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Insulin Activation of Mitogen-activated Protein Kinases Erk1,2 Is Amplified via β-adrenergic Receptor Expression and Requires the Integrity of the Tyr350 of the Receptor

2000; Elsevier BV; Volume: 275; Issue: 46 Linguagem: Inglês

10.1074/jbc.m004404200

1083-351X

Hsien‐yu Wang, Sergey V. Doronin, Craig C. Malbon,

Metabolism, Diabetes, and Cancer

Insulin activates a complex set of intracellular responses, including the activation of mitogen-activated protein kinases Erk1,2. The counterregulatory actions of insulin on catecholamine action are well known and include phosphorylation of the β2-adrenergic receptor on Tyr350, Tyr354, and Tyr364 in the C-terminal cytoplasmic domain, as well as enhanced sequestration of the β2-adrenergic receptor. Both β-adrenergic agonists and insulin provoke sequestration of β2-adrenergic receptors in a synergistic manner. In the current work, cross-talk between insulin action and β2-adrenergic receptors revealed that insulin activation of Erk1,2 was amplified via β2-adrenergic receptors. In Chinese hamster ovary cells, expression of β2-adrenergic receptors enhanced 5–10-fold the activation of Erk1,2 by insulin and prolonged the activation, the greatest enhancement occurring at 5 min post-insulin. The potentiation of insulin signaling on Erk1,2 was proportional to the level of expression of β2-adrenergic receptor. The potentiation of insulin signaling requires the integrity of Tyr350 of the β2-adrenergic receptor, a residue phosphorylated in response to insulin. β2-adrenergic receptors with a Y350F mutation failed to potentiate insulin activation of Erk1,2. Expression of the C-terminal domain of the β2-adrenergic receptor (Pro323-Leu418) in cells expressing the intact β2-adrenergic receptor acts as a dominant negative, blocking the potentiation of insulin activation of Erk1,2 via the β2-adrenergic receptor. Blockade of β2-adrenergic receptor sequestration does not alter the ability of the β2-adrenergic receptor to potentiate insulin action on Erk1,2. We propose a new paradigm in which a G-protein-linked receptor, such as the β2-adrenergic receptor, itself acts as a receptor-based scaffold via its binding site for Src homology 2 domains, facilitating signaling of the mitogen-activated protein kinase pathway by insulin. Insulin activates a complex set of intracellular responses, including the activation of mitogen-activated protein kinases Erk1,2. The counterregulatory actions of insulin on catecholamine action are well known and include phosphorylation of the β2-adrenergic receptor on Tyr350, Tyr354, and Tyr364 in the C-terminal cytoplasmic domain, as well as enhanced sequestration of the β2-adrenergic receptor. Both β-adrenergic agonists and insulin provoke sequestration of β2-adrenergic receptors in a synergistic manner. In the current work, cross-talk between insulin action and β2-adrenergic receptors revealed that insulin activation of Erk1,2 was amplified via β2-adrenergic receptors. In Chinese hamster ovary cells, expression of β2-adrenergic receptors enhanced 5–10-fold the activation of Erk1,2 by insulin and prolonged the activation, the greatest enhancement occurring at 5 min post-insulin. The potentiation of insulin signaling on Erk1,2 was proportional to the level of expression of β2-adrenergic receptor. The potentiation of insulin signaling requires the integrity of Tyr350 of the β2-adrenergic receptor, a residue phosphorylated in response to insulin. β2-adrenergic receptors with a Y350F mutation failed to potentiate insulin activation of Erk1,2. Expression of the C-terminal domain of the β2-adrenergic receptor (Pro323-Leu418) in cells expressing the intact β2-adrenergic receptor acts as a dominant negative, blocking the potentiation of insulin activation of Erk1,2 via the β2-adrenergic receptor. Blockade of β2-adrenergic receptor sequestration does not alter the ability of the β2-adrenergic receptor to potentiate insulin action on Erk1,2. We propose a new paradigm in which a G-protein-linked receptor, such as the β2-adrenergic receptor, itself acts as a receptor-based scaffold via its binding site for Src homology 2 domains, facilitating signaling of the mitogen-activated protein kinase pathway by insulin. G-protein-linked receptor(s) tyrosine kinase receptor(s) β2-adrenergic receptor(s) Src homology 2 1-phosphatidylinositol 3-kinase Chinese hamster ovary epidermal growth factor PI3K inhibitor LY294002 mitogen-activated protein kinase/extracellular signal-regulated kinase kinase green fluorescent protein 1,4-piperazinediethanesulfonic acid. G-protein-linked receptors (GPLRs)1 and growth factor receptors with intrinsic tyrosine kinase activity (TKR) represent two prominent pathways for cellular signaling (1Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4611) Google Scholar, 2Morris A.J. Malbon C.C. Physiol. Rev. 1999; 79: 1373-1430Crossref PubMed Scopus (396) Google Scholar). Study of the integration of signaling between GPLR and TKR pathways has recently revealed the existence of cross-talk at the most proximal point, receptor to receptor interaction with GPLRs acting as substrates for TKRs (3Hadcock J.R. Port J.D. Gelman M.S. Malbon C.C. J. Biol. Chem. 1992; 267: 26017-26022Abstract Full Text PDF PubMed Google Scholar, 4Karoor V. Baltensperger K. Paul H. Czech M.P. Malbon