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Molecular Endocrinology, doi:10.1210/me.2006-0090
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Molecular Endocrinology 20 (11): 2773-2783
Copyright © 2006 by The Endocrine Society

Insulin-Like Growth Factor-I Induces {alpha}1B-Adrenergic Receptor Phosphorylation through Gß{gamma} and Epidermal Growth Factor Receptor Transactivation

Tzindilú Molina-Muñoz, María Teresa Romero-Ávila and J. Adolfo García-Sáinz

Departamento de Biología Celular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, 04510 México D.F., México

Address all correspondence and requests for reprints to: J. Adolfo García-Sáinz, M.D., Ph.D., Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Apartado Postal 70-248, 04510 México D.F., México. E-mail: agarcia{at}ifc.unam.mx.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
IGF-I induces {alpha}1B-adrenoceptor ({alpha}1B-AR) phosphorylation. The effect of IGF-I was rapid and transient, reaching near-maximal values at 10 min and decreasing after 30 min; it was observed at low IGF-I concentrations (EC50 {approx} 10 ng/ml) and was associated to receptor desensitization as evidenced by a decreased {alpha}1B-adrenergic effect on intracellular calcium and production of inositol phosphates. The effect of IGF-I was markedly decreased in cells treated with pertussis toxin suggesting involvement of pertussis toxin-sensitive G proteins. Transfection of the carboxyl terminus of the ß-adrenergic receptor kinase or the {Delta}p85 mutant of phosphoinositide 3-kinase (PI3K) markedly decreased the {alpha}1B-AR phosphorylation induced by IGF-I without decreasing the receptor phosphorylation induced by noradrenaline. Inhibitors of PI3K and protein kinase C blocked IGF-I-induced {alpha}1B-AR phosphorylation. In addition, it was observed that AG1478, an inhibitor of the epidermal growth factor (EGF) receptor kinase, and BB-94, a metalloproteinase inhibitor, also diminished IGF-I-induced adrenoceptor phosphorylation.

The data clearly show that IGF-I triggers a complex signaling pathway, which leads to the phosphorylation and desensitization of a serpentine G protein-coupled receptor, suggesting the following hypothetical model: 1) stimulation of IGF-I receptors activate pertussis toxin-sensitive G proteins; 2) the growth factor action activates metalloproteinases, which catalyze heparin binding-EGF shedding, and transactivation of EGF receptors, and 3) dissociated Gß{gamma} subunits and phosphotyrosine residues seem to trigger PI3K activity, which leads to activation of protein kinase C, resulting in {alpha}1B-AR phosphorylation and desensitization.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE {alpha}1-ADRENOCEPTORS ({alpha}1-ARs) MEDIATE many physiological actions of adrenaline and noradrenaline, playing key roles in the functions of the cardiovascular, genitourinary, and central nervous systems (1, 2, 3, 4). They also participate in the pathogenesis of diseases such as hypertension or benign prostatic hypertrophy (1, 2, 3, 4). These receptors, i.e. the {alpha}1A-, {alpha}1B-, and {alpha}1D-AR subtypes, constitute a branch of the adrenergic subfamily of the seven-transmembrane domains G protein-coupled receptors. Knowledge of molecular events that control their function is of major physiological importance and current ideas indicate that phosphorylation, via G protein-coupled receptor kinases (GRKs) and second messenger-dependent kinases such as protein kinase C (PKC), is one of the earliest processes in their regulation (1, 2, 3, 4).

{alpha}1B-ARs have been more extensively studied than the other subtypes. GRK2 and GRK3 appear to be involved in phosphorylation of agonist-occupied {alpha}1B-ARs during homologous desensitization (5, 6), whereas PKC and phosphoinositide 3-kinase (PI3K) seem to play major roles in heterologous desensitization (1, 7).

Activation of nonadrenergic receptors has been observed to induce {alpha}1B-AR desensitization (heterologous desensitizations) and phosphorylation. Thus, activation of seven-transmembrane domains receptors coupled to Gq/11 [such as endothelin ETA (8) or bradykinin BK2 receptors (9)], or to Gi [such as lysophosphatidic acid receptors (10, 11), or receptors with intrinsic tyrosine kinase activity, such as insulin (12), epidermal growth factor (EGF), or platelet-derived growth factor (PDGF) receptors (13)] can also induce {alpha}1B-AR phosphorylation/desensitization. Recent evidence indicates that EGF receptor transactivation also seems to play a key general role in {alpha}1B-AR phosphorylation/desensitization (11, 45).

