2-Adrenergic Receptor Lacking the Cyclic AMP-Dependent Protein Kinase Consensus Sites Fully Activates Extracellular Signal-Regulated Kinase 1/2 in Human Embryonic Kidney 293 Cells: Lack of Evidence for Gs/Gi Switching

Stimulation of the 2-adrenergic receptor ( 2AR) in human embryonic kidney (HEK) 293 cells causes a transient activation of Extracellular Signal-Regulated Kinase (ERK) 1/2. One of the mechanisms proposed for this activation is a PKA-mediated phosphorylation of the 2AR that switches receptor coupling from Gs to Gi and triggers internalization of the receptor. To examine these phenomena, we characterized agonist activation of ERK1/2 in HEK293 cells by the endogenous 2AR and in HEK293 cells stably overexpressing either the wild-type 2AR or a substitution mutant 2AR (PKA ) that lacks the cyclic AMP-dependent protein kinase (PKA) consensus phosphorylation sites (S261A, S262A and S345A, S346A). As the baseline, we established that epinephrine stimulation of the endogenous 2AR in HEK293 cells (20–30 fmol/mg) caused a rapid and transient activation of ERK1/2 with an EC50 of 5 to 6 nM. In contrast, the potency of epinephrine stimulation of ERK1/2 in cells stably overexpressing WT 2AR and PKA (2–4 pmol of 2AR/mg) was increased by over 100-fold relative to HEK293 cells, the EC50 values being 20 to 60 pM. The nearly identical 100-fold shift in EC50 for ERK1/2 activation in the PKA and WT 2AR relative to that in the HEK293 showed that the PKA are fully capable of activating ERK1/2. We also found maximal activation of ERK1/2 in the overexpressing cell lines at concentrations of epinephrine that cause no internalization (i.e., the EC50 for internalization was 75 nM). Pertussis toxin pretreatment caused only a weak inhibition of epinephrine activation of ERK1/2 in the HEK293 (7–16%) and no inhibition in the PKA cells. Finally we found that the Src family kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (10 M) caused a 90% inhibition of epinephrine or forskolin activation of ERK1/2 in both cell lines. Our results indicate that the dominant mechanism of 2AR activation of ERK1/2 does not require PKA phosphorylation of the 2AR, receptor internalization or switching from activation of Gs to Gi but clearly requires activation of a Src family member that may be downstream of PKA. Early reports that purified 2AR or a synthetic peptide corresponding to the third intracellular loop of the 2AR could activate pure Gi in reconstituted preparations in vitro raised the possibility that the 2AR could activate Gi in vivo (Cerione et al., 1985; Rubenstein et al., 1991), although with reduced efficiency relative to Gs. It was then demonstrated that PKA phosphorylation of a IIIi loop peptide of the 2AR increased activation of Gi in reconstituted preparations and slightly reduced its activation of Gs (Okamoto et al., 1991), leading to the proposal that 2AR activation could switch from Gs to Gi after PKA phosphorylation of the IIIi loop. Recent support for this scheme was derived from studies showing that isoproterenol activation of ERK1/2 in HEK293 cells was blocked more than 85% by pretreatment with pertussis toxin, that isoproterenol activation of ERK1/2 was inhibited by H89, and that transient expression of a 2AR lacking the PKA consensus sites (S261A, S262A, S345A, S346A) inhibited isoproterenol activation of ERK1/2 by 40% (Daaka et al., 1997). Other studies are not consistent with the switching hypothesis. First, it has been shown that forskolin activates ERK1/2 consistent with a PKA-mediated pathway of activation downstream of the receptor (Daaka et al., 1997; Schmitt and Stork, 2000). Second, a recent study found that isoproterenol activation of ERK1/2 in HEK293 cells was not inhibited by pertussis toxin (Schmitt and Stork, This work was supported by National