C.C. J. Biol. Chem. 1995; 270: 25305-25308Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 5Baltensperger K. Karoor V. Paul H. Ruoho A. Czech M.P. Malbon C.C. J. Biol. Chem. 1996; 271: 1061-1064Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Insulin stimulates the phosphorylation of the β2-adrenergic receptor (β2AR) on tyrosyl residues Tyr350/354 and Tyr364, bothin vivo (3Hadcock J.R. Port J.D. Gelman M.S. Malbon C.C. J. Biol. Chem. 1992; 267: 26017-26022Abstract Full Text PDF PubMed Google Scholar, 4Karoor V. Baltensperger K. Paul H. Czech M.P. Malbon C.C. J. Biol. Chem. 1995; 270: 25305-25308Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) and in vitro (5Baltensperger K. Karoor V. Paul H. Ruoho A. Czech M.P. Malbon C.C. J. Biol. Chem. 1996; 271: 1061-1064Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) using recombinant, purified β2AR and insulin receptors. Tyrosyl residue 350, a prominent residue for insulin receptor-catalyzed phosphorylation, is embedded in a sequence motif (Tyr-Gly-Asn-Gly) that is similar to the motifs known to interact withCaenorhabditis elegans sem5 Src homology 2 (SH2) domains when phosphorylated (6Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1900) Google Scholar). Phosphorylation of sites on the β2AR by the insulin receptor and the insulin-like growth factor-1 receptor include a motif for TKR at Tyr364 (7Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2384) Google Scholar), the Grb2 binding site at Tyr350 (4Karoor V. Baltensperger K. Paul H. Czech M.P. Malbon C.C. J. Biol. Chem. 1995; 270: 25305-25308Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 8Geahlen R.L. Harrison M.L. Peptides and Protein Phosphorylation. CRC Press, Inc., Boca Raton, FL1989Google Scholar), and a potential Shc binding site at Tyr132 (4Karoor V. Baltensperger K. Paul H. Czech M.P. Malbon C.C. J. Biol. Chem. 1995; 270: 25305-25308Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 5Baltensperger K. Karoor V. Paul H. Ruoho A. Czech M.P. Malbon C.C. J. Biol. Chem. 1996; 271: 1061-1064Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 7Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2384) Google Scholar). For insulin action, activation of 1-phosphatidylinositol 3-kinase (PI3K) is an early event, following temporally the phosphorylation of the insulin receptor and insulin receptor substrate-1 (9White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar, 10Quon M. Butte A. Taylor S. Trends Endocrinol. Metab. 1994; 5: 369-376Abstract Full Text PDF PubMed Scopus (48) Google Scholar). In response to insulin stimulation, the p85 regulatory subunit of PI3K binds the IRS-1 via SH2 domain(s), activating the catalytic p110 subunit, which phosphorylates various phosphoinositides at the 3′ position of the inositol ring (11Rordorf-Nikolic T. Van Horn D.J. Chen D. White M.F. Backer J.M. J. Biol. Chem. 1995; 270: 3662-3666Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). Ample reports support the premise that PI3K and its 3′-phosphoinositide products are critical to intracellular trafficking of membrane-bound elements in general (12Schu P.V. Takegawa K. Fry M.J. Stack J.H. Waterfield M.D. Emr S.D. Science. 1993; 260: 88-91Crossref PubMed Scopus (806) Google Scholar) and of downstream elements of TKR signaling, particularly insulin (13Heller-Harrison R.A. Morin M. Guilherme A. Czech M.P. J. Biol. Chem. 1996; 271: 10200-10204Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). We have shown that insulin, much like β-adrenergic agonists, provoke rapid sequestration of β2AR in a synergistic manner (14Karoor V. Wang L. Wang H. Malbon C.C. J. Biol. Chem. 1998; 273: 33035-33041Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 15Malbon C.C. Karoor V. Cell. Signal. 1998; 10: 523-527Crossref PubMed Scopus (28) Google Scholar). We probed for possible cross-talk between insulin and β2ARs in the mitogen-activated protein kinase pathway. Remarkably, the activation of Erk1,2 by insulin in Chinese hamster ovary (CHO) cells was found to be amplified by β2AR expression, i.e. the higher the level of cellular complement of β2AR, the greater was the potentiation of insulin activation of Erk1,2. Further studies reveal that the ability of the β2AR to amplify the insulin response was dependent on the integrity of the Tyr350 residue, which is phosphorylated by the insulin receptor and constitutes a binding site for an SH2 domain to which Grb2, and other proteins, can bind (16Shih M. Malbon C.C. Cell. Signal. 1998; 10: 575-582Crossref PubMed Scopus (21) Google Scholar). A new paradigm is proposed in which G-protein-linked receptors function as a receptor-based scaffold via a binding site for SH2 domains, amplifying signaling via the mitogen-activated protein kinase pathway. CHO stably expressing wild-type and mutant β2ARs and human epidermoid carcinoma A431 cells were cultured in Dulbecco's modified Eagle's medium containing 5 and 10% fetal bovine serum, respectively (17George S.T. Berrios M. Hadcock J.R. Wang H.Y. Malbon C.C. Biochem. Biophys. Res. Commun. 1988; 150: 665-672Crossref PubMed Scopus (41) Google Scholar, 18Shih M. Lin F. Scott J.D. Wang H.Y. Malbon C.C. J. Biol. Chem. 1999; 274: 1588-1595Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 19Shih M. Malbon C.