As previously indicated, activation of receptors with intrinsic protein tyrosine kinase activity, induce {alpha}1B-AR phosphorylation/desensitization (12, 13). In the present work, we studied the effect of IGF-I on {alpha}1B-ARs. The study was inspired by the physiological importance of this factor and the peculiarities of its signaling. IGF-I mediates most of the actions of GH, including its anabolic and mitogenic activities (14, 15). It plays a critical role in normal fetal and postnatal growth and development. Inactivation of the Igf-I gene results in dwarfism at birth, retarded postnatal growth, and infertility in mice (14, 16, 17). Similarly, it has been observed that mutations in the GH receptor gene result in a new type of dwarfism indistinguishable from GH deficiency characterized by high values of serum GH (Laron syndrome) (15). IGF-I is a small 70-amino acid peptide (produced in liver and other organs), which signals through the IGF-I receptor, a transmembrane tyrosine kinase similar to the insulin receptor (17). Although the insulin receptor and that for IGF-I share some actions, they mediate different effects on metabolism and cell proliferation and differentiation. Such differences may in part be attributed to the tissue distribution of those receptors, but structural differences in their ß-subunits might also participate by determining the substrate specificity of their tyrosine kinases (17, 18, 19). Some tyrosine kinase receptors seem to couple to G proteins (20). IGF-I receptors couple to heterotrimeric G proteins, particularly of the Gi family (Refs. 21 and 22 ; reviewed in Ref. 20). It has also been suggested that IGF-I receptors recruit EGF receptors as a signaling pattern (14, 23). Our data clearly show that IGF-I triggers a complex signaling pathway, which leads to the phosphorylation and desensitization of {alpha}1B-ARs in a process that involves pertussis toxin-sensitive G proteins, transactivation of EGF receptors, and activation of PI3K and PKC. In addition, we observed that such pathway might also be shared by other receptors with endogenous tyrosine kinase activity.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
As shown in Fig. 1Go, left panel, IGF-I induced a clear increase in {alpha}1B-AR phosphorylation in a concentration-dependent fashion; the maximal increase was of {approx}3-fold with an EC50 value of 13 ± 2 ng/ml (mean ± SEM; n = 5). The effect was rapid, being clearly observable as early as 2 min after the addition of IGF-I, reached a plateau between 10 and 30 min, and decreased thereafter (Fig. 1Go, right panel).


Figure 1
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  		Fig. 1. Effect of IGF-I on {alpha}1B-AR Phosphorylation in Rat-1 Fibroblasts

Rat-1 fibroblasts were metabolically labeled with [32P]Pi, stimulated for 15 min with the agents indicated below, and lysed; {alpha}1B-ARs were immunoprecipitated and subjected to electrophoresis and autoradiography. Left panel, Concentration response curve. Cells were incubated for 15 min in the presence of the indicated concentration of IGF-I. Right panel, Time course of the effect. Cells were incubated for the times indicated in the presence of 100 ng/ml IGF-I. Plotted are the means, and vertical lines represent the SEM of four to five experiments using different cell preparations. Representative autoradiographs are shown above the graphs (32P) (entire-gel autoradiographs are presented in supplemental data 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org), and Western blot detection of immunoprecipitated {alpha}1B-ARs of samples run in parallel are also shown (WB).

 
The functional consequences of the IGF-I action on {alpha}1B-ARs were next examined. It was observed that IGF-I induced a small (nonsignificant) increase in the production of [3H]inositol phosphates (Fig. 2Go, upper left panel). Noradrenaline markedly increases [3H]inositol phosphate production, and this action was clearly decreased by pretreatment with IGF-I. The effect on intracellular calcium concentration was examined, and we observed that IGF-I did not induce any clear change by itself on the basal level (Fig. 2Go, right upper panel). As expected, noradrenaline induced an immediate sharp increase, which after a few seconds started to decrease toward near-basal levels (Fig. 2Go, lower left panel). When cells were pretreated with IGF-I for 15 min and then challenged with noradrenaline, the action of the adrenergic amine was markedly reduced (Fig. 2Go, right panels). Such action was not due to a general perturbation of the calcium signaling process as evidenced by the ability of lysophosphatidic acid to increase the intracellular calcium concentration in a marked and immediate fashion (Fig. 2Go, lower panel).


Figure 2
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  		Fig. 2. Effect of IGF-I on {alpha}1B-AR-Mediated [3H]Inositol Phosphate Production and Intracellular Calcium Concentration

Upper panels, Cells were labeled with [3H]inositol and challenged, and the production of [3H]inositol phosphates was determined by ion exchange chromatography as described in Materials and Methods. Cells were incubated in the absence of any agent (Basal) or presence of 100 ng/ml IGF-I, 10 µM noradrenaline (NA), or both agents (IGF-I + NA). Plotted are the means, and vertical lines represent the SEM of five experiments using different cells preparations. *, P < 0.001 vs. basal; **, P < 0.001 vs. noradrenaline alone. Lower panels, Cells were loaded with fura 2, and intracellular calcium was determined fluorometrically as described in Materials and Methods; representative tracings of cells preincubated in the absence (left) or presence of 100 ng/ml IGF-I (right) for 15 min (time 0); where indicated, 10 µM noradrenaline (NA) or 1 µM lysophosphatidic acid (LPA) was added.

