©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Adrenergic Receptor Subtypes Expressed in Chinese Hamster Ovary Cells Activate Differentially Mitogen-activated Protein Kinase by a p21 Independent Pathway (*)

(Received for publication, October 21, 1994; and in revised form, December 27, 1994)

Christos S. Flordellis (1)(§) Marie Berguerand (1)(¶) Patricia Gouache (1) Véronique Barbu (2) Haralambos Gavras (3) Diane E. Handy (3) Gilbert Béréziat (1) Joëlle Masliah (1)

From the  (1)URA CNRS 1283 and (2)Laboratoire de Biochimie, CHU Saint-Antoine, 75571 Paris Cedex 12, France and (3)Section of Hypertension and Atherosclerosis, Boston University School of Medicine, Boston, Massachusetts 02118

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Epinephrine stimulation of rat alpha, alpha, and alpha adrenergic receptor subtypes, expressed stably in Chinese hamster ovary (CHO) cells, caused a rapid, transient activation of mitogen-activated protein kinase (MAPK), with subtype-specific different efficiencies. The order of activation was CHO-2B CHO-2D CHO-2C. Pertussis toxin blocked the stimulation of MAPK enzymatic activity and the parallel MAPK phosphorylation, demonstrating that these responses are mediated by pertussis toxin-sensitive G(i) proteins.

Contrary to what has been reported for the alpha subtype expressed in rat-1 fibroblasts, epinephrine did not cause any detectable activation of p21 in the CHO transfectants.

Furthermore, combined application of epinephrine and phorbol myristate acetate had a potent cooperative but not additive effect in clones CHO-2D and CHO-2B but not in CHO-2C, suggesting that protein kinase C is probably differently involved in the signaling by the three alpha(2) receptor subtypes.

These results show that in CHO cells, the different alpha(2) adrenergic receptor subtypes utilize differential pathways to activate MAPK in a p21-independent way.


INTRODUCTION

At least three distinct alpha(2) adrenergic receptor subtypes, alpha, alpha, and alpha, mediate the cellular effects of epinephrine and norepinephrine in the rat(1, 2, 3, 4) . They are expressed in a tissue-specific manner and differ in their pharmacological, biochemical-molecular biological, and probably signal transduction properties(5, 6, 7, 8) .

alpha(2) adrenergic receptors mediate the action of epinephrine by regulating a variety of effectors including among others inhibition/stimulation of adenylyl cyclase through pertussis toxin-sensitive G proteins (9, 10) and stimulation of cytosolic phospholipase A(2)(11) . However, the function(s) and the physiological differences of the subtypes are virtually unknown, in part because their differential coupling to different intracellular signaling mechanisms has not been characterized. Studies on the coupling of alpha(2) adrenergic subtypes to adenylyl cyclase have shown that the rat alpha(2) subtypes couple differently to adenylyl cyclase in certain cells (12) and point to the possibility of further differences and increased plasticity in the coupling of subtypes to other effectors, including mitogen-activated protein kinase (MAPK). (^1)

MAPK, also known as extracellular signal-regulated kinase, is a central component of an evolutionarily conserved kinase cascade, which integrates various extracellular signals converging to it(13) . The cascade consists of three consecutive kinases, which relay and at the same time switch tyrosine phosphorylation signals, initiated from cell surface receptors, to serine/threonine phosphorylation of downstream serine/threonine protein kinases and other effectors that regulate mitogenesis, differentiation, and cellular metabolism.

Thus, upon activation, p74, a serine/threonine kinase, acts as MAPK kinase kinase (MEKK) phosphorylating and activating MAPK kinase, a dual specificity protein kinase, that activates MAPK by dual phosphorylation at closely spaced residues Thr-183 and Tyr-185. A parallel p74-independent pathway, including MEKK (instead of p74) that can phosphorylate and activate MAPK kinase, is thought to operate in the transmission of signals initiated by certain G protein receptors(14) . Although in most systems different isoforms of MAPK, mainly p42 and p44 MAPK, are coactivated by a variety of stimuli, p42 MAPK is selectively activated independently of p44 MAPK in some cells(15) . The activated MAPK activates, by phosphorylating consensus sites, downstream effectors, including p90 and phospholipase A(2) in the cytosol and transcription factors (c-jun, c-myc, c-fos, p62) in the nucleus, where MAPK translocates when activated (16) .