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12193-12197Crossref PubMed Scopus (91) Google Scholar, 20Shih M. Malbon C.C. J. Biol. Chem. 1996; 271: 21478-21483Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Prior to analysis for insulin action, the cells were serum-starved overnight (4Karoor V. Baltensperger K. Paul H. Czech M.P. Malbon C.C. J. Biol. Chem. 1995; 270: 25305-25308Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The CHO clones stably expressing the hamster β2AR were characterized earlier (17George S.T. Berrios M. Hadcock J.R. Wang H.Y. Malbon C.C. Biochem. Biophys. Res. Commun. 1988; 150: 665-672Crossref PubMed Scopus (41) Google Scholar). The C-terminal, cytoplasmic domain of the hamster β2AR (Pro323-Leu418) was cloned into the pCDNA3 expression vector and employed to stably transfect A431 clones. Cells were stimulated with epidermal growth factor (EGF) (50 ng/ml) or insulin (100 nm) for the indicated times and then lysed (150 mm NaCl, 5 mm EDTA, 50 mm NaF, 40 mm sodium pyrophosphate, 50 mmKH2PO4, 10 mm sodium molybdate, 2 mm sodium orthovanadate, 20 mm Tris-HCl, pH 7.4, 1% Triton X-100, 0.5% Nonidet P-40, 6 mmdithiothreitol, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 0.2 mm phenylmethylsulfonyl fluoride). Samples were subjected to SDS polyacrylamide gel electrophoresis and immunoblotting as described earlier (21Jho E.H. Davis R.J. Malbon C.C. J. Biol. Chem. 1997; 272: 24468-24474Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The blots were stained with antibodies purchased from Promega (Madison, WI) that specifically recognize only the dually phosphorylated, active forms of Erk1,2. For inhibitor studies, A431 cells and CHO clones stably transfected to express β2-adrenergic receptors were pretreated for 12 h in the absence or presence of one of the following agents: the PI3K inhibitor LY294002 (LY; 20 μm), the MEK inhibitor PD98059 (10 μm), or the Src inhibitor PP2 (50 nm). After an overnight exposure to an inhibitor, the cells were incubated with or without 100 nm insulin for 5 min, and the amount of Erk1,2 activated was measured. The number of β2AR was determined by radioligand binding. Intact A431 cells were incubated with 0.5 nm [125I]iodocyanopindolol (PerkinElmer Life Sciences) in the presence or absence of 10 μm propranolol at 23 °C for 90 min. The incubation buffer contained 50 mm Tris-HCl, pH 7.5, 10 mmMgCl2, and 150 mm NaCl. The cells were collected on GF/C membranes at reduced pressure and washed rapidly. The radioligand bound to the washed cell mass retained by the filter was quantified by use of a γ-counter (17George S.T. Berrios M. Hadcock J.R. Wang H.Y. Malbon C.C. Biochem. Biophys. Res. Commun. 1988; 150: 665-672Crossref PubMed Scopus (41) Google Scholar). Receptor sequestration was assayed using the hydrophilic, membrane-impermeable β-adrenergic antagonist [3H]CGP-12177 (18Shih M. Lin F. Scott J.D. Wang H.Y. Malbon C.C. J. Biol. Chem. 1999; 274: 1588-1595Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). A431 cells were preincubated with isoproterenol (10 μm) for periods up to 60 min or preincubated with insulin (100 nm) for 5 to 30 min. The cells were then resuspended in Dulbecco's modified Eagle's medium containing 20 mm HEPES, pH 7.4, and 70 nm [3H]CGP-12177 (PerkinElmer Life Sciences) at 4 °C for 6 h. The cells were diluted, collected on GF/C membranes, and washed rapidly. The radioligand bound to the washed cell mass retained on the filter was counted by use of liquid scintillation spectrometry. Nonspecific binding was defined as the radioligand binding insensitive to competition by the unlabeled, β-adrenergic antagonist propranolol (10 μm). A431 cells stably transfected with green fluorescence protein (GFP)-tagged β2-adrenergic receptor were treated with or without insulin in the absence or presence (overnight) of the PI3K inhibitor LY294002 (20 μm). The cells were incubated with or without 100 nm insulin for 5 min, fixed with 3% paraformaldehyde, and washed 3 times with modified Shields' medium (MSM)-Pipes buffer (18 mm MgSO4, 5 mmCaCl2, 40 mm KCl, 24 mm NaCl, and 5 mm Pipes, pH 6.8). The cells were analyzed by confocal laser scanning microscopy on an Odyssey instrument (Noran Instruments). The construct pCDNA3-β2AR-GFP, encoding a β2-adrenergic receptor fusion protein with GFP at its carboxyl terminus (22Gagnon A.W. Kallal L. Benovic J.L. J. Biol. Chem. 1998; 273: 6976-6981Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar), was a generous gift from Dr. J. L. Benovic. The confocal microscopy was performed at the University Microscopy Imaging Center at Stony Brook. Unless otherwise noted, the values presented are means ± S.E. The autoradiograms are representative of multiple (at least three) independent experiments. In all figures, an asterisk denotes a mean value with statistical significance (p ≤ 0.05) compared with the mean values of the control, time 0, or with the control group as indicated in the legend to the figure. Initial studies of the time-course for activation of Erk1,2 revealed a biphasic response to insulin stimulation of CHO clones. In CHOK cells, which express very low numbers of endogenous β2ARs, the activation of Erk1,2 displayed an initial peak of activation (as measured with phosphospecific antibodies for activated Erk1,2) in response to insulin at 0.5–1.0 min (Fig.1, inset), followed by a gradual decline in activation observed in analyses of multiple experiments performed on