 
Because the IGF-I receptor mediates some of its actions through activation of G proteins of the Gi family (20, 21, 22), we tested whether the {alpha}1B-AR phosphorylation induced by IGF-I was sensitive to the action of pertussis toxin. In the experiments shown in Fig. 3Go, cells were preincubated with pertussis toxin for 12 h to ensure complete ADP-ribosylation of toxin-sensitive G proteins (8). The effect of IGF-I on {alpha}1B-AR phosphorylation was markedly attenuated. As expected, the effect of noradrenaline on {alpha}1B-AR phosphorylation was not significantly altered by the pertussis toxin pretreatment and that of lysophosphatidic acid was markedly reduced (8, 10), which served as negative and positive controls, respectively (Fig. 3Go, left panel). Similar results were obtained after preincubation for 2 h with the toxin (data not shown). Then, we tested the possibility that the {alpha}1B-AR phosphorylation induced by other receptors with endogenous protein tyrosine kinase activity could also involve pertussis toxin-sensitive G proteins, and in fact this was the case. As is shown in Fig. 3Go, right panel, the {alpha}1B-AR phosphorylations induced by insulin, EGF, and PDGF were strongly diminished in cells pretreated with pertussis toxin. Nevertheless, it should be made clear that in no case was the effect completely abolished.


Figure 3
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  		Fig. 3. Effect of Pertussis Toxin Treatment

Left panel, Rat-1 fibroblast were preincubated for 12 h in the absence (dotted bars) or presence (crisscrossed bars) of 100 ng/ml pertussis toxin and challenged with 10 µM noradrenaline (NA), 100 ng/ml IGF-I, or 1 µM lysophosphatidic acid (LPA). Plotted are the means, and vertical lines represent the SEM of four to five experiments using different cell preparations. A representative autoradiograph is presented. Basal {alpha}1B-AR labeling was not affected by pertussis toxin treatment. *, P < 0.01 vs. basal labeling; **, P < 0.001 vs. IGF-I alone; ***, P < 0.001 vs. lysophosphatidic acid alone. Right panel, Rat-1 fibroblasts were preincubated for 12 h in the absence (dotted bars) or presence (crisscrossed bars) of 100 ng/ml pertussis toxin and challenged with 100 nM insulin, 100 ng/ml EGF, or 50 ng/ml PDGF. Plotted are the means, and vertical lines represent the SEM of four to five experiments using different cell preparations. A representative autoradiograph (32P) and Western blot (WB) detection of immunoprecipitated {alpha}1B-ARs of samples run in parallel are shown. *, P < 0.001 vs. basal labeling; **, P < 0.05 vs. insulin alone; ***, P < 0.01 vs. EGF alone; ****, P < 0.05 vs. PDGF alone.

 
Activation of Gi proteins by the lysophosphatidic acid LPA1 receptor induces {alpha}1B-AR phosphorylation via the shedding of membrane-bound heparin-binding EGF (HB-EGF) by metalloproteinases, which leads to the autocrine activation of EGF receptors, triggering activation of PI3K and PKC (11). The possibility that a similar process could be involved in the action of IGF-I was considered, and we tested the roles of metalloproteinases and protein kinases through the use of inhibitors. In agreement with previous findings, BB-94 (batimastat), a broad spectrum metalloproteinase inhibitor, markedly attenuated the {alpha}1B-AR phosphorylations induced by lysophosphatidic acid (11), noradrenaline (45), and IGF-I, but not that induced by the addition of EGF (Fig. 4Go, upper left panel). These data suggested that EGF receptor activation could participate in the process. AG1478, a selective inhibitor of the EGF receptor tyrosine kinase activity, diminished the {alpha}1B-AR phosphorylations induced by noradrenaline, EGF, and IGF-I, which is consistent with the previous data (Fig. 4Go, upper right panel). It was similarly observed that inhibitors of PI3K, i.e. wortmannin and LY294002, and inhibitors of PKC, i.e. staurosporin and Ro 318220, inhibited the {alpha}1B-AR phosphorylation induced by IGF-I. Inhibitors were without effect at the concentrations tested on basal receptor phosphorylation (data not shown). To show the participation of HB-EGF in these actions, we took advantage of the availability of a neutralizing anti-HB-EGF antibody. This antibody did not alter basal {alpha}1B-AR phosphorylation (11) or that induced by tetradecanoyl phorbol acetate but strongly inhibited the action of lysophosphatidic acid (11) (Fig. 5Go). The neutralizing antibody markedly reduced the {alpha}1B-AR phosphorylation induced by IGF-I, indicating a role of HB-EGF in this action.