Ligand activation of tyrosine kinase receptors (like the EGF receptor) stimulates MAPK via a pathway that involves accumulation of active GTP-bound p21. However, EGF activates also, in rat-1 and NIH 3T3 fibroblasts, a Ca-dependent signaling pathway capable of phosphorylating p42 MAPK in a p21-independent way(17) . In a similar way some G protein-coupled receptors (like the endogenous thrombin receptor and the lysophosphatidic acid receptor in hamster lung fibroblasts and rat-1 cells) use activated p21 in signaling to MAPK(18, 19) . Yet other G protein-linked receptors (like the platelet-activating factor receptor in CHO cells) utilize a p21-independent pathway(20) .

Recently it has been reported that the agonist-stimulated alpha adrenergic receptor activates MAP kinase activity in rat-1 fibroblasts through the activation of the p21 pathway (21) . However, it is not known if the activation of MAPK by alpha(2) adrenergic receptor stimulation is subtype-specific and, furthermore, if there are cell type-related differences in the signaling pathway employed by alpha(2) subtypes.

To characterize potential differences of the biochemical signals induced upon stimulation of cloned alpha(2) adrenergic receptor subtypes, we have expressed the rat alpha, alpha, and alpha subtypes in CHO cells and studied their coupling to MAPK.

We have found that in CHO cells alpha(2) adrenergic receptor subtypes couple to this effector with different efficacy and that they transmit their signal to MAPK activation through a p21-independent pathway probably involving different protein kinase C.


EXPERIMENTAL PROCEDURES

Materials

Pertussis toxin was from Seikagaku (Japan). Anti-p21 Y13-259 and Y13-238 monoclonal antibodies were purchased from Oncogene Science. Anti-MAPK antibodies were from Zymed (immunoblotting) and Santa Cruz (immunoprecipitation). A rabbit anti-mouse peroxidase-conjugated monoclonal antibody was from Biosys. The MAPK substrate peptide was from Upstate Biotechnology Inc. [^3H]Rauwolscine, [P]orthophosphate (5000 Ci/mmol), and anti-G protein antibodies were from DuPont NEN. [-P]ATP (>5000 Ci/mmol) and ECL (enhanced chemiluminescence) detection system were purchased from Amersham Corp. MEM alpha medium and dialyzed fetal calf serum were from Life Technologies, Inc. Trypsin-EDTA, penicillin, and streptomycin were from Eurobio. Polyethyleneimine cellulose F plates were from Merck and protein A-Sepharose from Pharmacia Biotech.(-)-Epinephrine bitartrate salt and all other chemicals used were from Sigma.

Cell Lines and Culture

The cell lines, which are analyzed in this study, have been established previously (7) by expressing stably in CHO cells rat alpha(2) adrenergic receptor cDNAs coding for alpha, alpha(7, 8) , and alpha subtypes(3) . The rat alpha receptor has distinct ligand binding properties in comparison with the human alpha receptor, but they are 90% homologous (2) (^2)and have been suggested to represent species homologs(2, 7) . cDNAs encoding the rat alpha, alpha, and alpha subtypes were placed under the transcriptional control of adenovirus major late promoter in the expression vector pMT2 and transfected CHO cells. Stable transfectants, CHO-2D, CHO-2B, CHO-2C, were selected and amplified using MEM alpha medium without ribo- and deoxyribonucleosides and amplified (CHO-2D and CHO-2C) using methotrexate as described(22) . CHO transfectants were grown in MEM alpha medium, without ribonucleosides and deoxyribonucleosides, supplemented with 10% dialyzed fetal calf serum, penicillin (100 IU/ml), and streptomycin (100 µg/ml). For the experiments, cells in 60-mm dishes were grown to near confluence, serum-starved for 24 h, and subsequently treated under various conditions.