separate occasions (Fig. 1, bar graph). The activation of Erk2 (p42Erk) in response to insulin exceeded that of Erk1 (p44ErK). When examined in CHO cells expressing a modest level of β2ARs (18.2 fmol/100,000 cells), the activation of Erk1,2 in response to insulin shows both a biphasic response, as well as an amplified response to stimulation by insulin. The Erk1,2 response to insulin showed an early (0.5–1.0 min) peak of activation followed by a robust later response, peaking at 5 min, declining to unstimulated levels within 10 min (not shown). The response for activation of p42Erk in the CHO-β2AR clones was 5–7-fold greater than that in the β2AR-deficient CHOK clones. The response for activation of p44Erk by insulin was similar to that observed for p42Erk,i.e. 5–7-fold greater in the CHO-β2AR clones as compared with the CHOK clones. The simplest interpretation of the data is that the expression of β2AR potentiates and prolongs the ability of insulin to activate Erk1,2, a novel hypothesis. To test the hypothesis that the expressed level of the G-protein-linked receptor β2AR regulates the temporal nature and magnitude of the Erk1,2 activation by insulin, we examined the Erk1,2 response to activation by insulin in cells that stably express varying levels of β2AR (Fig. 2). Four clones were included in the analysis that displayed few receptors with CHOK (wild-type; 0.6 ± 0.08 fmol/100,000 cells), low (βL; 4.5 ± 1.1 fmol/100,000 cells), middle (βM; 18.3 ± 2.3 fmol/100,000 cells), and high (βH; 27.9 ± 3.0 fmol/100,000 cells) levels of expressed β2AR ([125I]iodocyanopindolol binding; mean values ± S.E., n = 3). The activation of Erk1,2 was measured in response to insulin challenge after 5 min. Immunoblots of the cell lysates were stained with antibodies to the phosphospecific, activated forms of Erk1 and Erk2. In the absence of insulin, levels of activated Erk1,2 were very low. Often, as shown here, the basal levels of phosphorylated, activated Erk1,2 were suppressed in the clones expressing β2AR. The greater the expression of β2AR, the greater was the apparent suppression of basal levels of Erk1,2 activation. Challenge with EGF provokes an activation of Erk1,2 in wild-type cells with low levels of β2AR, whereas the EGF response is attenuated in the clones expressing significant levels of β2ARs. The Erk1,2 activation in response to insulin, in sharp contrast, is clearly amplified in cells expressing β2ARs. Although not strictly proportional, the activation of Erk1,2 by insulin, but not EGF, was amplified to a greater extent with increasing levels of β2AR expression. These data suggest that the β2AR appears to facilitate insulin signaling through the mitogen-activated protein kinase cascade to the level of Erk1,2.Figure 1The activation of Erk1,2 in response to insulin is potentiated by β2AR. CHOK cells and CHO clones stably transfected to express the β2AR (CHO-β2AR) were challenged with insulin (100 nm) for the time (min) indicated, and the activation of Erk1,2 was determined using antibodies specific for the dually phosphorylated, active forms of p44 and p42 Erk (phospho-Erk). The results of three independent assays in which the amount of the phosphoactivated forms of p44 and p42 Erk were quantified are summarized in the bar graphs.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 2The activation of Erk1,2 in response to insulin is increased with increasing expression of β2AR. CHO clones were stably transfected and selected to express different levels of β2AR, indicated as wild-type (WT, with very low expression of β2AR), low (βL), middle (βM), and high (βH) expressing clones. The activation of Erk1,2 was determined, as indicated in the legend to Fig. 1, in clones incubated in the absence (-) or presence of either 100 nm insulin (+Insulin) or 50 ng/ml epidermal growth factor (+ EGF) for 5 min. The results of three independent assays in which the amount of the phosphoactivated forms of p44 and p42 Erk were quantified are summarized in the bar graphs.View Large Image Figure ViewerDownload Hi-res image Download (PPT) It was important to evaluate whether co-stimulation of the insulin- and β2AR-stimulated pathways leads to activation of Erk1,2 and whether some adrenergic agonist activity derived from serum might contribute to the insulin response. Treating A431 human epidermoid carcinoma cells with isoproterenol (10 μm) or insulin (100 nm) stimulated the activation of both Erk1 and Erk2 (Fig.3). Insulin and isoproterenol, in combination, do not produce an additive response. Propranolol (10 μm) blocked isoproterenol-stimulated activation of Erk1,2 but not the response to stimulation by insulin. These data rule out the possibility that co-stimulation by some serum-derived agent via a β2AR-mediated pathway is an explanation for the enhanced insulin-stimulated activation of Erk1,2 in cells expressing β2AR. The diterpene activator of adenylyl cyclase forskolin stimulates cyclic AMP accumulation and also provoked a weak activation of p44Erk and an activation of p42Erk similar to that of isoproterenol. These same results were obtained in CHO clones stably transfected to express β2ARs (data not shown). The inverse agonist compound ICI118551 was without effect on Erk1,2 activation in these cells (data not shown). We extended these studies by examining the ability of insulin to activate Erk1,2 in A431 