Isolation of TCR genes with tumor-killing activity from tumor-infiltrating and circulating lymphocytes in a tumor rejection cynomolgus macaque model.

To develop effective adoptive cell transfer therapy using T cell receptor (TCR)-engineered T cells, it is critical to isolate tumor-reactive TCRs that have potent anti-tumor activity. In humans, tumor-infiltrating lymphocytes (TILs) have been reported to contain CD8+PD-1+ T cells that express tumor-reactive TCRs. Characterization of tumor reactivity of TILs from non-human primate tumors could improve anti-tumor activity of TCR-engineered T cells in preclinical research. In this study, we sought to isolate TCR genes from CD8+PD-1+ T cells among TILs in a cynomolgus macaque model of tumor transplantation in which the tumors were infiltrated with CD8+ T cells and were eventually rejected. We analyzed the repertoire of TCRα and ß pairs obtained from single CD8+PD-1+ T cells in TILs and circulating lymphocytes and identified multiple TCR pairs with high frequency, suggesting that T cells expressing these recurrent TCRs were clonally expanded in response to tumor cells. We further showed that the recurrent TCRs exhibited cytotoxic activity to tumor cells in vitro and potent anti-tumor activity in mice transplanted with tumor cells. These results imply that this tumor transplantation macaque model recapitulates key features of human TILs and can serve as a platform toward preclinical studies of non-human primate tumor models.

Regeneration of Tumor-Antigen-Specific Cytotoxic T Lymphocytes from iPSCs Transduced with Exogenous TCR Genes.

In the current adoptive T cell therapy, T cells from a patient are given back to that patient after ex vivo activation, expansion, or genetic manipulation. However, such strategy depends on the quality of the patient's T cells, sometimes leading to treatment failure. It would therefore be ideal to use allogeneic T cells as "off-the-shelf" T cells. To this aim, we have been developing a strategy where potent tumor-antigen-specific cytotoxic T lymphocytes (CTLs) are regenerated from T-cell-derived induced pluripotent stem cells (T-iPSCs). However, certain issues still remain that make it difficult to establish highly potent T-iPSCs: poor reprogramming efficiency of T cells into iPSCs and high variability in the differentiation capability of each T-iPSC clone. To expand the versatility of this approach, we thought of a method to produce iPSCs equivalent to T-iPSCs, namely, iPSCs transduced with exogenous T cell receptor (TCR) genes (TCR-iPSCs). To test this idea, we first cloned TCR genes from WT1-specific CTLs regenerated from T-iPSCs and then established WT1-TCR-iPSCs. We show that the regenerated CTLs from TCR-iPSCs exerted cytotoxic activity comparable to those from T-iPSCs against WT1 peptide-loaded cell line in in vitro model. These results collectively demonstrate the feasibility of the TCR-iPSC strategy.

Cigarette Smoke Extract and Its Cytotoxic Factor Acrolein Inhibit Nitric Oxide Production in Human Vascular Endothelial Cells.

Acrolein (ACR), a highly reactive α,ß-unsaturated aldehyde, is a major cytotoxic factor in nicotine- and tar-free cigarette smoke extract (CSE). There are conflicting results regarding endothelial functions despite the fact that both CSE and ACR cause cellular damage. Several lines of evidence indicate that CSE impairs endothelium-derived nitric oxide (NO)-dependent vasodilation by reducing the activity and protein expression of endothelial NO synthase (eNOS), whereas ACR elicits endothelium-dependent vasorelaxation by increasing the production of NO and expression of eNOS. To clarify whether CSE and its cytotoxic factor ACR cause endothelial dysfunction, this study examined the effects of CSE and ACR on human vascular endothelial EA.hy926 cells. CSE and ACR reduced the phosphorylation of eNOS at serine (Ser)1177 and total expression of eNOS. The CSE- and ACR-induced decrease in the phosphorylation and expression of eNOS was counteracted by glutathione (reduced form), an antioxidant. Basal NO production was inhibited by CSE, ACR, NG-nitro-L-arginine methyl ester (a competitive eNOS inhibitor), and nominally Ca2+-free solution supplemented with BAPTA-AM (a membrane permeable Ca2+ chelator). These results indicate that CSE and ACR increase oxidative stress, and reduce NO production by reducing the activity and total protein level of eNOS.

Extracellular Ca2+ promotes nitric oxide production via Ca2+-sensing receptor-Gq/11 protein-endothelial nitric oxide synthase signaling in human vascular endothelial cells.

This study examined the possible involvement of Ca2+-sensing receptor (CaSR) in nitric oxide (NO) production in human vascular endothelial cells. Extracellular Ca2+ elevated the intracellular Ca2+ concentration, the endothelial NO synthase (eNOS) phosphorylation level, and NO release from the cells. These responses were inhibited by a CaSR antagonist and a Gq/11 protein inhibitor. Application of an endothelial cell suspension induced vasorelaxation in isolated rat thoracic aorta precontracted by phenylephrine. Adding an NO scavenger to the organ bath abolished this vasorelaxation response. These results suggest that extracellular Ca2+ promotes NO generation via CaSR- and Gq/11 protein-mediated eNOS activation.

Characterization of tumour-infiltrating lymphocytes in a tumour rejection cynomolgus macaque model.