Measurement of cAMP Content

The assay for cAMP accumulation was carried out with cells in suspension as described previously (10) (^3), using a cAMP Kit (Amersham Corp.).

Ligand Binding

Membranes were prepared from cultured transfectants and ligand binding assays were carried out, with appropriate amounts of membrane protein, basically as described(23) . The K(D) values of clones CHO-2D and CHO-2C were found to be 21.3 and 0.12 nM, respectively. The B(max) value of the CHO-2D was 9.8 ± 1.16 pmol/mg of protein, of the CHO-2B was 3.0 pmol/mg of protein, and of the CHO-2C was 4.8 ± 0.68 pmol/mg of protein.

MAPK Activity

Confluent non-transfected and transfected CHO cells were incubated for 24 h in serum-free medium in 60-mm dishes. After stimulation with epinephrine, under different conditions, the cells were washed three times with 10 ml of ice-cold phosphate-buffered saline, scraped into 1 ml of 25 mM Hepes buffer pH 7.4 (containing 5 mM EDTA, 50 mM NaF, 100 µM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin), and homogenized by passing repeatedly through a 25-gauge needle. Insoluble material was eliminated by centrifugation of the lysate (100,000 times g, 20 min at 4 °C). The supernatant, containing the MAPK activity, was aliquoted and stored at -80 °C until use. 200 µg of supernatant proteins were precleared with protein A-Sepharose prior to immunoprecipitation with anti-MAPK antibody and protein A-Sepharose for 2 h at room temperature. The pellets were washed four times with lysis buffer and resuspended in kinase buffer (25 mM Hepes, pH 7.4, 50 mM MgCl(2)). An aliquot was incubated for MAPK activity determined as described elsewhere using 25 µg of a synthetic peptide (APRTPGGRR) corresponding to amino acids 95-98 of bovine myelin basic protein(24, 25) . Control incubations without synthetic peptide or without addition of cell lysate were also performed.

Immunoblotting Analysis

For the mobility shift of MAPK, cell lysates were prepared as in the assay for the MAPK activity, and proteins were separated on 8-15% continuous gradient SDS-PAGE gel (26) followed by immunoblotting.

The blots were incubated with the anti-MAPK antibody and then with peroxidase-conjugated rabbit anti-mouse antibody (1:600). Immunostained proteins were visualized using the ECL detection system.

For the Western blotting analysis of G proteins, membrane proteins were prepared as described(27) . Membrane proteins (50 µg) were separated by SDS-PAGE, electroblotted to nitrocellulose, and the filters were incubated with anti-G protein antibodies (diluted 1:1000) and processed as described above.

Analysis of p21-bound Guanine Nucleotides

Nearly confluent cells in 100-mm dishes were serum-starved for 8 h and then labeled with 125 µCi/ml [P]orthophosphate for an additional 16 h in serum-free phosphate-free medium. After agonist stimulation, incubations were terminated by washing three times 10 ml of ice-cold phosphate-buffered saline, and the cells were lysed in Triton X-114 buffer(25) . Membrane-bound p21 was recovered by detergent phase splitting and immunoprecipitated with monoclonal antibodies Y13-259 and Y13-238 coupled to rabbit anti-rat IgG/protein A-Sepharose complex.

Guanine nucleotides bound to p21 were eluted from the immunoprecipitate and analyzed by ascending thin layer chromatography on a polyethyleneimine cellulose plate as described elsewhere(28) . After autoradiography the spots corresponding to GTP and GDP were scraped off the TLC plates, and the radioactivity was determined by liquid scintillation.


RESULTS AND DISCUSSION

In initial experiments we analyzed the CHO transfectants by ligand binding and determined that they express comparable levels of alpha(2) adrenergic receptors in the range of 3-10 pmol/mg of membrane protein (see ``Experimental Procedures''). This was necessary as receptor density such as in our clones (pmol/mg protein) has been found to permit consistent and comparable analysis of alpha(2) adrenergic receptor-mediated responses(29) .

