Search

Endothelin type A receptor (ET A R) is internalized upon agonist stimulation; however, the mechanism thereof remains controversial. In this study, we characterized the endothelin-1 (ET-1)-induced internalization of ET A R expressed in Chinese hamster ovary cells. ET-1 elicited ET A R internalization and increase in intracellular Ca 2+ concentration. ET-1-induced ET A R internalization was completely inhibited by a reduction in intracellular and extracellular Ca 2+ levels and partially suppressed by inhibitors of protein kinase C (PKC) and extracellular signal-regulated kinases 1/2 (ERK1/2), both of which are downstream molecules in ET A R signaling. These results suggest that Ca 2+ mobilization, PKC, and ERK1/2 are involved in ET-1-induced ET A R internalization., (Copyright © 2019 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Endothelin (ET)-1 is involved in the vascular system, cell proliferation and apoptosis. ET receptors consist of ET type A receptor (ET A R) and ET type B receptor (ET B R). ET A R and ET B R generally exhibit opposite responses, although many exceptions exist. In the present study, we attempted to identify ET A R- or ET B R-specific binding proteins to understand the differences in ET A R- and ET B R-mediated responses after ET-1 stimulation. The 78-kDa glucose-regulated protein (GRP78) showed a stronger binding affinity towards ET B R than towards ET A R. Moreover, GRP78 overexpression promoted ET B R-mediated ERK activation and GRP78 silencing suppressed ET B R-mediated ERK activation. Furthermore, ET B R can localize GRP78 to the cell periphery. These results suggest that the interaction of ET B R with GRP78 affects ERK activation and GRP78 localization., (© 2019 Federation of European Biochemical Societies.)

Two types of G protein-coupled receptors for endothelin-1 (ET-1), ET type A receptor (ETAR) and ETBR, closely resemble each other, but upon ET-1 stimulation, they follow totally different intracellular trafficking pathways; ETAR is recycled back to plasma membrane, whereas ETBR is targeted to lysosome for degradation. However, the mechanisms for such different fates are unknown. Here we demonstrated that ETBR but not ETAR was ubiquitinated on the cell surface following ET-1 stimulation and that ETBR was internalized and degraded in lysosome more rapidly than ETAR. The mutant ETBR (designated "5KR mutant") in which 5 lysine residues in the C-tail were substituted to arginine was not ubiquitinated, and its rates of internalization and degradation after ET-1 stimulation became slower, being comparable with those of ETAR. Confocal microscopic study showed that following ET-1 stimulation, ETAR and 5KR mutant of ETBR were co-localized mainly with Rab11, a marker of recycling endosome, whereas ETBR was co-localized with Rab7, a marker of late endosome/lysosome. In the 5KR mutant, ET-1-induced ERK phosphorylation and an increase in the intracellular Ca(2+) concentration upon repetitive ET-1 stimulation were larger. A series of ETBR mutants (designated "4KR mutant"), in which either one of 5 arginine residues of the 5KR mutant was reverted to lysine, were normally ubiquitinated, internalized, and degraded, with ERK phosphorylation being normalized. These results demonstrate that agonist-induced ubiquitination at either lysine residue in the C-tail of ETBR but not ETAR switches intracellular trafficking from recycling to plasma membrane to targeting to lysosome, causing decreases in the cell surface level of ETBR and intracellular signaling., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The endothelin (ET) system consists of two G protein coupled-receptors (GPCRs), ET type A receptor (ETAR) and ET type B receptor (ETBR), and three endogenous ligands, ET-1, ET-2, and ET-3. Stimulation of ETRs with ET-1 induces an increase in intracellular Ca(2+) concentration that is involved in a diverse array of physiological and pathophysiological processes, including vasoconstriction, and cell proliferation. Store-operated Ca(2+) entry and receptor-operated Ca(2+) entry triggered by activation of ETRs are regulated or modulated by endoplasmic reticulum Ca(2+) sensor (stromal interaction molecule 1) and voltage-independent cation channels (transient receptor potential canonical channels and Orai1). The ET-1-induced Ca(2+) mobilization results from activation of heterotrimeric G proteins by ETRs. In contrast, GPCR biology including modulation of receptor function and trafficking is regulated by a variety of GPCR interacting proteins (GIPs) that generally interact with the C-terminal domain of GPCRs. The ETR signaling is also regulated by GIPs such as Jun activation domain-binding protein 1. This review focuses on the regulatory mechanisms of the ETR signaling with special attention to the components involved in Ca(2+) signaling and to GIPs in the signal transduction, modification, and degradation of ETRs.

