Stimulation of Mitogen-activated Protein Kinase by G Protein-coupled α2-Adrenergic Receptors Does Not Require Agonist-elicited Endocytosis

Abstract

Agonist-elicited receptor sequestration is strikingly different for the α2A-versus α2B-adrenergic receptor (α2-AR) subtypes; the α2B-AR undergoes rapid and extensive disappearance from the HEK 293 cell surface, whereas the α2A-AR does not (Daunt, D. A., Hurt, C., Hein, L., Kallio, J., Feng, F., and Kobilka, B. K. (1997)Mol. Pharmacol. 51, 711–720; Eason, M. G., and Liggett, S. B. (1992) J. Biol. Chem. 267, 25473–25479). Since recent reports suggest that endocytosis is required for some G protein-coupled receptors to stimulate the mitogen-activated protein (MAP) kinase cascade (Daaka, Y., Luttrell, L. M., Ahn, S., Della Rocca, G. J., Ferguson, S. S., Caron, M. G., and Lefkowitz, R. J. (1998) J. Biol. Chem. 273, 685–688; Luttrell, L. M., Daaka, Y., Della Rocca, G. J., and Lefkowitz, R. J. (1997) J. Biol. Chem. 272, 31648–31656; Ignatova, E. G., Belcheva, M. M., Bohn, L. M., Neuman, M. C., and Coscia, C. J. (1999)J. Neurosci. 19, 56–63), we evaluated the differential ability of these two subtypes to activate MAP kinase. We observed no correlation between subtype-dependent agonist-elicited receptor redistribution and receptor activation of the MAP kinase cascade. Furthermore, incubation of cells with K+-depleted medium eliminated α2B-AR internalization but did not eliminate MAP kinase activation, suggesting that receptor internalization is not a general prerequisite for activation of the MAP kinase cascade via Gi-coupled receptors. We also noted that neither dominant negative dynamin (K44A) nor concanavalin A treatment dramatically altered MAP kinase activation or receptor redistribution, indicating that these experimental tools do not universally block G protein-coupled receptor internalization. Agonist-elicited receptor sequestration is strikingly different for the α2A-versus α2B-adrenergic receptor (α2-AR) subtypes; the α2B-AR undergoes rapid and extensive disappearance from the HEK 293 cell surface, whereas the α2A-AR does not (Daunt, D. A., Hurt, C., Hein, L., Kallio, J., Feng, F., and Kobilka, B. K. (1997)Mol. Pharmacol. 51, 711–720; Eason, M. G., and Liggett, S. B. (1992) J. Biol. Chem. 267, 25473–25479). Since recent reports suggest that endocytosis is required for some G protein-coupled receptors to stimulate the mitogen-activated protein (MAP) kinase cascade (Daaka, Y., Luttrell, L. M., Ahn, S., Della Rocca, G. J., Ferguson, S. S., Caron, M. G., and Lefkowitz, R. J. (1998) J. Biol. Chem. 273, 685–688; Luttrell, L. M., Daaka, Y., Della Rocca, G. J., and Lefkowitz, R. J. (1997) J. Biol. Chem. 272, 31648–31656; Ignatova, E. G., Belcheva, M. M., Bohn, L. M., Neuman, M. C., and Coscia, C. J. (1999)J. Neurosci. 19, 56–63), we evaluated the differential ability of these two subtypes to activate MAP kinase. We observed no correlation between subtype-dependent agonist-elicited receptor redistribution and receptor activation of the MAP kinase cascade. Furthermore, incubation of cells with K+-depleted medium eliminated α2B-AR internalization but did not eliminate MAP kinase activation, suggesting that receptor internalization is not a general prerequisite for activation of the MAP kinase cascade via Gi-coupled receptors. We also noted that neither dominant negative dynamin (K44A) nor concanavalin A treatment dramatically altered MAP kinase activation or receptor redistribution, indicating that these experimental tools do not universally block G protein-coupled receptor internalization. adrenergic receptor mitogen-activated protein kinase enzyme-linked immunosorbent assay mercaptoethanesulfonic acid human embryonic kidney G protein-coupled receptor Dulbecco's modified Eagle's medium phosphate-buffered saline hemagglutinin The three subtypes of α2-adrenergic receptors (α2-AR)1 signal via the Gi/Go subfamily of G proteins to effect several