Measurement of the cAMP in agonist-stimulated CHO clones verified that the expressed subtypes are functionally linked, but in a different way, to adenylyl cyclase in CHO cells. Epinephrine activation of alpha and alpha subtypes inhibited forskolin-stimulated elevation of cAMP, whereas activation of alpha receptor produced a biphasic effect with a maximum inhibition at 100 nM, followed by stimulation at doses of 1-100 µM epinephrine (results not shown).

Activation and Phosphorylation of MAPK

Cells were stimulated with concentrations of epinephrine varying between 100 nM and 100 µM. Maximum activation of MAPK was observed at 10 µM epinephrine for clones CHO-2D and CHO-2C, whereas for clone CHO-2B the maximum effect was observed at 100 µM epinephrine. At a lower dose (10M) epinephrine was less effective. The dose of epinephrine (10 µM) that induces maximal activation of MAPK in CHO-2D has a very weak inhibitory effect on the accumulation of intracellular cAMP level. This indicates that there is probably no correlation between inhibition of cAMP and stimulation of MAPK in CHO cells (data not shown).

Kinetic analysis of MAPK activation in response to epinephrine (10 µM) showed almost identical time courses in the three clones with a rapid, transient increase in the MAPK activity. MAPK increase was measurable at 1 min after stimulation, reached a maximum at 5 min, and decreased rapidly thereafter, with continued epinephrine treatment, so that at 30 min MAP kinase activity was to nearly prestimulated levels. The time course of the activation was paralleled by an identical time course in the phosphorylation and subsequent mobility shift of p42 MAP kinase (results not shown). This uniform time course of activation of MAPK by the alpha(2) subtypes is similar to what has been found with other G-protein-coupled receptors (alpha-thrombin, lysophosphatidic acid, platelet-activating factor receptors) (18, 19, 20) in a variety of cells, as well as receptor tyrosine kinases in certain cell types (like EGF receptor in PC12 cells)(30) .

As shown in Fig. 1, epinephrine applied at 10 µM for 5 min activates MAPK in all three clones but with substantially different efficiencies. The order of activation is CHO-2B CHO-2D CHO-2C. No activation was observed in non-transfected CHO cells. The activation of MAPK by epinephrine is 80% of the effect of serum on MAPK activity in clone CHO-2D, 75% in clone CHO-2B, and 25% in CHO-2C. Yohimbine (10 µM) caused partial inhibition of activation of MAP kinase, whereas pretreatment of cells with pertussis toxin (100 ng/ml) for 4 h inhibited the epinephrine-induced activation completely in CHO-2C and almost completely in CHO-2D and CHO-2B cells (85 and 67% inhibition, respectively), without affecting basal MAPK activity (not shown). This proves that the activating effect of epinephrine through alpha(2) adrenergic receptor subtypes is mediated by pertussis toxin-sensitive G(i) proteins. Pertussis toxin had only a moderate inhibitory effect on the serum activation of MAP kinase, an expected result of many of the growth factors present in the serum signal to MAP kinase through tyrosine kinase receptors.


Figure 1: Analysis of MAP kinase activation. Cultures of parental CHO cells and transfectants, expressing the alpha (CHO-2D), alpha (CHO-2B), and alpha (CHO-2C) adrenergic receptor subtypes, were serum-deprived for 24 h. Then the cells were treated for 5 min with epinephrine (Epi, 10 µM) in the absence or presence of yohimbine (Yoh, 10 µM) or pertussis toxin (PTX, 100 ng/ml), or alternatively they were treated for 5 min with dialyzed fetal calf serum (FCS, 10%) in the absence or presence of pertussis toxin (100 ng/ml). Cell extracts were tested for MAPK activity as described under ``Experimental Procedures.'' The values of MAPK activity obtained under different conditions were normalized with respect to control cells, that is cells non-stimulated with any agonist. The results shown are the average of duplicate determinations from two separate experiments.



