| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
,1
* Institut für Pharmakologie, Charité Campus Benjamin Franklin, 14195 Berlin, Germany; Forschungsinstitut für Molekulare Pharmakologie, Campus Berlin-Buch, 13125 Berlin, Germany; and Abteilung für Klinische Pharmakologie, Ruhr-Universität Bochum, 44801 Bochum, Germany
1 To whom requests for reprints should be addressed at Institut für Pharmakologie, Charité Campus Benjamin Franklin, Thielallee 67-73, 14195 Berlin, Germany. E-mail: alexander.oksche{at}charite.de
Abstract
Endothelin-1 (ET-1) acts on two different G protein–coupled receptors, namely the endothelin A (ETA) and the endothelin B (ETB) receptors. Both receptor subtypes show differences in their tissue expression and signal transduction. In the present study, we compared the ability of ETA and ETB receptors to stimulate extracellular signal-regulated kinase 1/2 (ERK1/2). In addition, we analyzed the role of the extracellular N terminus for ERK1/2 activation, because the ETB receptor undergoes an agonist-dependent N-terminal proteolysis. ET-1 stimulation of HEK293 cells stably expressing the ETA receptor induced a monophasic, but sustained ERK1/2 activation, whereas the ETB receptor showed a biphasic ERK1/2 activation. A truncated mutant ETB receptor, lacking the proteolytically cleaved N terminus (
2-64 ETB) revealed only a monophasic and transient ERK1/2 activation. Treatment of HEK293 2-64 ETB cell clones with ET-1 and a synthetic NT27-64 peptide, corresponding to the N-terminally cleaved fragment of the ETB receptor and ET-1, did not restore the biphasic activation of ERK1/2. A chimeric ETB receptor in which the N terminus was replaced by the N terminus of the ETA receptor elicited biphasic ERK1/2 activation. The presented data suggest that an intact N terminus of the ETB receptor is necessary for the second phase of ERK1/2 activation. However, it appears that the length of the N terminus rather than a specific sequence motif is required for biphasic ERK1/2 activation.
Key Words: endothelin • glycosylation • G protein-coupled receptor • MAPK
Introduction
Endothelins (ET-1, ET-2, and ET-3) are vasoactive peptides (1), which exert their action via two G protein–coupled receptors: the endothelin A (ETA) and the endothelin B (ETB) receptor (2, 3). The ETA receptor is mainly expressed in vascular smooth muscle cells, and its stimulation induces a long-lasting vasoconstriction (4). The ETB receptor is predominantly expressed in endothelial cells, and its activation causes a transient vasodilation (5). Although both endothelin receptors share 64% amino acid sequence identity, marked differences in the activation of G proteins exist. Whereas the ETA receptor activates G proteins of the Gq/11 and G12/13, the ETB receptor stimulates G proteins of the Gi and Gq/11 families (6–8). In addition, the ETB receptor displays several unique features that are not shared by the ETA receptor. For example, only the ETB receptor undergoes an agonist-dependent proteolysis of its N terminus. Although both endothelin receptor subtypes possess a cleavable signal peptide (ETA receptor: 16 aa, ETB receptor: 26 aa) that is removed by a signal peptidase in the endoplasmic reticulum lumen during receptor biosynthesis (9–11), only the ETB receptor reveals a further proteolytic cleavage within the extracellular N terminus (10). This cleavage occurs 38 amino acids C-terminally of the signal peptidase cleavage site, between arginine 64 and serine 65 (R64/S65) and is mediated by a metalloprotease in an agonist-dependent manner (12). Because the proteolytically released N-terminal peptide fragment harbors the only N-linked glycosylation site (N59/A60/S61), agonist-mediated proteolysis results in a truncated, nonglycosylated receptor. However, the physiologic significance of this process remains elusive.
Both, ETA and ETB receptors lead to an activation of extracellular signal-regulated kinase 1/2 (ERK1/2). Because ERK1/2 is important regulator of cellular proliferation, migration, and differentiation, we analyzed whether ETA and ETB receptors differ in their ability to activate ERK1/2. In addition, we studied whether N-terminal proteolysis of the ETB receptor is involved in ERK1/2 activation.
