RESUMEN
OBJECTIVE: The extracellular N terminus of the endothelin B (ETB) receptor is cleaved by a metalloprotease in an agonist-dependent manner, but the physiological role of this N-terminal proteolysis is not known. In this study, we aimed to determine the functional role of the ETB receptor and of its N-terminal cleavage in vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: VSMCs expressing either the full-length ETB receptor or an N-terminally truncated ETB receptor (corresponding to the N-terminally cleaved receptor) were analyzed for ligand-induced mitogen-activated protein kinase activation and expression of contractile proteins. In VSMCs expressing the full-length ETB receptor, IRL1620 (an ETB-selective agonist) induced a biphasic extracellular signal-regulated kinase 1/2 (ERK1/2) activation and increased expression of contractile proteins (smooth muscle myosin-1 [SM-1]/SM-2, SM22alpha, and alpha-actin). Interestingly, the second phase of ERK1/2 activation required metalloprotease activity, epidermal growth factor (EGF) receptor transactivation, and predominantly activation of Gi proteins. In contrast, in VSMCs expressing N-terminally truncated ETB receptors, IRL1620 did not elicit EGF transactivation and failed to increase contractile protein expression. CONCLUSIONS: This study is the first to show that stimulation of full-length ETB receptors promotes expression of contractile proteins and may thus participate in the differentiation of VSMCs.
Asunto(s)
Proteínas Contráctiles/metabolismo , Receptores ErbB/genética , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Péptido Hidrolasas/metabolismo , Receptor de Endotelina B/química , Receptor de Endotelina B/metabolismo , Activación Transcripcional , Animales , Células Cultivadas , Endotelinas/farmacología , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/fisiología , Proteínas Fluorescentes Verdes/genética , Humanos , Músculo Liso Vascular/citología , Mutación , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Ratas , Receptor de Endotelina B/agonistas , Receptor de Endotelina B/genética , Proteínas Recombinantes de Fusión/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
Endothelin-1 (ET-1) acts on two different G protein-coupled receptors, namely the endothelin A (ET(A)) and the endothelin B (ET(B)) receptors. Both receptor subtypes show differences in their tissue expression and signal transduction. In the present study, we compared the ability of ET(A) and ET(B) 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 ET(B) receptor undergoes an agonist-dependent N-terminal proteolysis. ET-1 stimulation of HEK293 cells stably expressing the ET(A) receptor induced a monophasic, but sustained ERK1/2 activation, whereas the ET(B) receptor showed a biphasic ERK1/2 activation. A truncated mutant ET(B) receptor, lacking the proteolytically cleaved N terminus (delta2-64 ET(B)) revealed only a monophasic and transient ERK1/2 activation. Treatment of HEK293 delta2-64 ET(B) cell clones with ET-1 and a synthetic NT27-64 peptide, corresponding to the N-terminally cleaved fragment of the ET(B) receptor and ET-1, did not restore the biphasic activation of ERK1/2. A chimeric ET(B) receptor in which the N terminus was replaced by the N terminus of the ET(A) receptor elicited biphasic ERK1/2 activation. The presented data suggest that an intact N terminus of the ET(B) 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.
Asunto(s)
Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/química , Receptor de Endotelina B/metabolismo , Línea Celular , Endotelina-1/farmacología , Activación Enzimática/efectos de los fármacos , Glicosilación , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Transducción de Señal/efectos de los fármacos , TransfecciónRESUMEN
The extracellular N terminus of the endothelin B (ET(B)) receptor is susceptible to limited proteolysis (cleavage at R64 downward arrow S65), but the regulation and the functional consequences of the proteolysis remain elusive. We analyzed the ET(B) receptor or an ET(B)-GFP fusion protein stably or transiently expressed in HEK293 cells. After incubation of cells at 4 degrees C, only the full-length ET(B) receptor was detected at the cell surface. However, when cells were incubated at 37 degrees C, N-terminal cleavage was observed, provided endothelin 1 was present during the incubation. Cleavage was not inhibited by internalization inhibitors (sucrose, phenylarsine oxide). However, in cells incubated with both internalization inhibitors and metalloprotease inhibitors (batimastat, inhibitor of TNFalpha-convertase) or metal chelators (EDTA, phenanthroline), the cleavage was blocked, indicating that metalloproteases cleave the agonist-occupied ET(B) receptor at the cell surface. Functional analysis of a mutant ET(B) receptor lacking the first 64 amino acids ([Delta2-64]ET(B) receptor) revealed normal functional properties, but a 15-fold reduced cell surface expression. The results suggest a role of the N-terminal proteolysis in the regulation of cell surface expression of the ET(B) receptor. This is the first example of a multispanning membrane protein, which is cleaved by a metalloprotease, but retains its functional activity and overall structure.