Figure 4
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  		Fig. 4. Effect of Metalloproteinase and Protein Kinase Inhibitors

Upper left panel, Rat-1 fibroblasts were incubated for 30 min in the absence (dotted bars) or presence of 10 µM BB-94 (Batimastat) (crisscrossed bars), and then challenged with 10 µM noradrenaline (NA), 1 µM lysophosphatidic acid (LPA), 100 ng/ml EGF, or 100 ng/ml IGF-I. A representative autoradiograph is presented. Basal {alpha}1B-AR labeling was not affected by BB-94. *, P < 0.01 vs. basal labeling; **, P < 0.05 vs. noradrenaline alone; ***, P < 0.01 vs. lysophosphatidic acid alone; ****, P < 0.05 vs. IGF-I alone. Upper right panel, Rat-1 fibroblasts were incubated for 30 min in the absence (dotted bars) or presence of 5 µM AG1478 (crisscrossed bars), and then challenged with 10 µM noradrenaline (NA), 100 ng/ml EGF, or 100 ng/ml IGF-I. A representative autoradiograph (32P) and Western blot (WB) detection of immunoprecipitated {alpha}1B-ARs of samples run in parallel are shown. Basal {alpha}1B-AR labeling was not affected by AG1478. *, P < 0.001 vs. basal labeling; **, P < 0.01 vs. noradrenaline alone; ***, P < 0.001 vs. EGF alone; ****, P < 0.001 vs. IGF-I alone. Lower panels, Rat-1 fibroblasts were incubated for 30 min in the absence of any agent or presence of 100 nM wortmannin (+WT), 1 µM LY 294002 (+LY), 1 µM staurosporin (+ST), or 1 µM Ro 318220 (+Ro), and then challenged with 100 ng/ml IGF-I or vehicle (basal). A representative autoradiograph (32P) and Western blot (WB) detection of immunoprecipitated {alpha}1B-ARs of samples run in parallel are shown. The inhibitors did not alter basal {alpha}1B-AR labeling. *, P < 0.001 vs. basal labeling; **, P < 0.01 vs. IGF-I alone.

 

Figure 5
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  		Fig. 5. Effect of a Neutralizing Anti-HB-EGF Antibody

Rat-1 fibroblasts were incubated in the absence or presence of 5 µg/ml of anti-HB-EGF antibody (Anti-HB) for 15 min, and then challenged with in the indicated agents for another 15 min, and receptor phosphorylation was determined as indicated in Materials and Methods. Agents were as follows: 1 µM lysophosphatidic acid (LPA), 1 µM tetradecanoyl phorbol acetate (TPA), and 100 ng/ml IGF-I. A representative autoradiograph (32P) and Western blot (WB) detection of immunoprecipitated {alpha}1B-ARs of samples run in parallel are shown. *, P < 0.001 vs. basal labeling; **, P < 0.001 vs. lysophosphatidic acid alone; ***, P < 0.01 vs. IGF-I alone.

 
We took a further step by using molecular biological approaches. Cos-1 cells were used, because they allowed cotransfection experiments (11). We observed expression of {alpha}1B-ARs in the range of 1–1.5 fmol/mg of membrane protein in these cells as observed previously (11). These receptors were functional as evidenced by [3H]inositol phosphate production (data not shown) and were phosphorylated in response to noradrenaline and IGF-I (Fig. 6Go, upper left panel).


Figure 6
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  		Fig. 6. Effect of Cotransfection with the Carboxyl Terminus of ß-ARK, Wild-Type p85 PI3K, or the Dominant-Negative Mutant {Delta}p85 PI3K on IGF-I-Mediated {alpha}1B-AR Phosphorylation

Cos-1 cells were cotransfected with the {alpha}!B-AR and one of the following: ß-galactosidase (control cells) (upper left panel), the carboxyl terminus of ß-ARK (upper right panel), wild-type p85{alpha} (lower left panel), or {Delta}p85{alpha} (lower right panel). Incubations were for 5 min for 10 µM noradrenaline (NA) and 15 min for 100 ng/ml IGF-I. Plotted are the means ± SEM of five to six determinations using different cell preparations. A representative autoradiograph is shown for each condition. Western blot (WB) detection of transfected proteins is presented in supplemental data 2 (published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). *, P < 0.001 vs. basal.