Materials and Methods
Peptide Synthesis.
Synthesis of ET-1 and of the NT27-64 peptide (corresponds to the N-terminal fragment of the ETB receptor released by a metalloprotease) were carried out by the solid-phase, standard 9-fluorenylmethoxy-carbonyl chemistry method as described recently (13).
Generation of ETB Receptor/GFP Expression Constructs.
The plasmids pcDNA3.ETB, pEGFP.ETB, and p2-64.ETB encoding the human full-length ETB receptor, the human ETB/GFP fusion protein, and a mutant ETB receptor with a truncated extracellular N terminus (corresponding to the N-terminally cleaved ETB receptor), respectively, have been described previously (12, 13). Plasmids encoding a chimeric ETA receptor in which the N terminus is replaced by the N terminus of the ETB receptor (ANB·GFP), and a chimeric ETB receptor in which the N terminus is replaced by that of the ETA receptor (BNA·GFP) were generated by site-directed mutagenesis of the pEGFP.ETB and pEGFP.ETA plasmids (13). A SnaBI (underlined) site in the coding region of both plasmids was introduced using the following primers: for the ETB receptor: 5'-GAGA CTTT CAAA TACG TAAA CACT GTGA TA- 3'; reverse: 5'-TATC ACGT GTTT ACGT ATTT GAAA GTCTC- 3'; for the ETA receptor: 5'-TCAG CTTT CAAA TACG TAAA CACG GTTG TG- 3'; reverse: 5'- CACA ACCG TGTT TACG TACG TATT TGAA AGCT GA- 3'. The resulting plasmids pETA·GFP.SnaBI and pETB·GFP.SnaBI were then subjected to endonuclease treatment with SnaBI. Because within the promoter region of pETA·GFP.SnaBI and pETB·GFP.SnaBI, a second SnaBI site is present, the SnaBI digest results in fragments of 560 base pairs and 5454 base pairs (ETA receptor) and 634 base pairs and 5433 base pairs (ETB receptor). Subsequently, the 560 base-pairs fragment of pETA·GFP.S-na BI was ligated with the 5433 base-pairs fragment of pETB·GFP.SnaBI, and the 5454 base-pairs fragment of pETA·GFP.SnaBI was ligated with the 634 base-pairs fragment of pETB·GFP.SnaBI. The sequences of the resulting plasmids pBNA·GFP and pANB·GFP were verified by DNA sequencing using the Big Dye Terminator kit (Applied Biosystems, Weiterstadt, Germany).
Cell Culture and Transient Transfections.
HEK293 cell clones stably expressing the ETB·GFP, BNA·GFP, ANB·GFP, or the 2-64 ETB·GFP receptor were maintained as described (12). Growth arrest was induced by culturing cells for 48 hrs in a serum-free quiescent medium. The protocol for the transient transfection of HEK293 cells was as described previously (14).
Immunoblots.
Detection of total ERK1/2 and phospho-ERK1/2 in immunoblots was performed as described (12, 15). In brief, HEK293 cells were grown on 35-mm Petri dishes, stimulated with ET-1 (100 nM) for up to 3 hrs and finally lysed in Laemmli buffer. Proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gels, blotted to nitrocellulose filters (Schleicher und Schuell, Dassel, Germany) and probed with affinity-purified polyclonal phospho-ERK1/2 (detects phosphoERK1/2) or ERK1/2 antibodies (detects phosphorylated and non-phosphorylated forms of ERK1/2) (both 1:1000) (Cell Signaling Technology, Danvers, MA).