Institutes of Health grant GM31208 (to R.B.C.). ABBREVIATIONS: 2AR, 2-adrenergic receptor; HEK, human embryonic kidney; ERK, extracellular signal-regulated kinase; PKA, cyclic AMP-dependent protein kinase; WT, wild-type; CGP-12177, 4-[3-[(1,1-dimethylethyl)amino]2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2one; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; DMEM, Dulbecco’s modified Eagle’s medium; HA, hemagglutinin; AT, ascorbate/thiourea; HE, HEPES/EDTA; PAGE, polyacrylamide gel electrophoresis; ICI-118551, ( )-1-[2,3-(dihydro-5,7-methyl-1H-inden-4-yl)oxy]3-[(methylethyl)amino]-2-butanol; EGF, epidermal growth factor. 0026-895X/02/6205-1094–1102$7.00 MOLECULAR PHARMACOLOGY Vol. 62, No. 5 Copyright © 2002 The American Society for Pharmacology and Experimental Therapeutics 1695/1016546 Mol Pharmacol 62:1094–1102, 2002 Printed in U.S.A. 1094 at A PE T Jornals on O cber 9, 2017 m oharm .aspeurnals.org D ow nladed from 2000). Third, we have shown that pertussis toxin pretreatment does not alter epinephrine-induced desensitization of HEK293 cells and S49 lymphoma cells, as would be expected if a Gs to Gi switch occurred (Clark et al., 1986; Seibold et al., 2000). Fourth, isoproterenol activation of ERK1/2 does not occur in the kin and cyc mutants of S49 mouse lymphoma cells (Wan and Huang, 1998). Reports on the role of internalization of the 2AR in ERK1/2 activation, studied through the use of transient expression of dominant negative dynamin (K44A) and mutant arrestins, have also been inconsistent. Several studies suggested that internalization was required for ERK1/2 activation by the 2AR in HEK293 cells (Daaka et al., 1998; Luttrell et al., 1999b) and COS-7 cells (Pierce et al., 2000), whereas others found that internalization was not required for 2AR activation of ERK1/2 in COS-1 cells (DeGraff et al., 1999). Several lines of evidence suggest a role for Src in activation of ERK1/2 by the 2AR: i) isoproterenol activation caused the formation of a multiprotein complex of receptor, -arrestin, and Src, and dominant negative mutants of Src reduced ERK1/2 activation (Daaka et al., 1997; Luttrell et al., 1999b; Zou et al., 1999; Miller et al., 2000); ii) Src was shown to be activated by Gs and Gi in reconstituted in vitro preparations suggesting direct activation of Src by G proteins (Ma et al., 2000); and iii) it was suggested that Src binds to tyrosine 350 of the 2AR after phosphorylation of this residue by an unidentified tyrosine kinase (Fan et al., 2001). Obviously there seems to be considerable complexity in 2AR activation of ERK1/2, and many issues are unresolved. In this article, we examine the role of receptor switching, Gi-internalization, and Src family kinases in 2AR activation of ERK1/2 in HEK293 cells. We had previously generated a mutant 2AR in which the two PKA consensus sites were eliminated by substitution of serines 261, 262, 345, and 346 with alanine, and this mutant receptor, termed PKA , was stably overexpressed in the HEK293 cell line. We reported previously that the coupling efficiency of this mutant and the EC50 for activation of adenylyl cyclase were nearly identical to those of WT 2AR overexpressed at comparable levels (Seibold et al., 1998, 2000). Stable overexpression of the PKA and the WT 2AR ( 100-fold relative to endogenous receptor) caused a dramatic left-shift in the EC50 for epinephrine activation of adenylyl cyclase. This property of the 2AR allows the analysis of mutant receptors overexpressed in HEK293 cells because the response of the endogenous receptor is overwhelmed if expression is high enough. Because the EC50 for epinephrine activation of adenylyl cyclase in cells expressing either the PKA or WT 2AR is left-shifted relative to the endogenous receptor, it follows that activation of ERK1/2 