 
The carboxyl terminus of GRK2 [ß-adrenergic receptor kinase (ß-ARK)] is a powerful tool to study the role of G protein subunits in vivo, because it is capable of binding and sequestering the ß{gamma} subunits of G proteins (24). In cells cotransfected with the carboxyl terminus of ß-ARK and {alpha}1B-ARs, the adrenoceptor phosphorylation induced by noradrenaline was not altered, whereas that induced by IGF-I was markedly diminished (Fig. 6Go, upper right panel). To further confirm the role of PI3K, we took advantage of the dominant-negative mutant of the regulatory subunit ({Delta}p85). Cotransfection of the adrenoceptor with this PI3K mutant did not alter the {alpha}1B-AR phosphorylation induced by noradrenaline but markedly reduced the effect of IGF-I on this parameter (Fig. 6Go, lower right panel). Wild-type p85 PI3K altered neither the effect of noradrenaline nor that of IGF-I (Fig. 6Go, lower left panel). Expression of {alpha}1B-ARs, the carboxyl terminus of ß-ARK, {Delta}p85 and p85 was confirmed by Western blot analysis (see supplemental data 2, published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org).

The effect of IGF-I on {alpha}1B-AR phosphorylation and desensitization was also studied in DDT1 MF-2 cells, which endogenously express both IGF-I receptors and {alpha}1B-ARs. As shown in Fig. 7Go, in DDT1 MF-2 cells, noradrenaline induced a marked increase in intracellular calcium; IGF-I did not alter this parameter by itself but markedly reduced the effect of noradrenaline. In addition, we observed that IGF-I also induced {alpha}1B-AR phosphorylation in these cells and that such effect is blocked by pertussis toxin treatment, AG1478, BB-94, and the anti-HB-EGF neutralizing antibody (Fig. 8Go). Neither the antibody nor the inhibitors alter basal {alpha}1B-AR phosphorylation (data not shown).


Figure 7
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  		Fig. 7. Effect of IGF-I on {alpha}1B-AR-Mediated Increase in Intracellular Calcium Concentration

DDT1 MF-2 cells were loaded with fura 2, and intracellular calcium was determined fluorometrically as described in Materials and Methods; representative tracings of cells preincubated in the absence (left) or presence of 100 ng/ml IGF-I (right) for 15 min (time 0); where indicated, 10 µM noradrenaline (NA) was added. Representative tracings are presented in the upper panels, and the mean ± SEM of four to five determinations using different cell preparations are plotted in the lower panel. *, P < 0.001 vs. basal; **, P < 0.001 vs. noradrenaline alone.

 

Figure 8
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  		Fig. 8. Effect of IGF-I on {alpha}1B-AR Phosphorylation in DDT1 MF-2 Cells

Cells were metabolically labeled with [32P]Pi, stimulated for 15 min with the agents indicated below, and lysed; {alpha}1B-ARs were immunoprecipitated and subjected to electrophoresis and autoradiography. Cells were treated with pertussis toxin (+PTX) at 100 ng/ml 12 h before the experiment was performed; the inhibitors, 5 µM AG1478 (+AG), 10 µM BB-94 (+BB-94), and 5 µg/ml neutralizing anti-HB-EGF antibody (+Anti-HB), were added 15 min before the cells were challenged with 100 ng/ml IGF-I. A representative autoradiograph (32P) and Western blot (WB) detection of immunoprecipitated {alpha}1B-ARs of samples run in parallel are shown. *, P < 0.001 vs. basal; **, P < 0.001 vs. IGF-I alone; ***, P < 0.01 vs. IGF-I alone.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In the present paper, we documented the ability of IGF-I to induce {alpha}1B-AR phosphorylation and desensitization, rapidly and at relatively low concentrations. This action of IGF-I showed similarities and differences with that of insulin (12, 13). Insulin induced a hardly detected functional desensitization of {alpha}1B-ARs in Rat-1 fibroblast, although clear functional desensitization was evidenced in DDT1-FM2 cells (12). In contrast, as shown here, IGF-I clearly desensitized {alpha}1B-ARs in both cells lines. The data comparing these different cell lines indicated that the cellular context plays a role in defining the functional consequences of the hormone action. Many factors may account for this difference, among them the following: the relative density of the receptors for these hormones, the specificity of their substrates and their availability, and the relative coupling to the G proteins present in the cells. The data further show similarities and differences in action between insulin and IGF-I.