Results and Discussion
To analyze whether ETA and ETB receptors differ in their ability to activate ERK1/2, we studied HEK293 cells stably expressing the full-length ETB·GFP receptor or the ETA·GFP receptor. HEK293 cells were chosen for these experiments because nontransfected cells lack endogenous expression of endothelin receptors (no specific binding of 125I-ET-1 and no ET-1–induced ERK1/2 activation; data not shown). Cell clones expressing ETA·GFP or ETB·GFP receptors were stimulated with ET-1 for up to 3 hrs, then lysed, and samples were subjected to immunoblot analysis using phosphoERK1/2 and ERK1/2 antibodies. Lysates derived from HEK293 cells expressing ETA·GFP showed a monophasic, but sustained increase in ERK1/2 phosphorylation (Fig. 1A
). For lysates derived from cells expressing the ETB·GFP receptor, a biphasic ERK1/2 activation was found (Fig. 1B). The early phase of ERK1/2 activation reached a peak at 3–5 min and slowly declined thereafter within 30–60 min. The second phase was observed after 80 min of agonist application and persisted for up to 180 min (Fig. 1B). To study the role of the N-terminal proteolysis on ERK1/2 activation of the ETB receptor, we also analyzed ERK1/2 phosphorylation in HEK293 cells stably expressing the 2-64 ETB·GFP receptor. ET-1 stimulation of 2-64 ETB·GFP did not show biphasic, but only monophasic ERK1/2 activation (Fig. 1C).
|
2-64 ETB·GFP receptor were incubated with a synthetic peptide (NT27-64, 100 µM; Fig. 2A). This peptide corresponds to the proteolytically cleaved N-terminal fragment of the ETB receptor, with the exception that it lacks an N-linked glycosylation at asparagine 59. Treatment of HEK 2-64 ETB·GFP cell clones with the synthetic NT27-64 peptide and ET-1 did not restore the biphasic activation of ERK1/2 (Fig. 2A).
|
), similar to that seen for the wild-type ETB receptor, and the ANB·GFP chimera elicited a monophasic, but sustained ERK1/2 activation as observed for the wild-type ETA receptor (Fig. 2C). The data suggest that the intact, extracellular N terminus of the ETB receptor is involved in biphasic ERK1/2 activation. Curiously, replacement of the extracellular N terminus of the ETB receptor by the N terminus of the ETA receptor did not alter biphasic ERK1/2 activation. This is notable because the extracellular N termini of the ETA and ETB receptors display only a little amino acid sequence identity. The data demonstrate that ETA and ETB receptors differ considerably in their ability to stimulate ERK1/2 activation. Whereas the ETA receptor shows a monophasic, but sustained ERK1/2 activation, the ETB receptor elicits a biphasic ERK1/2 activation. Although an intact N terminus of the ETB receptor is necessary for biphasic ERK1/2 activation, the extracellular ETB receptor’s N terminus itself is not sufficient for the second phase of ERK1/2 activation, because it can be replaced by the N terminus of the ETA receptor. Regarding the low level of amino acid identity between the N termini of the ETA and the ETB receptor, it appears that the length of the N terminus rather than a specific sequence motif is required for biphasic ERK1/2 activation. In future experiments, the mechanisms involved in biphasic ERK1/2 activation of the full-length ETB receptor have to be studied in more detail. In addition, further studies are planned to demonstrate whether differences in the pattern of ERK1/2 activation between ETA and ETB receptors also result in different cellular responses, such as proliferation and differentiation. This is highlighted by studies on the pheochromocytoma cell line PC12: whereas treatment of these cells with epidermal growth factor resulted in monophasic ERK1/2 activation and proliferation, incubation of cells with nerve growth factor elicited biphasic ERK1/2 activation and differentiation (16).
Acknowledgments
We thank Dr. Gisela Papsdorf for the generation of HEK293 clones and Monika Bigalke for technical assistance.
Footnotes
Supported by the Deutsche Forschungsgemeinschaft (FG 341), the Sonnenfeld-Stiftung, and the Fonds der Chemischen Industrie.
Received for publication September 30, 2005. Accepted for publication November 17, 2005.
References
)q. Eur J Biochem 268:5449–5459, 2001.[Medline]
mediate differentiation of vascular smooth muscle cells. J Biol Chem 276: 19540–19547, 2001.This article has been cited by other articles:
|
|
 |
|
  F. R. C. Giachini, S. M. Zemse, F. S. Carneiro, V. V. Lima, Z. N. Carneiro, G. E. Callera, A. Ergul, R. C. Webb, and R. C. Tostes Interleukin-10 attenuates vascular responses to endothelin-1 via effects on ERK1/2-dependent pathway Am J Physiol Heart Circ Physiol, February 1, 2009; 296(2): H489 - H496. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