Receptor-operated Ca²⁺ entry (ROCE) via transient receptor potential canonical channel 6 (TRPC6) is important machinery for an increase in intracellular Ca²⁺ concentration triggered by the activation of G(q) protein-coupled receptors. TRPC6 is phosphorylated by various protein kinases including protein kinase A (PKA). However, the regulation of TRPC6 activity by PKA is still controversial. The purpose of this study was to elucidate the role of adenylate cyclase/cAMP/PKA signaling pathway in the regulation of G(q) protein-coupled endothelin type A receptor (ET(A)R)-mediated ROCE via TRPC6. For this purpose, human embryonic kidney 293 (HEK293) cells stably coexpressing human ET(A)R and TRPC6 (wild type) or its mutants possessing a single point mutation of putative phosphorylation sites for PKA were used to analyze ROCE and amino acids responsible for PKA-mediated phosphorylation of TRPC6. Ca²⁺ measurements with thapsigargin-induced Ca²⁺-depletion/Ca²⁺-restoration protocol to estimate ROCE showed that the stimulation of ET(A)R induced marked ROCE in HEK293 cells expressing TRPC6 compared with control cells. The ROCE was inhibited by forskolin and papaverine to activate the cAMP/PKA pathway, whereas it was potentiated by Rp-8-bromoadenosine-cAMP sodium salt, a PKA inhibitor. The inhibitory effects of forskolin and papaverine were partially cancelled by replacing Ser28 (TRPC6(S28A)) but not Thr69 (TRPC6(T69A)) of TRPC6 with alanine. In vitro kinase assay with Phos-tag biotin to determine the phosphorylation level of TRPC6 revealed that wild-type and mutant (TRPC6(S28A) and TRPC6(T69A)) TRPC6 proteins were phosphorylated by PKA, but the phosphorylation level of these mutants was lower (approximately 50%) than that of wild type. These results suggest that TRPC6 is negatively regulated by the PKA-mediated phosphorylation of Ser28 but not Thr69.

The purpose of this study is to identify transient receptor potential canonical (TRPC) channels responsible for receptor-operated Ca(2+) entry (ROCE) triggered by activation of endothelin type A receptor (ET(A)R) and to clarify the importance of calmodulin (CaM) / inositol 1,4,5-trisphosphate (IP(3)) receptor binding (CIRB) domain at the C terminus of TRPC channels in ET(A)R-activated channel regulation. In HEK293 cells coexpressing ET(A)R and one of seven TRPC isoforms, ET(A)R stimulation induced ROCE through TRPC3, TRPC5, TRPC6, and TRPC7. The TRPC3- and TRPC6-mediated ROCE was inhibited by selective inhibitors of G(q) protein, phospholipase C (PLC), and CaM. The CIRB domain deletion mutants of TRPC3 and TRPC6 failed to induce ET(A)R-mediated ROCE. Either deletion of the CIRB domain or pharmacological inhibition of CaM did not inhibit the targeting of these channels to the plasma membrane. These results suggest that 1) TRPC3, TRPC5, TRPC6, and TRPC7 can function as ET(A)R-operated Ca(2+) channels; 2) G(q) protein, PLC, and CaM are involved in TRPC3- and TRPC6-mediated ROCE; 3) ET(A)R-mediated activation of TRPC3 and TRPC6 requires the CIRB domain; and 4) abolition of ET(A)R-induced ROCE by CIRB domain deletion and CaM inhibition is due to loss of CaM binding to the channels but not loss of cell surface TRPC3 and TRPC6.

Endothelin type A receptor (ET(A)R) plays an important role in some cardiovascular disorders where ET(A)R levels are increased. However, regulatory mechanisms for ET(A)R levels are unknown. Here, we identified Jun activation domain-binding protein 1 (Jab1) as an ET(A)R-interacting protein by yeast two-hybrid screening of human heart cDNA library using carboxyl terminal tail (C-tail) of ET(A)R as a bait. The interaction was confirmed by glutathione S-transferase pull-down assay, co-immunoprecipitation in HEK293T cells expressing ET(A)R-myc and FLAG-Jab1, and confocal microscopy. Jab1 knockdown increased whole cell and cell surface levels of ET(A)R and ET-1-induced ERK1/2 phosphorylation in HEK293T cells expressing ET(A)R, whereas Jab1 overexpression decreased them. Jab1 overexpression accelerated disappearance rate of ET(A)R after protein synthesis inhibition as an index of a degradation rate. ET(A)R was constitutively ubiquitinated, and the level of ubiquitination was enhanced by Jab1 overexpression. Long-term ET-1 stimulation markedly accelerated the rate of ET(A)R degradation and increased the amount of Jab1 bound to ET(A)R with a maximal level of 500% at 3h. In the absence of ET-1 stimulation, the level of ET(B)R was lower than that of ET(A)R and the degradation rate of ET(B)R was markedly faster than that of ET(A)R. Notably, the amount of Jab1 bound to ET(B)R and ubiquitination level of ET(B)R were markedly higher than those for ET(A)R. Taken together, these results suggest that the amount of Jab1 bound to ETR regulates the degradation rate of ET(A)R and ET(B)R by modulating ubiquitination of these receptors, leading to changes in ET(A)R and ET(B)R levels., (Copyright 2009 Elsevier Inc. All rights reserved.)