The changes in enzymatic activity of MAP kinase were paralleled by modifications of MAP kinase phosphorylation. Fig. 2shows that epinephrine induced a different degree of shift in the mobility of p42 MAP kinase in the different clones: minor shift in CHO-2C, moderate in CHO-2D, and pronounced in CHO-2B cells. In this last clone specifically, retarded migration of an additional electrophoretic zone can be observed, most likely representing the 44-kDa (p44) form of MAP kinase. The slower migrating forms of p42/p44 on anti-MAPK immunoblots represent the threonine and tyrosine phosphorylated and activated form of MAPK(31) . Incubation of the same filter with anti-phosphotyrosine antibody showed that the shifted electrophoretic bands are tyrosine-phosphorylated (not shown). Consistent with the enzymatic activity of MAPK, the retardation in the electrophoretic mobility of p42/p44 MAPK is prevented partially by yohimbine and completely by pertussis toxin treatment.


Figure 2: Phosphorylation of MAP kinase in CHO transfectants. Cell extracts were prepared from cells treated under the conditions described in the legend of Fig. 1, and proteins were resolved by SDS-PAGE followed by immunoblotting with the anti-p42 MAPK antibody (see ``Experimental Procedures''). The position of the inactive unphosphorylated MAPK is indicated by p42, whereas the phosphorylated activated form of p42 MAPK displays reduced electrophoretic mobility and is indicated by pp42. Abbreviations are the same as listed in the legend to Fig. 1.



The substantial quantitative differences in the MAPK activation observed among the CHO clones in this study cannot be attributed to differences in receptor number, since the three CHO clones express comparable levels of receptor protein. They also express similar levels of G(i) protein, as shown by immunoblot analysis of G subunits (Fig. 3). In this context it should be noted that, in the course of these experiments, while preparing the cells by serum deprivation for the MAP kinase assays, we have observed a reproducible induction of the levels of G(i) proteins. The immunoblots of Fig. 3show clearly the induction of both G and G proteins (the G(i) isoforms expressed in CHO cells) (32) when parental or transfected CHO cells were serum-depleted for 24 h.


Figure 3: Immunoblot analysis of Galphai proteins. Parental non-transfected CHO cells and transfectants were grown in medium supplemented with 10% fetal calf serum. Membrane proteins were prepared from the cells under these conditions (+) or after serum deprivation for 24 hours(-). The proteins were separated by SDS-PAGE, transferred onto nitrocellulose membrane, and revealed by antibody AS7 recognizing the Galphai(1) and Galphai(2) subunits and antibody EC2 specific for the Galphai(3) and Galpha(0) subunits.



The functional significance of these changes in conditioning the signal transduction apparatus is currently under investigation. We report these preliminary findings here in order to show that the G(i) levels change under serum deprivation in the same direction (increase) in all clones studied. We reason therefore that the found differences in MAPK activation rather reflect different coupling efficiency of different subtypes to the same signaling pathway to MAPK or reflect the employment by the subtypes of different signaling pathways regulating MAPK activation such as p21 activation, protein kinase C activation, or Ca mobilization(17) .

p21 Stimulation in CHO Transfectants

Agonist stimulation of alpha subtype has been reported to activate rapidly p21 in rat-1 fibroblasts(21) . In order to test if the same happens in CHO cells we determined the activation of p21 of clones CHO-2D and CHO-2B, in which effective activation of MAPK was found, by measuring the GTP-bound form of p21 after epinephrine stimulation. It can be seen in the thin layer chromatogram of Fig. 4that epinephrine at 10 µM, a concentration that induced maximal activation of MAPK, did not cause any detectable increase of GTP-bound p21 in these cells 5 min after stimulation. CHO cells transfected with insulin receptor were used as a positive control of p21 activation(33) ; 1 µM insulin produced a 2-fold increase of GTP bound to p21 (Fig. 4) 5 min after stimulation. This result shows that in CHO cells the alpha(2) adrenergic receptors activate MAPK through a p21-independent pathway.