downstream signaling events (1Limbird L.E. FASEB J. 1988; 2: 2686-2695Crossref PubMed Scopus (277) Google Scholar). Since all three receptor subtypes appear to couple to the same effectors, it is of interest to explore other differences among these subtypes in an effort to understand the functional relevance of subtype diversity. Two α2-AR subtypes manifest differences in agonist-induced receptor redistribution from the cell surface as follows: the α2B-AR becomes rapidly and extensively internalized following agonist occupancy, whereas the α2A-AR does not readily redistribute to an intracellular compartment following agonist occupancy (2Daunt D.A. Hurt C. Hein L. Kallio J. Feng F. Kobilka B.K. Mol. Pharmacol. 1997; 51: 711-720Crossref PubMed Scopus (175) Google Scholar, 3Eason M.G. Liggett S.B. J. Biol. Chem. 1992; 267: 25473-25479Abstract Full Text PDF PubMed Google Scholar). The α2C-AR exists both on the surface and in an intracellular compartment at steady state (2Daunt D.A. Hurt C. Hein L. Kallio J. Feng F. Kobilka B.K. Mol. Pharmacol. 1997; 51: 711-720Crossref PubMed Scopus (175) Google Scholar), confounding quantitative assessment of α2C-AR redistribution. Recent reports suggest that internalization is required for some G protein-coupled receptors (GPCRs) to stimulate the MAP kinase cascade (4Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar, 5Luttrell L.M. Daaka Y. Della Rocca G.J. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31648-31656Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 6Ignatova E.G. Belcheva M.M. Bohn L.M. Neuman M.C. Coscia C.J. J. Neurosci. 1999; 19: 56-63Crossref PubMed Google Scholar). The purpose of the current studies was to determine if the differing profiles of agonist-induced internalization of the α2A-AR and α2B-AR subtypes are paralleled by differing rates or extents of MAP kinase activation and whether agents that interfere with agonist-elicited receptor redistribution alter MAP kinase activation. HEK 293 cells were maintained in DMEM containing 10% fetal calf serum at 37 °C in a 5% CO2 incubator. Permanent transfectants were generated by LipofectAMINE-mediated co-transfection of the cells with plasmids containing the indicated receptors and a neomycin resistance gene. Cells that survived selection in medium containing 500 μg/ml G418 were screened for expression of the expected receptor by binding of the radiolabeled α2-AR antagonist, [3H]rauwolscine. Clonal cell lines with varying levels of α2-AR expression were kept for further study. The experiments reported here were performed on an α2A-AR expressing cell line that contains 7–8 pmol of receptor per mg of protein and an α2B-AR-expressing cell line that contains 2–4 pmol/mg. Permanent transfectants of HEK 293 cells were plated on 60-mm dishes and allowed to multiply until they reached a density of 60–80%. The cells were then serum-deprived overnight. On the day of the experiment, the medium was replaced with fresh serum-free DMEM, DMEM supplemented with 250 μg/ml concanavalin A, or K+-depleted medium as described below to block clathrin-mediated endocytosis. After the pretreatment, the indicated drugs were added directly to the medium on the cells and were swirled to mix. After the indicated times, the cells were washed once with Dulbecco's phosphate-buffered saline containing 1 mmMgCl2 and 0.5 mm CaCl2(PBS/Ca2+/Mg2+) and then lysed in SDS sample buffer (62.5 mm Tris-HCl, pH 6.8; 2% w/v SDS; 10% glycerol; 50 mm dithiothreitol) supplemented with 1 mmsodium orthovanadate (Sigma), 10 units/ml leupeptin (Sigma), and 10 units/ml aprotinin (Bayer Corp., Kankakee, IL). The lysates were transferred to an Eppendorf tube on ice. When all samples were collected, they were bath sonicated for 30–40 s, then placed in a heating block at 90 °C, allowed to warm to 95 °C, and incubated at 95 °C for 5 min. The lysates were then spun in a microcentrifuge at room temperature for 5 min to remove