 16–24 h. The autoradiograms were scanned using a laser densitometer. The amount of c-fos mRNA was quantified relative to the amount of 18s tRNA on the same filter.

Immunoprecipitation

After treatment, cultures on 100-mm² plates were rinsed with ice-cold PBS containing 1 mm sodium orthovanadate. Cells were incubated with lysis buffer (1% Nonidet P-40, 25 mm HEPES (pH 7.5), 50 mm NaCl, 50 mm NaF, 5 mm EDTA, 10 nm okadaic acid, 1 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, and 10 μg/ml of antipain, aprotinin, and leupeptin) for 10–15 min on the ice. Insoluble material was removed by centrifugation at 12,100 × g for 20 min. The amount of cell lysate was normalized by protein content in each experiment using a kit from Bio-Rad (Richmond, CA). The lysate was incubated with an appropriate amount of antibody and agitated for 2 h. The lysate was further incubated with 20–30 μl of protein A/G plus-agarose with agitation for 1 h. The beads containing the immunoprecipitates were washed three times with lysis buffer, once with distilled water, and once with washing buffer (0.1 m NaCl, 1 mm EDTA, 20 mm Tris-HCl, pH 7.5), and subjected to PI 3-kinase and MAP kinase assays or analysis of Ras-bound GTP.

Analysis of Ras-Bound GTP and GDP

Agonist-stimulated change in Ras-bound GTP was performed following a method described previously (36). Quiescent cells were labeled with 0.1 mCi/ml of [³²P]orthophosphate in phosphate-free DMEM for 12 h. After stimulation with agonists for the indicated times, cells were washed with cold PBS for three times and cells were lysed as described above. Ras proteins were recovered by immunoprecipitation with an anti-Ras polyclonal antibody. After extensively washing as described above, the immunoprecipitates were suspended in 20 μl of reaction mixture containing 20 mm HEPES, pH 7.5, 20 mm EDTA, 2% SDS, 0.5 mm GDP, and 0.5 mm GTP. The suspension was heated at 90 C for 3 min and centrifuged for 5 min. The bound nucleotides were separated by TLC on polyethyleneimine-cellulose plate with 0.75 m KH₂PO₄ for development. GDP and GTP were visualized using unlabeled standards. Ras-associated GTP was calculated from the ratio of GTP/(GDP+GTP). The radioactivity was quantitated with a PhosphorImager system (Molecular Dynamics, Sunnyvale, CA).

In Vitro Assays of PI 3-Kinase

For measurement of PI 3-kinase activity, cell lysates (1 mg protein) were incubated with antibody against the p85-α subunit of PI 3-kinase (2 μg/mg protein) as described above. Assay of PI 3-kinase activity was conducted as described previously (36). Briefly, the washed pellets were resuspended in 50 μl of kinase reaction buffer (20 mm Tris-HCl, pH 7.5, 100 mm NaCl, and 0.5 mm EGTA) and incubated at 25 C for 10 min after addition of 0.5 μl of 20 mg/ml phosphatidylinositol dissolved in chloroform to make micelles of PI. Assays were initiated with addition of 5 μl of ATP solution (0.4 m ATP, 0.1 m MgCl₂, and 1 μCi/ml [γ³²P]ATP) and incubated at room temperature for 30 min. During this period of time the formation of phosphatidylinositol phosphate was linear (data not shown). The reaction was stopped upon addition of 100 μl of chloroform-methanol-11.6 n HCl (100:200:2). After centrifugation, the lower organic phase was taken for TLC on silica gel plates from J. T. Baker Inc. (Phillipsburg, NJ) and developed in chloroform-methanol-25% ammonium hydroxide-water (43:38:5:7). The plates were exposed to Kodak XAR-5 film at −70 C with an intensifying screen for 16–24 h or were visualized after development with a PhosphorImager system.

In Vitro Assay of MAP Kinase Activity

Assay of MAP kinase activity was performed following a method described previously (36). Confluent cells were incubated in the absence of serum overnight and treated with norepinephrine or other agonists for various times as indicated. The cells were lysed in 0.4 ml lysis buffer as described above. For MAP kinase activity assay, cell lysates (400 μg protein) were incubated with antibody against ERK1 (2μ g/mg protein) and washed as above. The washed immunocomplexes were resuspended in 50 μl of kinase buffer (25 mm HEPES, pH 7.5, 10 mm MgCl₂, 1 mm dithiothreitol, 0.5 mm EGTA, 40 μm ATP, 1μ Ci of [γ³²P]ATP), and MBP (1 mg/ml) as a substrate. The reaction mixture was incubated for 10 min at 30 C because preliminary experiments indicated that MBP-induced phosphorylation is linear for 20–30 min. The reaction was stopped by spotting 10 μl of reaction mixture onto p-81 phosphocellulose paper (Whatman LabSales, Hillsboro, OR), which was then washed in 75 mm phosphoric acid with constant stirring for 1 h and transferred to another washing overnight. The papers were washed with acetone for 5 min and dried. ³²P, which represented the phosphorylation of MBP by MAP kinase, was quantitated by scintillation spectrophotometry. Alternatively, reaction mixtures were loaded and separated on 14% SDS-PAGE, and the dried gels were exposed to Kodak XAR-5 film at −70 C with an intensifying screen for 16–24 h for visualization.

Data Analysis

Data are presented as mean ± sem, and treatment effects were compared by one-way ANOVA or Student’s paired t test (two tailed). P < 0.05 was taken as level of significance.

Acknowledgments

We thank Geoffrey Johnston for providing expression vectors encoding human α_1A-, α_1B-, andα _1D-adrenergic receptors and control vectors; Joan H. Brown for providing the dominant negative mutant of p21^Ras expression and control vectors; Henry R. Bourne for providing the αt and control constructs; and Wataru Ogawa for the dominant negative mutant of PI 3-kinase p85α and control vector.

This work was supported by NIH Grant HL-41315 and by a preclinical grant from Pfizer Inc.

¹

Supported by a Pharmaceutical Research and Manufacturers of America Foundation Fellowship for Careers in Clinical Pharmacology.