It is generally assumed that seven-transmembrane domains receptors are the G protein-coupled receptors. Obviously, this is an oversimplification. It is known that some receptors with a single transmembrane domain, including those with tyrosine kinase activity, can also interact with G proteins, which in turn mediate some of their actions (reviewed in Ref. 20). In this context, these receptors should be considered also as G protein coupled (20). Despite all of this information, it was surprising that pertussis toxin inhibited IGF-I-induced {alpha}1B-AR phosphorylation to such an extent. This led us to explore and discover that the actions of insulin, EGF, and PDGF on this parameter, were also sensitive to pertussis toxin. It is important to emphasize that pertussis toxin did not block completely the effects of these growth factors, which indicates that G proteins are not the sole mediators of the effects and that, likely, the tyrosine kinase activity of these receptors and the formation of phosphotyrosine residues on their target proteins might also play a role (see below). This is similar to what we have observed with lysophosphatidic acid-induced {alpha}1B-AR phosphorylation, which involves the conjunction of two signaling pathways (11).

Our present results allow us to conclude that IGF-I-induced {alpha}1B-AR phosphorylation also involves transactivation of EGF receptors by an autocrine loop, involving metalloproteinase activation and shedding of HB-EGF. There is already some evidence indicating that cross talk of IGF-I receptors and EGF receptors, through autocrine loops, could be relevant for some actions (14, 23). Thus, IGF-I induced activation of MAPKs can be blocked by inhibitors of the EGF receptor tyrosine kinase, in Cos-7 cells (23). It has also been shown that this effect is abolished by [Glu52]diphtheria toxin (an inhibitor of HB-EGF) and by 1,10-phenanthroline (a metal chelator and broad inhibitor of metalloproteinases) (23). Ullrich and co-workers (25, 26, 27) made the landmark observation that activation of seven-transmembrane domains G protein-coupled receptors induces the transactivation of EGF receptors, through the shedding of HB-EGF by metalloproteinases, and that such effect is involved in some of their actions. Our present findings and those of Roudabush et al. (23) indicate that such concept likely can be extended to receptor tyrosine kinases and other receptors that couple to G proteins. It is very probable that such process might have physiological (cell proliferation and differentiation) and pathological (cancer) implications, particularly in the case of IGF-I whose landmark action is a potent inhibition of apoptosis (14).

The ability of pertussis toxin to block the here-described effects indicated that G proteins, likely of the Gi subfamily, were involved in the action of IGF-I, but gave no clue to whether {alpha}-subunits, the Gß{gamma} complexes, or both mediate the effect. The experiments in which the carboxyl terminus of ß-ARK was expressed strongly suggest that Gß{gamma} complexes mediate to a large extent this effect. Our data also indicate the participation of two kinases, PI3K and PKC.

PI3K is a family of enzymes comprising three major classes (I–III) that have been implicated in very diverse cellular functions including cell cycle progression, cell growth, cell adhesion, survival and intracellular trafficking of proteins, among many others (28, 29). It was initially considered that class IA PI3Ks mediated actions of receptors with intrinsic tyrosine kinase activity, whereas class IB (PI3K{gamma}) mediated actions of G protein-coupled receptors via Gß{gamma} subunits (30, 31, 32). However, it was soon observed that PI3K{gamma} has a very discrete pattern of expression and that therefore it is not present in the vast majority of cells (30, 31, 32) and that G protein-coupled receptors can induce tyrosine phosphorylation-independent activation of class IA PI3Ks by G protein ß{gamma} dimers (33). Cos-1 cells express PI3K{alpha} and PI3Kß (30). Our data using either pertussis toxin treatment or transfection of ß-ARK strongly suggest that Gß{gamma} activate PI3K {alpha}/ß isoforms. PI3K {Delta}p85 is a dominant-negative mutant (34) that occupies phosphotyrosine residues but lacks the p110-binding site, i.e. it blocks the phosphotyrosine binding sites normally occupied by endogenous PI3K. The data in cells expressing {Delta}p85 suggested that the phosphotyrosine-p85 association was important, indicating that both phosphotyrosine residues and Gß{gamma} play roles. This is consistent with the observation that PI3K isoforms consisting of p85 and p110ß are synergically activated by the Gß{gamma} subunits and phosphotyrosine-containing peptides (33).

Inhibitors of PKC and PI3K essentially blocked the effect of IGF-I, suggesting that these kinases acted in a sequence and not through independent pathways. PI3K is a modulator of PKC activity through synthesis of 3-phosphorylated phosphoinositides. These lipids modulate phosphoinositide-dependent protein kinase 1 (PDK-1), which phosphorylates the activation loop of some PKC isoforms; in addition, 3-phosphorylated phosphoinositides have also been reported to activate novel and atypical PKC isoforms (35, 36). Therefore, it is likely that PI3K may activate PKC through PDK-1, the generation of 3-phosphoinositides, or both processes.