debris. The supernatants were assayed in Bio-Rad's protein assay for relative protein concentration, and equivalent amounts of protein were loaded on a 10% SDS-polyacrylamide gel for electrophoresis. The gel was run for 160 mA-h and then transferred overnight onto nitrocellulose in transfer buffer (20% methanol; 0.19 m glycine; 25 mmTris base) at 33 mV. MAP kinase activation was evaluated using an antibody that recognizes dually phosphorylated (Thr/Tyr) MAP kinase (Promega catalog number V6671) and normalized to total MAP kinase using an antibody that recognizes MAP kinase regardless of its phosphorylation state (New England Biolabs catalog number 9102). To assess activated MAP kinase content, the nitrocellulose blot was incubated in blocking buffer (1× TBS; 0.1% Tween 20; 5% w/v nonfat dry milk) for 1 h at room temperature and then probed with rabbit polyclonal antibody (from Promega) to dually phosphorylated MAP kinase, diluted 1/500 in blocking buffer, for 1 h at room temperature. The blot was washed three times for 5 min each with TBST (2.42 g/liter Tris base; 8.0 g/liter sodium chloride; 0.1% Tween 20; pH 7.6) and then probed with donkey anti-rabbit horseradish peroxidase-linked secondary antibody (1/2000 dilution in blocking buffer) (Amersham Pharmacia Biotech) for 1 h at room temperature. The wash protocol was repeated, and the immunoreactive bands were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech). The blots were then stripped with stripping buffer (62.5 mm Tris-HCl, pH 6.8; 2% SDS; 100 mm 2-mercaptoethanol) for 30 min at 65 °C and reprobed with antibody to total MAP kinase (New England Biolabs) at a 1/500 dilution in blocking buffer overnight at 4 °C, followed by donkey anti-rabbit secondary antibody as described above. To semi-quantify MAP kinase activation, the enhanced chemiluminescence (ECL) images were scanned into Adobe Photoshop with a UMAX Astra 600 scanner, and the band intensities were measured using NIH Image software. Background pixel density was subtracted from each band's pixel density. The corrected pixel density for active MAP kinase was divided by the corrected pixel density for total MAP kinase to obtain the normalized levels of activated MAP kinase reported in the figures as “active/total.” The introduction of the HA epitope into the amino terminus of α2-ARs expressed in HEK 293 cells provided a tool for quantitation of receptors expressed at the cell surface using a cell surface ELISA, as described previously (2Daunt D.A. Hurt C. Hein L. Kallio J. Feng F. Kobilka B.K. Mol. Pharmacol. 1997; 51: 711-720Crossref PubMed Scopus (175) Google Scholar, 7Schoneberg T. Liu J. Wess J. J. Biol. Chem. 1995; 270: 18000-18006Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). On day 1, HEK 293 cells stably expressing the receptors of interest were plated on poly-d-lysine-coated 96-well culture plates at a density of 20,000 cells/well. On day 2, the cells were serum-starved overnight. On the morning of day 3, the cells were treated in the absence or presence of agonist as indicated in the figure legends and then washed twice in PBS/Ca2+/Mg2+ to stop the drug treatment and fixed in 4% paraformaldehyde containing 0.12 m sucrose for 20 min at room temperature. The wells were washed three times with PBS/Ca2+/Mg2+ and then incubated for 30 min at 37 °C in DMEM containing 10% sheep serum to block nonspecific antibody binding in subsequent incubations. The 12CA5 antibody directed against the HA epitope was diluted 1/100 in blocking medium and applied for 1 h at 37 °C. The cells were then washed 3 times for 5 min each in PBS/Ca2+/Mg2+ at room temperature. The sheep anti-mouse secondary antibody (Amersham Pharmacia Biotech) was applied at a 1/2500 dilution in blocking medium for 1 h at 37 °C. The wells were then washed three times for 5 min at room temperature in PBS/Ca2+/Mg2+, as above. The colorimetric substrate o-phenylenediamine dihydrochloride (1 mg/ml, Pierce) was