cells that were challenged simultaneously with either 1 or 10 μm isoproterenol (Fig.4). The response to insulin alone is provided for comparison. Treatment with 1 μmisoproterenol alone provokes activation of Erk1,2. Increasing the concentration of insulin leads to further activation of Erk1,2. Increasing the concentration of isoproterenol alone to 10 μm leads to additional activation of that produced by 1 μm. Challenging the A431 cells with 10 μmisoproterenol in combination with increasing concentrations of insulin leads to a dampening of the activation of Erk1,2 especially at the lower concentrations of insulin (1 and 10 nm). These data suggest that the ability of isoproterenol to activate Erk1,2 is intrinsically lower than that of insulin to activate Erk1,2. In combination, insulin and isoproterenol do not activate Erk1,2 in an additive manner but rather appear to compete for the activation process in a weakly competitive manner. The observations both in vivo (4Karoor V. Baltensperger K. Paul H. Czech M.P. Malbon C.C. J. Biol. Chem. 1995; 270: 25305-25308Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 23Karoor V. Malbon C.C. J. Biol. Chem. 1996; 271: 29347-29352Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) and in vitro (5Baltensperger K. Karoor V. Paul H. Ruoho A. Czech M.P. Malbon C.C. J. Biol. Chem. 1996; 271: 1061-1064Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) demonstrate that insulin treatment leads to the phosphorylation of the β2AR on tyrosyl residues, particularly Tyr350. Phosphorylation of Tyr350 creates a binding site for SH2 domains that can act as a protein module for the docking and regulation of a wide spectrum of adaptor molecules, as well as interesting molecules such as Src and PI3K (6Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1900) Google Scholar). The ability of insulin to counterregulate the ability of the β2AR to activate adenyl cyclase is dependent upon the availability of the Tyr350 for phosphorylation. Mutation of this tyrosyl residue abolishes the counterregulation of β2AR-stimulated cyclic AMP accumulation (4Karoor V. Baltensperger K. Paul H. Czech M.P. Malbon C.C. J. Biol. Chem. 1995; 270: 25305-25308Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 24Karoor V. Malbon C.C. Adv. Pharmacol. 1998; 42: 425-428Crossref PubMed Scopus (23) Google Scholar). Similarly, the ability of insulin to provoke profound sequestration of the β2AR is dependent upon the availability of Tyr350, i.e. the Y350F mutant fails to display insulin-induced sequestration (14Karoor V. Wang L. Wang H. Malbon C.C. J. Biol. Chem. 1998; 273: 33035-33041Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). We tested whether mutation of the wild-type β2AR to Y350F, likewise, might influence the ability of insulin to signal to the level of Erk1,2 activation (Fig. 5). As displayed in the immunoblot of whole-cell extracts stained with antibodies to the phosphospecific, activated forms of Erk1,2, insulin stimulates an enhanced activation of Erk1,2 in CHO cells expressing the wild-type (Tyr350) β2AR. To address the nature of the effect of expressing a mutant receptor on the insulin response, we selected clones that express nearly equivalent levels of β2AR (3.5 ± 0.7 and 3.0 ± 0.6 fmol/100,000 cells for mutant Y350F and wild-type β2AR-expressing cells, respectively). The effect of the Y350F mutation was clear; the activation of Erk1,2 by insulin in the cells expressing the mutant receptors was largely abolished. Analysis of data from multiple experiments confirms the data displayed in the immunoblot. Mutation of Tyr350 of β2AR blocks the ability of insulin to counterregulate β2AR-mediated activation of adenylyl cyclase (3Hadcock J.R. Port J.D. Gelman M.S. Malbon C.C. J. Biol. Chem. 1992; 267: 26017-26022Abstract Full Text PDF PubMed Google Scholar, 4Karoor V. Baltensperger K. Paul H. Czech M.P. Malbon C.C. J. Biol. Chem. 1995; 270: 25305-25308Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) and to stimulate sequestration of the β2AR (14Karoor V. Wang L. Wang H. Malbon C.C. J. Biol. Chem. 1998; 273: 33035-33041Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), as well as the ability of the insulin receptor to phosphorylate Tyr350both in vivo (4Karoor V. Baltensperger K. Paul H. Czech M.P. Malbon C.C. J. Biol. Chem. 1995; 270: 25305-25308Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) and in vitro (5Baltensperger K. Karoor V. Paul H. Ruoho A. Czech M.P. Malbon C.C. J. Biol. Chem. 1996; 271: 1061-1064Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Taken together, these results further implicate the β2AR acting as a receptor-based scaffold for insulin signaling in which phosphorylation of the Tyr350 residue of the β2AR creates a binding site for SH2 domains in response to insulin, a key element to activation of Erk1,2. The Tyr350 residue is located in the C-terminal, cytoplasmic tail of the β2AR. If the β2AR is acting as a template or scaffold for insulin signaling to the level of Erk1,2, then expression of the C-terminal domain of the receptor that is not localized to the plasma membrane may influence this novel ability of the β2AR to potentiate insulin signaling. Human epidermoid A431 cells were employed because of their relatively high native expression of β2AR. A431 clones were stably transfected with an expression vector (pCDNA3) harboring