PKC is a large family of enzymes with differences in sensitivity to activators and substrate specificity (37), and there is a large body of experimental evidence indicating that PKC phosphorylates {alpha}1B-ARs. However, there is no direct evidence concerning isoform(s) that participate in {alpha}1B-AR phosphorylation. Nevertheless, we have observed that, in Rat-1 cells, {alpha}, {delta}, and {epsilon} isoforms of PKC coimmunoprecipitate with {alpha}1B-ARs under basal conditions and that such an association is dynamically increased by cell treatment with phorbol esters or hormones that increase receptor phosphorylation (38). These data indicate that several PKC isoforms participate in this process and suggest the possibilities that they may play roles at different steps or that functional redundancy may exist.

In summary, our data indicate that stimulation of IGF-I receptors activates pertussis toxin-sensitive G proteins. IGF-I action induces an autocrine loop with activation of metalloproteinases, HB-EGF shedding, and transactivation of EGF receptors. Gß{gamma} dimers in synergism with the synthesis of phosphotyrosine residues seem to trigger PI3K activity, which leads to activation of PKC and this to {alpha}1B-AR phosphorylation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
(–)-Noradrenaline, IGF-I, lysophosphatidic acid, staurosporin, wortmannin, and protease inhibitors were obtained from Sigma-Aldrich (St. Louis, MO). LY294002 and Ro 318220 were from Calbiochem (La Jolla, CA). Pertussis toxin was purified from vaccine concentrates as described (39). DMEM, fetal bovine serum, trypsin, antibiotics, and other reagents used for cell culture were from Invitrogen Life Technologies (San Diego, CA). [32P]Pi (8500–9120 Ci/mmol), myo-[2,3-3H]inositol (22.9 Ci/mmol) and [3H]prazosin (74.4 Ci/mmol) were from PerkinElmer Life Sciences (Wellesley, MA). Sepharose-coupled protein A was from Upstate Biotechnology (Lake Placid, NY), whereas BB-94 (Batimastat) was generously provided to us by Dr. S. Mobashery (40) (Wayne State University, Detroit, MI). DNA purification kits were from Qiagen (Hilden, Germany). Anti-GRK2 antibodies and anti-p85 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Chemiluminescence kits were from Pierce (Rockford, IL). Anti-heparin binding-EGF neutralizing antibodies were from R&D Systems (Minneapolis, MN).

Cell Lines and Expression Plasmids
Rat-1 fibroblasts transfected with hamster {alpha}1B-AR, generously provided to us by Drs. R. J. Lefkowitz, M. G. Caron, and L. Allen (Duke University, Durham, NC), were cultured in glutamine-containing high-glucose DMEM supplemented with 10% fetal bovine serum, 300 µg/ml neomycin analog, G-418 sulfate, 100 µg/ml streptomycin, 100 U/ml penicillin, and 0.25 µg/ml amphotericin B at 37 C under a 95% air/5% CO2 atmosphere as described previously (8, 11, 12). Receptor density in this cell line was in the range of 1–1.5 pmol/mg membrane protein. Cos-1 and DDT1 MF-2 cells were obtained from American Type Culture Collection (Manassas, VA). These cells were cultured as described above but in the absence of G-418 sulfate. Transient transfection into Cos-1 cells was performed using diethylaminoethyl-dextran (41) as described (11). Cells at 90% confluence were transfected with a total of 6 µg plasmid for each 10-cm petri dish, usually using 3 µg plasmid containing the {alpha}1B-AR cDNA and 3 µg plasmid containing cDNAs coding the carboxyl terminus of ß-ARK, wild-type PI3K, a dominant-negative PI3K mutant, or ß-galactosidase (pCH110). Experiments with transient transfected cells were performed 48 h after transfection. Transfection efficiency (~60%) was determined in parallel dishes transfected with pCH110 and evaluated by ß-galactosidase activity. Expression of proteins of interest was checked by Western blot analysis or [3H]prazosin binding. Hamster {alpha}1B-AR cDNA (42), kindly provided to us by Dr. R. J. Lefkowitz, was subcloned into pcDNA3 as described (11). Plasmids SR{alpha}-p85 containing the cDNA of wild-type of p85 and SR{alpha}-{Delta}p85 containing cDNA of the dominant-negative mutant p85{alpha} subunit of PI3K, lacking the p110 binding site, were kindly provided by Drs. W. Ogawa and M. Kasuga (Kobe University School of Medicine, Kobe, Japan) (34). Plasmid pRK5 expressing the carboxyl terminus (ß{gamma}-binding domain) of ß-ARK was kindly donated by Dr. W. Koch (Duke University, Durham, NC) (24).