added and incubated for 20 min to 1 h at 15 °C. The color development was stopped by the addition of an equal volume of 1 m sulfuric acid. The absorbance at 490 nm in each well was read on a microtiter plate reader. Generally, four replicates of each treatment were performed per experiment. Surface receptors were biotinylated on ice with disulfide-cleavable biotin (sulfo-NHS-SS-biotin; Pierce) and treated with a hydrophilic reducing agent, mercaptoethanesulfonic acid (MESNA), after termination of the surface biotinylation reaction. Receptors that are inside the cell at the time of MESNA treatment are protected from reduction and therefore subsequently isolated from the detergent extract by adsorption to streptavidin-agarose. Our protocol was adapted from a previously published procedure (8Whistler J.L. von Zastrow M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9914-9919Crossref PubMed Scopus (318) Google Scholar). For each experiment, HEK 293 clonal cell lines were plated on 60-mm dishes coated with poly-d-lysine and allowed to grow to 70–80% confluence. Cells were serum-starved for 16–18 h prior to each experiment. On the day of the experiment, cells were first treated with serum-free DMEM with or without 250 μg/ml concanavalin A or with K+-depleted medium as described below. Subsequent steps were performed at 4 °C as follows: cells were washed twice with ice-cold phosphate-buffered saline (PBS) containing 1 mmMgCl2 and 0.5 mm CaCl2 or with K+ depletion buffer and then incubated with 100 μg/ml sulfo-NHS-SS-biotin in PBS/Ca2+/Mg2+ or K+ depletion buffer for 30 min at 4 °C. The cells were washed twice with PBS/Ca2+/Mg2+ and once with serum-free DMEM or with K+ depletion buffer at 4 °C. Dishes were then incubated at 37 °C by placement on a in a For agonist activation, the medium was replaced with 37 °C serum-free DMEM or K+ depletion buffer containing 100 for 5 min at 37 °C cells medium replaced with warm medium without The incubation was by of the 37 °C medium with ice-cold DMEM or K+ depletion The culture dishes were to 4 °C, washed twice with ice-cold PBS/Ca2+/Mg2+, and then incubated with 250 mm MESNA in PBS/Ca2+/Mg2+ for two to via The cells were then washed twice with ice-cold serum-free DMEM, and the reducing effect of MESNA was then by incubation with 5 in PBS/Ca2+/Mg2+ for 20 min at 4 °C. receptors to MESNA of biotinylation were as To total receptors on these cell 60-mm per was treated with MESNA following biotinylation at 4 °C to the of surface receptor biotinylation that MESNA To biotinylated cells were lysed in buffer (Sigma), 1 (Sigma), 25 mm 5 mm 20 mm pH and by 5 a was by in a microcentrifuge at 4 °C for 1 The as the was incubated with for 1 h at room temperature. The was and the were washed 3 times with a dilution of the buffer in mm glycine; 5 mm 20 mm pH m were from in buffer containing 15 μg/ml The was by SDS-polyacrylamide gel transferred to and probed with a dilution in blocking buffer of antibody from to the sheep anti-mouse secondary antibody (Amersham Pharmacia Biotech) was at a dilution in blocking proteins were by (Amersham Pharmacia Biotech). were by and NIH Image as described above. To the of the band in the cells were to MESNA 4 is subtracted as from all other band and then the band in the treated or after 5 min at 37 is divided by the band and the is by The depletion protocol for blocking receptor endocytosis was adapted from previously published K. B. J. Biol. PubMed Scopus Google Scholar, A. T. T. G. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). Clonal cell expressing α2-AR subtype were incubated at 37 °C for 5 min in DMEM and in a they were once in K+ depletion buffer mm 50 mm pH 1 mm 1 mm and then incubated in the same buffer for 1 h at 37 °C. cells were treated with of depletion MAP kinase or biotinylation then as described above. Clonal cell lines expressing α2-AR subtype were incubated with 250 μg/ml concanavalin A (Sigma), fresh in serum-free DMEM, for 30 min at 37 °C, as described previously J. Biol. Chem. Full Text PDF PubMed Google Scholar). MAP kinase or biotinylation then as described above. Clonal cell lines expressing α2-AR subtype were washed once and then incubated for 1 h at 37 °C with DMEM containing as described previously K. B. J. Biol. PubMed Scopus Google Scholar). treatment and then as HEK 293 cells were plated on plates or culture dishes and with the α2B-AR and dominant negative dynamin provided by or as a was to with a or of and MAP kinase activation were assayed as described above. Two wells of a plate were for each Two experimental were to agonist-elicited receptor redistribution of α2-AR cell performed on HEK 293 cells stably expressing α2A-AR and α2B-AR was to assess of receptor from the cell surface 1 surface (2Daunt D.A. Hurt C. Hein L. Kallio J. Feng F. Kobilka B.K. Mol. Pharmacol. 1997; 51: 711-720Crossref PubMed Scopus (175) Google Scholar, 3Eason M.G. Liggett S.B. J. Biol. Chem. 1992; 267: 25473-25479Abstract Full Text PDF PubMed Google that the α2A-AR and α2B-AR differing profiles of agonist-induced internalization. the α2A-AR of surface receptors following to agonist the α2B-AR subtype a of epitope the same internalization also by the of surface receptors that in a to an For these we a agent, sulfo-NHS-SS-biotin 1 on the surface at the of a treatment protocol was by treatment with a reducing that the and the from proteins at the cell MESNA after biotinylation 1 time an assessment of the of biotinylated receptor that is to at time incubation of the cells at 37 °C in the absence or presence of agonist MESNA of surface biotinylation of receptor redistribution. in 1 the α2A-AR is not extensively to a compartment at 37 °C, in the presence of the agonist whereas the α2B-AR is rapidly internalized from MESNA and internalization is enhanced by agonist studies in activation of MAP kinase T. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, T. Luttrell L.M. Lefkowitz R.J. PubMed Scopus Google Scholar, M. G. J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, A. M. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Recent suggest that G protein-coupled receptors internalization to activate MAP kinase, via (4Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar, 5Luttrell L.M. Daaka Y. Della Rocca G.J. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31648-31656Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). To assess whether receptor internalization is required for MAP kinase activation by we the differential internalization profiles of the α2A-AR and α2B-AR subtypes and ability to activate MAP kinase in transfectants of HEK 293 cell in 2, the of MAP kinase activation by these two receptor subtypes is measured by the of dually phosphorylated 1 and by both subtypes was to not indicating that α2A-AR and α2B-AR are MAP kinase via a Gi-coupled as observed previously J. G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). We are that the of MAP kinase by in is to activation of the expressed α2A-AR or α2B-AR in the HEK 293 as activation of MAP kinase is also by the α2-AR agonist and is by the and not Furthermore, HEK 293 cells did not activation by prior to introduction of the the of some clonal HEK 293 cell lines reported in the (4Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar), we no for of MAP kinase in HEK 293 as MAP kinase is not activated by and the to was not by the antagonist, The that both α2A-AR and α2B-AR subtypes stimulate MAP kinase that receptor is for the α2B-AR but not for α2A-AR is not required for MAP kinase activation. with is the that MAP kinase activation at a time agonist-elicited receptor from the surface, for is To assess directly the of internalization on MAP kinase activation, we four experimental previously to block internalization by as follows: sucrose K. B. J. Biol. PubMed Scopus Google Scholar), with concanavalin A J. Biol. Chem. Full