the entire cytoplasmic C-terminal domain of the β2AR (BAC1). Expression was confirmed for the A431 cells stably transfected to express BAC1 by accumulation of the peptide (M r = 11,000) in sufficient quantities to permit ready detection in immunoblots of the whole-cell extracts from these cells, probed with an antibody to BAC1 (Fig.6 A). Insulin challenge of A431 cells yields a significant activation of Erk1,2 (Fig. 6 B). The expression of BAC1 in A431 clones resulted in a total loss of the ability of insulin to signal to the level of Erk1,2. Probing immunoblots of extracts from the A431 clones with antibodies specific for phosphotyrosine (Anti-pY) revealed, indeed, that BAC1 was tyrosine-phosphorylated in response to insulin (Fig. 5 A). Expression of BAC1 had no effect on the ability of isoproterenol (10 μm) to stimulate a normal elevation of intracellular cyclic AMP accumulation by A431 cells (data not shown). Several slower migrating species that are recognized by the BAC1 antibody and are phosphorylated in response to insulin are likely to represent palmitoylated forms of BAC1 (25Bouvier M. Chidiac P. Hebert T.E. Loisel T.P. Moffett S. Mouillac B. Methods Enzymol. 1995; 250: 300-314Crossref PubMed Scopus (26) Google Scholar). Thus, with respect to the activation of Erk1,2 by insulin in cells expressing native β2AR, BAC1 acts as a dominant-negative molecule that is phosphorylated in response to insulin stimulation and suppresses the ability of insulin to signal. PI3K plays an important role in many cellular process, including intracellular trafficking of molecules (26Carpenter C.L. Cantley L.C. Curr. Opin. Cell Biol. 1996; 8: 153-158Crossref PubMed Scopus (576) Google Scholar, 27Galetic I. Andjelkovic M. Meier R. Brodbeck D. Park J. Hemmings B.A. Pharmacol. Ther. 1999; 82: 409-425Crossref PubMed Scopus (96) Google Scholar). The microbial product wortmannin and the LY294002 compound both inhibit PI3K and many facets of intracellular trafficking. Inhibitors of P13K block various aspects of insulin action, and we evaluated whether the LY294002 compound would influence the ability of the β2AR to enhance insulin activation of Erk1,2 and perhaps influence the sequestration of β2ARs (Fig.7). Insulin, as well as isoproterenol, stimulates the sequestration of β2AR (14Karoor V. Wang L. Wang H. Malbon C.C. J. Biol. Chem. 1998; 273: 33035-33041Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). The cellular localization of the β2AR was defined by use of a GFP-tagged version of the β2AR, characterized earlier (18Shih M. Lin F. Scott J.D. Wang H.Y. Malbon C.C. J. Biol. Chem. 1999; 274: 1588-1595Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 22Gagnon A.W. Kallal L. Benovic J.L. J. Biol. Chem. 1998; 273: 6976-6981Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Using A431 cells stably transfected with an expression vector harboring the GFP-tagged β2AR, in tandem with confocal laser scanning microscopy, we observed the GFP-tagged β2AR display a pattern of distribution largely confined to the cell membrane (Fig. 7, panel a). Challenge with insulin induces a dramatic sequestration of the β2AR from the cell membrane to intracellular locales (Fig. 7, panel b), as previously observed (14Karoor V. Wang L. Wang H. Malbon C.C. J. Biol. Chem. 1998; 273: 33035-33041Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). In the presence of the LY294002 inhibitor (20 μm), in contrast, the ability of insulin to induce sequestration of the β2AR was abolished (Fig. 7,panel c). Analysis of β2AR internalization using the hydrophilic, beta-adrenergic antagonist ligand [3H]CGP-12177, confirms in an independent manner that inhibition of PI3K with the LY compound suppresses insulin-stimulated β2AR sequestration, measured at 5 min post-insulin (TableI). Taken together, these data suggest that the ability of insulin, but not the ability of isoproterenol, to stimulate sequestration of the β2AR was dependent upon PI3K,i.e. inhibition of PI3K by LY compound suppresses sequestration of β2AR in response to insulin. These novel data prompted us to probe further the possible role for β2AR internalization in the ability of β2AR to potentiate insulin activation of Erk1,2.Table IInhibition of PI3 kinase by LY294002 suppresses the sequestration of β2AR in response to insulin but not in response to isoproterenolTreatmentSequestration of β2AR%30-min Incubation None (Control) 0 + Isoproterenol (10 μm)25.8 ± 1.8 + Isoproterenol (10 μm) and propranolol (10 μm) 0 + Isoproterenol (10 μm) and LY294002 (20 μm)27.6 ± 2.45-min Incubation None (Control) 0 + Insulin (100 μm)19.5 ± 1.6 + Insulin (100 nm) and LY294002 (20 μm)04.9 ± 0.7A431 cells were treated in the absence (Control) and presence of either 10 μm isoproterenol or 100 nm insulin. The extent of β2AR sequestration was determined using the hydrophilic, cell-impermeable beta-adrenergic antagonist radioligand [3H]CGP-12177. The incubation with isoproterenol was for 30 min, whereas the incubation with insulin was measured at 5 min, as employed in the experiments shown in Fig. 6. The isoproterenol-stimulated sequestration was blocked by addition of the beta-adrenergic antagonist propranolol (10 μm). Aliquots of cells were pretreated with the PI3K inhibitor LY294002 (20 μm) and then stimulated with either isoproterenol or insulin. The LY compound suppresses