should show a similar left-shift if the mechanism involves receptor activation of Gs. The prediction of the switching model in which the PKA-phosphorylated 2AR is proposed to activate Gi is that there should be no left shift in ERK1/2 activation relative to the endogenous receptor; rather, there should be an inhibition of the activation by endogenous 2AR (Daaka et al., 1997). The potency of agonist activation of ERK1/2 in HEK293 cells by transiently expressed wild-type or PKA mutant 2ARs was not examined in previous studies (Daaka et al., 1997; Schmitt and Stork, 2000). In this article, we demonstrate that the stably overexpressed PKA mutant of the 2AR shows the predicted enormous left shift in the potency of activation of ERK1/2 by epinephrine relative to that of the endogenous low level of expression in the HEK293 and that the shift is nearly identical to that found with overexpressed wild-type 2AR. This demonstrated that the PKA consensus sites were not required for full activation of ERK1/2. Our data also show only a minor role for Gi in epinephrine activation of ERK1/2, no requirement for internalization (because the EC50 for epinephrine-induced internalization in the PKA was about 1000-fold higher than that for activation of ERK1/2), and a requirement for Src family kinase activation. Materials and Methods Materials. Antibodies to ERK1/2 and phospho-ERK1/2 were purchased from Cell Signaling Technology Inc. (Beverly, MA). [P]NAD and [H]CGP-12177 were obtained from PerkinElmer Life Sciences (Boston, MA). Pertussis toxin was from List Biological Laboratories Inc. (Campbell, CA). Enhanced chemiluminescence reagents and Hyperfilm were from Amersham Biosciences (Piscataway, NJ). Agonists and antagonists were purchased from Sigma-Aldrich. BA85 nitrocellulose was from Schleicher & Schuell (Keene, NH). Cell culture reagents were purchased from Mediatech (Herndon, VA). The Src family kinase inhibitor PP2 was from Alexis Corporation (Läufelfingen, Switzerland). Cell Culture. HEK293 cells purchased from the American Type Culture Collection (Manassas, VA) were grown in 5% CO2 at 37°C in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum. The stably transfected HEK293 cell lines used in this study expressing either WT or mutant receptors have been described previously (January et al., 1997; Seibold et al., 1998; Seibold et al., 2000), and were as follows: double epitope-tagged wild-type receptor (HA2AR-His6) and the double epitope-tagged PKA (S261A, S262A, S345A, S346A), both with the hemagglutinin (HA) tag on the N terminus and His6 tag on the C terminus; and HA-tagged wild-type (HA2AR). The stably transfected cell lines were cultured in medium containing 400 g/ml G418. The levels of 2AR in these transfect lines were 2 to 4 pmol/mg membrane protein, whereas the level of endogenous 2AR was 30 to 40 fmol/mg. Cell Treatment and Preparation of Solubilized Extract. Cells were grown to confluence in growth medium in 12-well plates coated with poly(L-lysine). The medium was removed 18 h before treatment, cells were incubated for 30 min with serum-free DMEM, and that medium was replaced with 2 ml of serum-free DMEM with or without 100 ng/ml of pertussis toxin. Cells were treated with hormones or carrier as indicated at 37°C with continuous rocking; pretreatments with 2AR antagonists were for 2 min before agonist treatment, and PP2 was added 1 h before agonist treatment. Epinephrine was stored in 10 mM ascorbate/100 mM thiourea pH 7 (AT). Stock solutions were diluted 100-fold when additions were made to cells such that the final concentration of AT in all incubations (control and treated) was 0.1 mM ascorbate/1.0 mM thiourea. PP2 was dissolved and stored in DMSO and was diluted 100-fold for cell treatments. To terminate treatments, the medium was removed and 