Figure 4: Activity of p21 in parental CHO cells and CHO cells expressing alpha(2) adrenergic receptor subtypes. Confluent P-labeled CHO-R cells were stimulated with 1 µM insulin for 5 min. P-Labeled CHO-2D and CHO-2B were stimulated with 10 µM epinephrine (Epi) for 5 min. Cell lysates were immunoprecipitated with anti-p21 monoclonal antibodies. Guanine nucleotides bound to p21 were eluted from the immunoprecipitates and separated by thin layer chromatography as described under ``Experimental Procedures.'' The positions of origin (Ori) and GTP and GDP standards are indicated. The percentage of GTP bound to p21 is calculated from the ratio of GTP/GDP + GTP. In CHO-R, the percent of GTP bound to p21was stimulated by insulin to 185% of the control value.



p21-independent activation of MAPK is not without precedent. It has been described among others for platelet-activating factor receptor in CHO cells(20) .

However, the finding of stimulation of MAPK activity by all alpha(2) adrenergic receptor subtypes without activation of p21 is to our knowledge the first concerning the alpha(2) receptor. It is in contrast to the p21-mediated activation of MAPK by the human alpha and the m(2) muscarinic receptor subtype in rat-1 cells (21, 34) or by the fluoroaluminate AlF(4) in PC 12 cells(35) , and it shows that activated p21, like other signaling effectors, can be employed by the same receptors in an alternative way depending on the cell type(36) .

Alternatively the inability to detect p21 activation by epinephrine in our study may mean that a very slight, undetectable increase in the GTP-p21 is sufficient for the activation of MAP kinase by alpha(2) adrenergic subtypes in CHO cells.

We examined the possibility of protein kinase C as a signaling pathway in the activation of p42 MAPK by epinephrine. Fig. 5demonstrates that stimulation of protein kinase C by PMA activated MAPK in the three clones. Furthermore, combined application of epinephrine (10 µM) and PMA had a potent cooperative, but not additive, effect in clones CHO-2D and CHO-2B but not in CHO-2C. These results suggest that protein kinase C is probably involved in a different fashion in the signaling by the three alpha(2) adrenergic receptor subtypes.


Figure 5: Effect of PMA pretreatment on epinephrine-induced activation of MAPK. MAPK activity was determined as described in Fig. 1in cells treated with 10 µM epinephrine (Epi) for 5 min, 100 nM PMA for 10 min, or a combination of 10 µM epinephrine and 100 nM PMA for 10 min.



In conclusion, our results show that alpha(2) adrenergic receptor subtypes expressed in CHO cells utilize differently biochemical pathways signaling to MAPK in a p21-independent way.


FOOTNOTES

*
This work was supported in part by the Association de Recherche contre le Cancer and by the Hellenic Central Health Committee. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: URA 1283, CHU Saint-Antoine, 27, rue Chaligny, 75571 Paris Cedex 12, France. Tel.: 16 1 40 01 13 39; Fax: 16 1 40 01 14 95.

Recipient of a fellowship from the Association Française de Lutte contre la Mucoviscidose.

(^1)
The abbreviations used are: MAPK, mitogen-activated protein kinase; CHO, Chinese hamster ovary; PMA, phorbol myristate acetate; EGF, epidermal growth factor; MEM, minimum Eagle's medium; PAGE, polyacrylamide gel electrophoresis.

(^2)
C. S. Flordellis, M. Berguerand, P. Gouache, V. Barbu, H. Gavras, D. E. Handy, G. Béréziat, and J. Masliah, unpublished data.

(^3)
H. Paris, personal communication.


ACKNOWLEDGEMENTS

We are grateful to Dr. Gisèle Cherqui (Unité INSERM 402) for providing CHO-R cells and for helpful discussion. We also thank Dr. Bertrand Saunier (Unité INSERM 90) for sharing his experience on MAPK blotting experiments, Dr. François Audubert for discussion on the manuscript, and Isabelle Leniobey for secretarial assistance.


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