insulin-stimulated sequestration of the β2AR. The data are the mean values ± S.E. of three replicate experiments. Total binding was 29.4 ± 3.2 fmol/100,000 cells. Open table in a new tab A431 cells were treated in the absence (Control) and presence of either 10 μm isoproterenol or 100 nm insulin. The extent of β2AR sequestration was determined using the hydrophilic, cell-impermeable beta-adrenergic antagonist radioligand [3H]CGP-12177. The incubation with isoproterenol was for 30 min, whereas the incubation with insulin was measured at 5 min, as employed in the experiments shown in Fig. 6. The isoproterenol-stimulated sequestration was blocked by addition of the beta-adrenergic antagonist propranolol (10 μm). Aliquots of cells were pretreated with the PI3K inhibitor LY294002 (20 μm) and then stimulated with either isoproterenol or insulin. The LY compound suppresses insulin-stimulated sequestration of the β2AR. The data are the mean values ± S.E. of three replicate experiments. Total binding was 29.4 ± 3.2 fmol/100,000 cells. To probe further the potentiation of insulin signaling to Erk1,2 by β2AR, we tested inhibitors of Src, MEK, and PI3K. The studies were performed in both A431 cells and CHO clones stably transfected to express β2AR. Recent data have implicated the non-receptor tyrosine kinase Src in the biology of β2AR (28Luttrell L.M. Daaka Y. Lefkowitz R.J. Curr. Opin. Cell Biol. 1999; 11: 177-183Crossref PubMed Scopus (608) Google Scholar); for this reason the PP2 inhibitor of Src was tested first. Treatment with the PP2 inhibitor (50 nm) failed to influence the ability of cells expressing β2AR to potentiate insulin activation of Erk1,2 (Fig.8). The MEK inhibitor PD98059 (29Lazar D.F. Wiese R.J. Brady M.J. Mastick C.C. Waters S.B. Yamauchi K. Pessin J.E. Cuatrecasas P. Saltiel A.R. J. Biol. Chem. 1995; 270: 20801-20807Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar) (10 μm, overnight), in sharp contrast, abolishes all activation of Erk1,2, suggesting that the potentiation of insulin-stimulated Erk1,2 activation by β2AR is, in fact, mediated via MEK. The LY294002 compound, which inhibits 1-phosphatidylinositol 3-kinase, was without effect on the ability of β2AR to potentiate Erk1,2 activation by insulin (Fig. 8), although LY294002 was found to suppress insulin-stimulated β2AR sequestration (Fig. 7). We wondered whether inhibition of PI3K activity and blockade of β2AR sequestration might alter either the time-course or decay of the activation of Erk1,2 by insulin. A431 cells pretreated with the LY compound for 2 h prior to challenge with insulin display the same onset, activation, and decay of the response as cells not treated with LY294002 (Fig. 9). The results suggest that insulin and isoproterenol can both activate Erk1,2 independently of each other but that their activation in combination is non-additive. To test further the notion that these two agents may compete on one level with each other for the activation of Erk1,2, we investigated the effects of pretreating A431 cells with isoproterenol for 30 min on the ability of a subsequent challenge of insulin to activate Erk1,2. Cells were challenged with isoproterenol for 30 min and then challenged directly without or with increasing concentrations of insulin (Fig. 10). When challenged sequentially, the ability of isoproterenol to counterregulate the activation of Erk1,2 by insulin was revealed. The activation of Erk1,2 by insulin was diminished in cells challenged 30 min prior with isoproterenol. When challenged simultaneously, this ability of isoproterenol to counterregulate insulin action was not so obvious (Figs. 3 and 4). The counterregulatory effects of catecholamines on insulin action are well known. We reveal the activation of Erk1,2 to be the target of both insulin and beta-adrenergic agonists, with beta-adrenergic agonists and insulin, at one level, competing for β2AR (Fig.11). Because the extent of Erk1,2 activation is less for beta-adrenergic agonists than for insulin, this competition dictates the final level of Erk1,2 activity, which in turn can modulate other members of the mitogen-activated protein kinase network. On another level, the current work identifies a novel role for the β2AR and perhaps other GPLRs, i.e. acting as a receptor-based scaffold enhancing the signaling of other pathways. Expression of the β2AR clearly can potentiate the ability of insulin to activate Erk1,2. The evidence to support this notion is as follows: activation of Erk1,2 by insulin is potentiated severalfold and prolonged in cells expressing elevated levels of β2AR; the extent of the potentiation of insulin action on Erk1,2 correlates with the amount of β2AR expressed; mutation of the β2AR tyrosyl residue (Y350F) that is both phosphorylated in response to insulin and creates a binding site for SH2 domains abolishes the potentiation of insulin action; the C-terminal, cytoplasmic domain of the β2AR (BAC1), when expressed in cells, is phosphorylated in response to insulin and acts as a dominant negative with respect to enhanced activation of Erk1,2 by insulin; and, blockade of β2AR internalization does not suppress the ability of the β2AR to potentiate insulin activation of Erk1,2. Several well known GPLRs themselves activate the Erk1,2 pathway (2Morris A.J. Malbon C.C. Physiol. Rev. 1999; 79: 1373-1430Crossref PubMed Scopus (396) Google Scholar). In some cases, the activation of Erk1,2 requires internalization of the GPLR, whereas in others the activation of Erk1,2 proceeds in the absence of internalization (30Della Rocca G.J. Mukhin Y.V. Garnovskaya M.N. Daaka Y. Clark G.J. Luttrell L.M. Lefkowitz R.J. Raymond J.R. J. Biol. Chem. 1999; 274: 4749-4753Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 31Della Rocca G.J. van Biesen T. Daaka Y. Luttrell D.K. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 19125-19132Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar). The ability of the β2AR to cross-talk to and potentiate insulin action on Erk1,2 also does not require large-scale β2AR sequestration. GPLR-based scaffold functions have been implicated for direct effects of GPLR-agonists action on the mitogen-activated protein kinase network (28Luttrell L.M. Daaka Y. Lefkowitz R.J. Curr. Opin. Cell Biol. 1999; 11: 177-183Crossref PubMed Scopus (608) Google Scholar). We propose that the potentiation of insulin activation of Erk1,2 is yet another example of a GPLR-based scaffold. The β2AR, upon phosphorylation in response to insulin (3Hadcock J.R. Port J.D. Gelman M.S. Malbon C.C. J. Biol. Chem. 1992; 267: 26017-26022Abstract Full Text PDF PubMed Google Scholar, 4Karoor V. Baltensperger K. Paul H. Czech M.P. Malbon C.C. J. Biol. Chem. 1995; 270: 25305-25308Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 5Baltensperger K. Karoor V. Paul H. Ruoho A. Czech M.P. Malbon C.C. J. Biol. Chem. 1996; 271: 1061-1064Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 23Karoor V. Malbon C.C. J. Biol. Chem. 1996; 271: 29347-29352Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 24Karoor V. Malbon C.C. Adv. Pharmacol. 1998; 42: 425-428Crossref PubMed Scopus (23) Google Scholar), possess a binding site for SH2 domains (5Baltensperger K. Karoor V. Paul H. Ruoho A. Czech M.P. Malbon C.C. J. Biol. Chem. 1996; 271: 1061-1064Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) that can interact with a variety of adaptor molecules and enzymes, including PI3K and dynamin that are involved in β2AR sequestration (16Shih M. Malbon C.C. Cell. Signal. 1998; 10: 575-582Crossref PubMed Scopus (21) Google Scholar). The sequestration of β2AR in response to agonist requires G-protein-linked receptor kinase-catalyzed phosphorylation, arrestin binding, and the involvement of clathrin, dynamin, and Src. The activation of Erk1,2 by insulin as compared with isoproterenol shares many common features (Fig. 11) and some interesting differences. Insulin, as well as beta-adrenergic agonists, stimulate phosphorylation of the β2AR, the former by intrinsic tyrosine kinase activity of the insulin receptor and the latter by G-protein-linked receptor kinases. Both beta-adrenergic agonists and insulin can sequester β2AR into a vesicle-associated form and activate Erk1,2 independently. The sequestration by insulin, but not that by isoproterenol, is sensitive to LY. Blockade of insulin-induced sequestration by LY294002, however, does not block activation of Erk1,2 by insulin. The extent of the activation of Erk1,2 is greater for stimulation by insulin than it is for beta-adrenergic agonists. Pretreatment with isoproterenol for 30 min leads to a diminished capacity of insulin to activate Erk1,2, suggesting that at one level the two pathways compete for β2AR. Taking these observations into account, we speculate that the β2AR acts as a receptor-based scaffold for the MEK kinase, MEK, and/or Erk1,2 elements of the pathway, targeting one or more of these elements to the cell membrane. Although insulin clearly sequesters β2AR, the activation of Erk1,2 proceeds even when the internalization is blocked, suggesting that the tyrosine-phosphorylated β2AR may organize the elements leading to Erk1,2 activation. Scaffold proteins, such as the A-kinase anchoring protein 250 gravin, have been shown only recently to play an integral role in the signaling of GPLRs (16Shih M. Malbon C.C. Cell. Signal. 1998; 10: 575-582Crossref PubMed Scopus (21) Google Scholar,18Shih M. Lin F. Scott J.D. Wang H.Y. Malbon C.C. J. Biol. Chem. 1999; 274: 1588-1595Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 19Shih M. Malbon C.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12193-12197Crossref PubMed Scopus (91) Google Scholar, 20Shih M. Malbon C.C. J. Biol. Chem. 1996; 271: 21478-21483Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Expression of the β2AR potentiates and prolongs the insulin signaling event, and mutation of the Tyr350 residue abolishes the binding site for SH2 domains, as well as the potentiation. Insulin stimulates the tyrosine phosphorylation and inactivation of the β2AR with respect to activation of adenyl cyclase (and cyclic AMP accumulation), while simultaneously providing a scaffold via a binding site for SH2 domains on the β2AR that can facilitate a second wave of signaling to the activation of Erk1,2. This proposal complements the notion that GPLRs can act as scaffolds for transactivation of receptor tyrosine kinases (2Morris A.J. Malbon C.C. Physiol. Rev. 1999; 79: 1373-1430Crossref PubMed Scopus (396) Google Scholar, 28Luttrell L.M. Daaka Y. Lefkowitz R.J. Curr. Opin. Cell Biol. 1999; 11: 177-183Crossref PubMed Scopus (608) Google Scholar), only in this case the tyrosine kinase creates a GPLR-based scaffold via protein phosphorylation.