0.5 ml of solubilization buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 0.8% dodecyl maltoside, 1 mM EDTA, pH 7, 20 mM tetrasodium pyrophosphate, 10 mM NaF, 10 g/ml benzamidine, 10 g/ml trypsin inhibitor, 10 g/ml leupeptin, and 0.1 M okadaic acid) was added to each well. The plates were placed on ice, and the solubilized cells were pipetted into 1.5-ml microcentrifuge tubes. The tubes were rocked at 4°C for 30 min, and then centrifuged at 12,500 rpm for 15 min. The supernatants were removed and frozen at 80°. Measurement of ERK1/2 and Phospho-ERK1/2. To measure activation of ERK1/2 by Western blotting, 20 l of the solubilized extracts were resolved by SDS-PAGE (10% gels) and blotted onto BA-85 nitrocellulose. The blots were blocked for 1 h in 5% nonfat dried milk in wash buffer (150 mM NaCl, 50 mM Tris pH 7.5, and 0.1% Tween 20). After two 10-min washes, the blots were incubated 2-Adrenergic Receptor Activation of ERK1/2 1095 at A PE T Jornals on O cber 9, 2017 m oharm .aspeurnals.org D ow nladed from overnight at 4°C on a rocker with a 1:1000 dilution of anti-phosphoERK, washed twice for 10 min each, and incubated for 1 h at room temperature with a 1:5000 dilution of secondary antibody (goat antirabbit HRP-conjugate, Bio-Rad, Hercules, CA) After 2 washes, the blots were exposed to ECL reagents for 1 min, dried, and exposed to Hyperfilm (Amersham Biosciences) for 15 sec to 2 min. The blots were then stripped and reprobed identically with anti-ERK. Western blots were quantitated using Scion Image software (Scion Corp., Frederick, MD). All results with the anti-phospho-ERK were normalized to the corresponding anti-ERK. Membrane Preparation and Adenylyl Cyclase Assay. Cells were plated into 100-mm dishes that had been precoated with poly(Llysine). Treatments were administered at 37°C and were stopped by removal of media followed by six washes with 5 ml of ice-cold HE buffer (20 mM HEPES, pH 8.0, 1 mM EDTA, pH 7). The cells were scraped into HE containing 10 g/ml leupeptin and 0.1 M okadaic acid and homogenized with seven strokes in a type B Dounce homogenizer (Bellco Glass, Vineland, NJ). The homogenates were layered onto sucrose step gradients (23 and 43% prepared in HE buffer) and centrifuged at 25,000 rpm in a Beckman SW28.1 rotor for 35 min. The fraction at the 23/43% sucrose interface was removed, frozen in liquid nitrogen, and stored at 80°C. Adenylyl cyclase activity was measured as described previously (Seibold et al., 2000). ADP Ribosylation. Membranes from control or pertussis toxintreated cells were incubated for 30 min at 30°C with 10 M NAD, 0.5 mM ATP, 0.2 mM GDP, 5 mM MgCl2, 1 mM EDTA, pH 7, 20 mM Tris, pH 7.5, 5 mM dithiothreitol, 5 mM thymidine, 8 mM creatine phosphate, 8 U/ml creatine phosphokinase, and 5 Ci/tube [P]NAD. The incubation mix was diluted in 5 ml of 20 mM Tris, pH 7.5, and centrifuged for 15 min at 30,000 rpm in a Beckman 50Ti rotor (Beckman Coulter, Fullerton, CA). The pellets were each dissolved in SDS sample buffer and resolved on 12% SDS-PAGE gels. The proteins were transferred to nitrocellulose and exposed to a Storm Phosphorimager screen (Molecular Dynamics, Sunnyvale, CA). The phosphorylated bands were quantitated using ImageQuant (Molecular Dynamics). Internalization. The procedure for measuring internalization of the 2AR has been described in detail previously (Seibold et al., 2000). Cells in 12-well dishes were treated with epinephrine or AT for 5 min. Cells were then washed five times with ice-cold, serumfree DMEM, placed on ice, then incubated with 5 to 10 nM [H]CGP12177 with or without 1 M alprenolol in serum-free DMEM. Dishes were then incubated for 1 h on ice, washed twice with ice-cold PBS to remove [H]CGP-12177, and cells released by trypsin were transferred to scintillation vials for counting.