Subtype-Specific Endothelin-A and Endothelin-B Receptor Desensitization Correlates with Differential Receptor Phosphorylation

Abstract

In the rat cardiovascular system endothelin-1 (ET-1) elicits prolonged physiologic responses mediated by the ETA receptor, whereas the effects mediated by the ETB receptor are transient. The molecular mechanisms for the subtype-specific responses are not yet clear. However, post-translational modifications such as phosphorylation and palmitoylation may play an important role. In Sf9 cells overexpressing the human ETA and ETB receptors, both subtypes are palmitoylated. However, only the ETB but not the ETA receptor is phosphorylated in a ligand-dependent manner. Because phosphorylation is believed to play an important role in ligand-dependent receptor inactivation, we analyzed whether the differential phosphorylation of the ETA and ETB receptors reflects a differential mechanism of receptor inactivation. Using a modified inositol phosphate accumulation assay, we analyzed CHO cells that expressed the ETA or ETB receptor. The ETB receptor was deactivated almost completely within 5 min after agonist stimulation, whereas stimulation of the ETA receptor resulted in sustained activation, i.e., > 90% of the initial activity was maintained after 5 min of ligand stimulation and > 30% after 20 min. A strong correlation was observed between the time course of ETA receptor inactivation and ETA receptor internalization. The endogenous ETA receptor in Rat-1 cells produced a prolonged stimulation of phospholipase C similar to that seen in CHO cells. Therefore, the sustained signaling activity of the ETA receptor is not a property only of recombinant cell lines. Together, our data suggest rapid ETB receptor inactivation due to phosphorylation and delayed ETA receptor inactivation by internalization. These mechanisms adequately reflect the differential response patterns of the ET receptors under physiologic conditions.

Distribution of Endothelin Receptor Subtypes ETA and ETB in the Rat Kidney

“…In the present study we observed an additional band of z60 kDa. This observation is in line with the findings of Cramer et al (1997) who characterized the antiserum in transfected cells expressing the human ET B receptor and in isolated kidney epithelial cells. In this study, bands of z41 and 45 kDa were obtained in cells overexpressing the ET B receptor protein, whereas in preparations of renal epithelial cells an additional band of higher molecular mass was present.…”

Comparison of human ETA and ETB receptor signalling via G-protein and β-arrestin pathways

“…It has been shown that, following agonist activation, the ET B receptor is rapidly desensitised by phosphorylation (Cramer et al, 1998), internalised (Wu-Wong et al, 1995) and targeted to the late endosomal/lysosomal pathway for degradation (Bremnes et al, 2000;Oksche et al, 2000), a mechanism that effectively removes endothelin peptides, for example, from the plasma. Efficacious recruitment of β-arrestin by agonists activating the ET B receptor, as observed in this study, is therefore consistent with the hypothesis that this subtype acts as a clearing receptor (Johnström et al, 2005;Kelland et al, 2010).…”

“…We found that ET-1 was equally potent in the ET A -mediated β-arrestin recruitment ass ay as the ET B . However, whilst the ET A subtype is also internalised following activation, this occurs more slowly than for the ET B receptor (Cramer et al, 1998) and it has been demonstrated that ET-1 remains associated with the receptor for up to 60 minutes after internalisation (Wang et al, 2000). This has been proposed to explain in part the long-lasting vascular responses to ET-1 mediated by the ET A receptor.…”

Muscular ETBReceptors Develop Postnatally and Are Differentially Distributed in Specific Segments of the Rat Vasculature

“…Our findings show that in the rat, ET B receptors on vascular smooth muscle cells develop postnatally in a tissue-specific manner and are present on vascular smooth muscle cells throughout the vascular system in the adult rat. ET B receptors are known to desensitize rapidly after agonist binding (Cramer et al 1998), and repeated agonist exposure leads to a rapid decrease in the vasoconstriction elicited by ET B receptor-selective ligands (Sharifi and Schiffrin 1996). These characteristics make them ideal scavenging receptors, blunting the vasoconstrictor response of locally elevated levels of ET-1.…”