This study examines the influence of receptor expression level on signaling pathways activated via endothelin type A receptor (ET(A)R) expressed in Chinese hamster ovary cells at 32,100 (ET(A)R-high-CHO) and 893 (ET(A)R-low-CHO) fmolmg protein(-1). Endothelin-1 (ET-1) elicited a sustained increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), which was dependent on G(q/11) protein, phospholipase C (PLC), Na(+)/H(+) exchanger (NHE), and p38 mitogen-activated protein kinase (p38MAPK) in ET(A)R-high-CHO, whereas the sustained [Ca(2+)](i) increase was negligible in ET(A)R-low-CHO. Functional study with Cytosensor(TM) microphysiometer showed that ET-1 evoked an NHE1-mediated increase in extracellular acidification rate (ECAR) in ET(A)R-high-CHO and ET(A)R-low-CHO. In ET(A)R-high-CHO, the ECAR response at 30 min after ET-1 stimulation was insensitive to G(q/11) and PLC inhibitors, but sensitive to the p38MAPK inhibitor. In ET(A)R-low-CHO, the ECAR response at 30 min was sensitive to these inhibitors. Western blot analysis demonstrated that ET-1-induced p38MAPK phosphorylation in ET(A)R-low-CHO but not in ET(A)R-high-CHO was mediated via G(q/11) and PLC. The G(q/11)/PLC-independent p38MAPK phosphorylation in ET(A)R-high-CHO was suppressed by expression of the C terminus of G(alpha12) protein to disrupt receptor-G(12) protein coupling. These results provide evidence for multiple signaling pathways of ET(A)R that were activated via at least the G(q/11)/PLC/NHE, G(12)/p38MAPK/NHE, and G(q/11)/PLC/p38MAPK/NHE cascades in an expression level-dependent manner.

This study examines the functional role of Na+/H+ exchanger (NHE) in Ca2+ influx mediated by human endothelin type A receptor (ET(A)R) expressed in Chinese hamster ovary (CHO) cells. Endothelin-1 (ET-1) increased extracellular acidification rate (ECAR), which was abolished by 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an NHE inhibitor. EIPA and KB-R7943, a Na+/Ca2+ exchanger (NCX) inhibitor, inhibited ET-1-induced sustained increases in intracellular Ca2+ concentration ([Ca2+]i), and EIPA had no effect on [Ca2+]i after KB-R7943 treatment. ET-1-elicited sustained [Ca2+]i increase was suppressed by reducing extracellular Na+ concentration. These results suggest that possible coupling of NHE with NCX via Na+ transport is involved in ET(A)R-mediated sustained [Ca2+]i increase.

Endothelin ET(A) receptor couples to Gq/11 protein that transduces a variety of receptor signals to modulate diverse cellular responses including Ca2+ mobilization. Stimulation of endothelin ETA receptor with endothelin-1 is generally believed to induce an increase in intracellular Ca2+ concentration ([Ca2+]i) via Gq/11 protein. Here we provide the first convincing evidence that endothelin-1 elicited Gq/11 protein-dependent and -independent 'decrease' in [Ca2+]i via Na+/Ca2+ exchanger (NCX) in Chinese hamster ovary (CHO) cells stably expressing human endothelin ETA receptor. In the cells treated with 1 microM thapsigargin, an inhibitor of endoplasmic Ca2+ pump, that induces an increase in [Ca2+]i via capacitative Ca2+ entry, endothelin-1 induced a decrease in [Ca2+]i which was partially inhibited by YM-254890, a specific inhibitor of Gq/11, indicating that Gq/11-dependent and independent pathways are involved in the decrease. The endothelin-1-induced decrease in [Ca2+]i was markedly suppressed by 3',4'-dichlorobenzamil hydrochloride, a potent NCX inhibitor, and also by a replacement of extracellular Na+ with Li+, which was not transported by NCX, indicating a major role of NCX operating in the forward mode in the endothelin-1-induced decrease in [Ca2+]i. Molecular approach with RT-PCR demonstrated the expression of mRNA for NCX1, NCX2 and NCX3. These results suggest that stimulation of endothelin ETA receptor with endothelin-1 activates the forward mode NCX through Gq/11-dependent and -independent mechanisms: the NCX exports Ca2+ out of the cell depending on Na+ gradient across the cell membrane, resulting in the decrease in [Ca2+]i.