Phosphorylation of {alpha}1B-ARs
The antibody used and procedures employed to study {alpha}1B-AR phosphorylation in Rat-1 fibroblasts, DDT1 MF-2, and Cos-1 cells have been described previously in detail (8, 11, 12). In brief, the day after transfection, cells were collected by trypsinization and transferred to six-well dishes. Cells were maintained overnight in phosphate-free DMEM without serum. The following day, cells were maintained in phosphate-free DMEM for 1 h and then incubated in 1 ml of the same medium containing [32P]orthophosphate (50 µCi/ml, Rat-1 cells; 75 µCi/ml, Cos-1 cells; and 150 µCi/ml, DDT1 MF-2 cells) for 3–5 h at 37 C. Labeled cells were stimulated as indicated, washed with ice-cold PBS, and solubilized with 0.5 ml ice-cold solubilization buffer containing 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% SDS, 10 mM NaF, 1 mM Na3VO4, 10 mM ß-glycerophosphate, 10 mM sodium pyrophosphate, 1 mM p-serine, 1 mM p-threonine, 1 mM p-tyrosine, and protease inhibitors (11, 12). Cell lysates were centrifuged at 12,700 x g for 15 min at 4 C, and supernatants were incubated with anti-{alpha}1B-AR antiserum and protein A-Sepharose overnight at 4 C. After four washes with 50 mM HEPES, 50 mM NaH2PO4, 100 mM NaCl (pH 7.2), 1% Triton X-100, 0.1% SDS, and 100 mM NaF, the immune complexes were boiled for 5 min in SDS-sample buffer containing 5% ß-mercaptoethanol, and subjected to SDS-PAGE. Gels were dried and exposed for 18–24 h, and the level of receptor phosphorylation was assessed with a Molecular Dynamics (Sunnyvale, CA) PhosphorImager and ImageQuant software (Amersham Biosciences, Piscataway, NJ). Data fell within the linear range of detection of the apparatus and were plotted using Prism 3 from GraphPad Software (San Diego, CA). Equal loading of immunoprecipitated receptor was checked by Western blotting (data omitted for clarity; see Ref. 12).

[Ca2+]i Measurements
Cells were incubated overnight in DMEM without serum and antibiotics and were loaded with 4 µM fura 2-acetoxymethyl ester in Krebs-Ringer-HEPES containing 0.05% BSA (pH 7.4) for 1 h at 37 C. Cells were detached by gentle trypsinization and fluorescence measurements were carried out as described previously (8, 12) with an Aminco-Bowman (Rochester, NY) Series 2 Spectrometer with excitation monochromator set at 340 and 380 nm; a chopper interval of 0.5 sec was used, and the emission monochromator was set at 510 nm. [Ca2+]i was calculated as described (43) using the software provided by Aminco-Bowman; traces were directly exported to the graphs.

[3H]Inositol Phosphate Production
Cells were labeled with [3H]inositol (5 µCi/ml) for 18–24 h in inositol-free DMEM containing 1% fetal bovine serum. On the day of the experiment, cells were washed twice with Krebs-Ringer-HEPES buffer containing 1.3 mM CaCl2 and preincubated for 20 min in 2 ml of the same buffer containing 20 mM LiCl, at 37 C in a 5% CO2 atmosphere. Incubations were for 15 min and were terminated by the addition of 2 ml chloroform/methanol (1:2 vol/vol); samples were thoroughly mixed and centrifuged in a clinical centrifuge. The aqueous phase was recovered and total [3H]inositol phosphates were separated by Dowex AG1-X8 chromatography (44).

Statistical analysis between comparable groups was performed using ANOVA with Newman-Keuls analysis and was effected with software included in the GraphPad Prism program.


   ACKNOWLEDGMENTS
 
We express our gratitude to Drs. Lefkowitz, Caron, Allen, Ogawa, Kasuga, and Koch for the indicated cells and plasmids and Dr. Mobashery for BB-94.


   FOOTNOTES
 
This research was partially supported by grants from Dirección General de Asuntos del Personal Académico (IN200206) and Consejo Nacional de Ciencia y Tecnología (45837Q).

The authors have nothing to disclose.

First Published Online June 27, 2006

Abbreviations: {alpha}1-AR, {alpha}1-Adrenoceptor; ß-ARK, ß-adrenergic receptor kinase; EGF, epidermal growth factor; GRK, G protein-coupled receptor kinase; HB-EGF, heparin-binding EGF; PDGF, platelet-derived growth factor; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C.

Received for publication February 21, 2006. Accepted for publication June 21, 2006.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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