Text PDF PubMed Google Scholar), to K+ medium K. B. J. Biol. PubMed Scopus Google Scholar), and with dominant negative (K44A) dynamin E. L. J. Biol. PubMed Scopus Google Scholar, J. Biol. PubMed Scopus Google Scholar). sucrose a in HEK 293 cell as it MAP kinase activation to without agonist addition and to a in HEK 293 cells as in transfectants expressing α2-AR A concanavalin A, is to internalization of by surface to J. Biol. Chem. Full Text PDF PubMed Google Scholar). concanavalin A was in blocking α2B-AR via the redistribution of biotinylated α2B-AR into a compartment not of of α2B-AR internalization a of the of the α2B-AR subtype K. S. L.E. Mol. Pharmacol. Google Scholar). the of concanavalin A as a tool for the of internalization of subtype in of MAP kinase. A experimental of dominant negative dynamin was as a tool to block agonist-elicited internalization. as in the biotinylation of dynamin by did not eliminate α2B-AR suggesting that these receptors internalized via a 3 endocytosis of G protein-coupled receptors also noted for and receptor subtypes von Zastrow M. J. Biol. 1999; PubMed Scopus Google Scholar, C. C.J. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). of dynamin with the α2B-AR did not alter MAP kinase activation by the α2B-AR 3 A experimental that receptor internalization via in K+-depleted was in the between receptor internalization and MAP kinase activation. in A, to K+-depleted medium biotinylated surface α2B-AR redistribution into a with of α2B-AR internalization. as in 4 α2B-AR readily MAP kinase in to the same that internalization is not required for the α2B-AR to stimulate MAP kinase. the of MAP kinase activation to of α2B-AR internalization by incubation of cells in K+-depleted is with internalization as a of of MAP kinase The studies two lines of that α2-ARs do not internalization to MAP kinase activation, in to recent reports internalization as a prerequisite for the MAP kinase cascade by the α2A-AR and α2B-AR subtypes both MAP kinase activation the of α2B-AR in the surface 1 and in an compartment 1 incubation of HEK 293 cells expressing the α2B-AR subtype in K+-depleted medium receptor internalization 4 but does not eliminate MAP kinase activation 4 The for the between and previously reported for other G protein-coupled receptors is not The is that different different to stimulate MAP kinase. it is also to that from studies at in on expression of receptors (4Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (465) Google or on expression of dominant negative of dynamin or to block internalization of or receptors (4Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). Our suggest that agonist-elicited redistribution of the α2B-AR does not and dynamin does not a for the of endocytosis in MAP kinase activation. different by different for internalization is not a of on dynamin for agonist-elicited endocytosis also observed for and receptors von Zastrow M. J. Biol. 1999; PubMed Scopus Google Scholar, C. C.J. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). in studies the of dynamin on MAP kinase activation, expression of dynamin altered events in addition to in receptor with J. Caron M.G. Ferguson S.S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google or in of from the surface J. Biol. PubMed Scopus Google Scholar), and it is these events that a in MAP kinase activation by but not Our that G protein-coupled receptor internalization is not a general prerequisite for activation of the MAP kinase cascade via Gi-coupled receptors previously with receptors Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). Our also suggest that MAP kinase activation by activation of MAP kinase by the α2A-AR that by the α2B-AR and incubation in K+-depleted medium also activation of the 3 studies whether different events the of MAP kinase signaling and other as of activation of or of We for the and of stably HEK 293 cell lines and all of the for