Endothelin A and Endothelin B Receptors Differ in Their Ability to Stimulate ERK1/2 Activation

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 (ET_A) and the endothelin B (ET_B) receptor (2, 3). The ET_A receptor is mainly expressed in vascular smooth muscle cells, and its stimulation induces a long-lasting vasoconstriction (4). The ET_B 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 ET_A receptor activates G proteins of the G_q/11 and G_12/13, the ET_B receptor stimulates G proteins of the G_i and G_q/11 families (6–8). In addition, the ET_B receptor displays several unique features that are not shared by the ET_A receptor. For example, only the ET_B receptor undergoes an agonist-dependent proteolysis of its N terminus. Although both endothelin receptor subtypes possess a cleavable signal peptide (ET_A receptor: 16 aa, ET_B receptor: 26 aa) that is removed by a signal peptidase in the endoplasmic reticulum lumen during receptor biosynthesis (9–11), only the ET_B 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, ET_A and ET_B 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 ET_A and ET_B receptors differ in their ability to activate ERK1/2. In addition, we studied whether N-terminal proteolysis of the ET_B receptor is involved in ERK1/2 activation.

Materials and Methods

Results and Discussion

To analyze whether ET_A and ET_B receptors differ in their ability to activate ERK1/2, we studied HEK293 cells stably expressing the full-length ET_B·GFP receptor or the ET_A·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 ET_A·GFP or ET_B·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 ET_A·GFP showed a monophasic, but sustained increase in ERK1/2 phosphorylation (Fig. 1A). For lysates derived from cells expressing the ET_B·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 ET_B receptor, we also analyzed ERK1/2 phosphorylation in HEK293 cells stably expressing the Δ2-64 ET_B·GFP receptor. ET-1 stimulation of Δ2-64 ET_B·GFP did not show biphasic, but only monophasic ERK1/2 activation (Fig. 1C).

Because only the full-length ET_B receptor induced biphasic ERK1/2 activation, the presence of the N-terminal fragment could provide a key for switching from biphasic to monophasic ERK1/2 activation. To elucidate, whether the proteolytically released N-terminal peptide functions as a soluble ligand or coactivator in ET-1–induced ERK1/2 activation, HEK293 cell clones expressing the Δ2-64 ET_B·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 ET_B receptor, with the exception that it lacks an N-linked glycosylation at asparagine 59. Treatment of HEK Δ2-64 ET_B·GFP cell clones with the synthetic NT27-64 peptide and ET-1 did not restore the biphasic activation of ERK1/2 (Fig. 2A).

To study whether the length of the ET_B receptor or a certain amino acid composition within the ET_B receptor’s N terminus is required for biphasic ERK1/2 activation, we analyzed a chimeric ET_B receptor in which the extracellular N terminus was replaced by that of the ET_A receptor (BNA·GFP) and also a chimeric ET_A receptor in which the N terminus was replaced by that of the ET_B receptor (ANB·GFP). The BNA·GFP chimera induced a biphasic ERK1/2 phosphorylation on ET-1 stimulation (Fig. 2B), similar to that seen for the wild-type ET_B receptor, and the ANB·GFP chimera elicited a monophasic, but sustained ERK1/2 activation as observed for the wild-type ET_A receptor (Fig. 2C). The data suggest that the intact, extracellular N terminus of the ET_B receptor is involved in biphasic ERK1/2 activation. Curiously, replacement of the extracellular N terminus of the ET_B receptor by the N terminus of the ET_A receptor did not alter biphasic ERK1/2 activation. This is notable because the extracellular N termini of the ET_A and ET_B receptors display only a little amino acid sequence identity.

The data demonstrate that ET_A and ET_B receptors differ considerably in their ability to stimulate ERK1/2 activation. Whereas the ET_A receptor shows a monophasic, but sustained ERK1/2 activation, the ET_B receptor elicits a biphasic ERK1/2 activation. Although an intact N terminus of the ET_B receptor is necessary for biphasic ERK1/2 activation, the extracellular ET_B 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 ET_A receptor. Regarding the low level of amino acid identity between the N termini of the ET_A and the ET_B 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 ET_B 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 ET_A and ET_B 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).

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Figure 2.

The second phase of ERK1/2 activation requires the presence of glycosylated endothelin receptors N terminus. HEK293 cell clones expressing the N-terminally truncated mutant Δ2-64 ET_B·GFP (A) were incubated with a synthetic, nonglycosylated N-terminal peptide (NT27-64, 100 μM) for 30 mins before the stimulation of cells with ET-1 for up to 180 mins. HEK293 cells stably expressing a chimeric ET_B receptor, in which the N-terminus was replaced by that of the ET_A receptor (BNA·GFP) (B) or a chimeric ET_A receptor, in which the N terminus was replaced by that of the ET_B receptor (ANB·GFP) (C) were stimulated with 100 nM ET- 1 for the indicated times. Lysates of cells were processed for immunoblotting as described in the legend to Figure 1.