Immunotherapy has emerged as a promising and effective treatment for cancer, yet the clinical benefit is still variable, in part due to insufficient accumulation of immune effector cells in the tumour microenvironment. Better understanding of tumour-infiltrating lymphocytes (TILs) from nonhuman primate tumours could provide insights into improving effector cell accumulation in tumour tissues during immunotherapy. Here, we characterize TILs in a cynomolgus macaque tumour model in which the tumours were infiltrated with CD4+ and CD8+ T cells and were eventually rejected. The majority of CD4+ and CD8+ TILs exhibited a CD45RA-CCR7- effector memory phenotype, but unlike circulating T cells, they expressed CD69, a marker for tissue-resident memory T (TRM) cells. CD69-expressing CD8+ TILs expressed high levels of the cytotoxic molecule granzyme B and the co-inhibitory receptor PD-1. Consistent with the TRM cell phenotype, CD8+ TILs minimally expressed CX3CR1 but expressed CXCR3 at higher levels than circulating CD8+ T cells. Meanwhile, CXCL9, CXCL10 and CXCL11, chemokine ligands for CXCR3, were expressed at high levels in the tumours, thus attracting CXCR3+CD8+ T cells. These results indicate that tumour-transplanted macaques can be a useful preclinical model for studying and optimizing T cell accumulation in tumours for the development of new immunotherapies.

Cytotoxic T Lymphocytes Regenerated from iPS Cells Have Therapeutic Efficacy in a Patient-Derived Xenograft Solid Tumor Model.

Current adoptive T cell therapies conducted in an autologous setting are costly, time consuming, and depend on the quality of the patient's T cells. To address these issues, we developed a strategy in which cytotoxic T lymphocytes (CTLs) are regenerated from iPSCs that were originally derived from T cells and succeeded in regenerating CTLs specific for the WT1 antigen, which exhibited therapeutic efficacy in a xenograft model of leukemia. In this study, we extended our strategy to solid tumors. The regenerated WT1-specific CTLs had a strong therapeutic effect in orthotopic xenograft model using a renal cell carcinoma (RCC) cell line. To make our method more generally applicable, we developed an allogeneic approach by transducing HLA-haplotype homozygous iPSCs with WT1-specific TCR α/ß genes that had been tested clinically. The regenerated CTLs antigen-specifically suppressed tumor growth in a patient-derived xenograft model of RCC, demonstrating the feasibility of our strategy against solid tumors.

Long non-coding RNA MANCR is a target of BET bromodomain protein BRD4 and plays a critical role in cellular migration and invasion abilities of prostate cancer.

Androgen receptor (AR)-negative castration-resistant prostate cancer (CRPC) is highly aggressive and is resistant to most of the current therapies. Bromodomain and extra terminal domain (BET) protein BRD4 binds to super-enhancers (SEs) that drive high expression of oncogenes in many cancers. A BET inhibitor, JQ1, has been found to suppress the malignant phenotypes of prostate cancer cells, however, the target genes of JQ1 remain largely unknown. Here we show that SE-associated genes specific for AR-negative CRPC PC3 cells include genes involved in migration and invasion, and that JQ1 impairs migration and invasion of PC3 cells. We identified a long non-coding RNA, MANCR, which was markedly down-regulated by JQ1, and found that BRD4 binds to the MANCR locus. MANCR knockdown led to a significant decrease in migration and invasion of PC3 cells. Furthermore, RNA sequencing analysis revealed that expression of the genes involved in migration and invasion was altered by MANCR knockdown. In summary, our data demonstrate that MANCR plays a critical role in migration and invasion of PC3 cells.

Ca2+ signal is involved in endothelin-1-induced internalization of endothelin type A receptor expressed in Chinese hamster ovary cells.

Endothelin type A receptor (ETAR) is internalized upon agonist stimulation; however, the mechanism thereof remains controversial. In this study, we characterized the endothelin-1 (ET-1)-induced internalization of ETAR expressed in Chinese hamster ovary cells. ET-1 elicited ETAR internalization and increase in intracellular Ca2+ concentration. ET-1-induced ETAR internalization was completely inhibited by a reduction in intracellular and extracellular Ca2+ 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 ETAR signaling. These results suggest that Ca2+ mobilization, PKC, and ERK1/2 are involved in ET-1-induced ETAR internalization.

Glutathione and cysteines suppress cytotoxicity of gas phase of cigarette smoke by direct reacting with unsaturated carbonyl compounds in the gas phase.

Unsaturated carbonyl compounds, such as acrolein (ACR) and methyl vinyl ketone (MVK), are environmental pollutants, and are contained in smoke, automobile exhaust, and heated oil. We have previously reported that major cytotoxic factors in the gas phase of cigarette smoke are ACR and MVK. ACR and MVK induce cell damage by reactive oxygen species generation via protein kinase C and NADPH oxidases, and antioxidants, such as glutathione (GSH) and N-acetylcysteine (NAC), can effectively suppress their cytotoxic activities. In this study, we attempted to elucidate the molecular mechanism(s) for suppression of ACR- and MVK-induced cytotoxic activities by these antioxidants. GSH, NAC, L- and D-cysteines completely suppressed cell damage induced by gas phase extract of cigarette smoke. The results of HPLC and mass spectrometry showed that GSH and NAC directly reacted with ACR and MVK. Cysteines and cysteine derivatives suppressed ACR-induced GAPDH carbonylation, a representative protein for carbonylation. The current results suggest that GSH, NAC, and cysteines directly reacted with ACR and MVK, and suppressed these unsaturated carbonyl compounds-induced cell damage by inhibition of protein carbonylation.

[Molecular mechanism for ET-1-induced insulin resistance in skeletal muscle cells].

Insulin resistance is a condition where the sensitivity to insulin of the tissues expressing insulin receptor (InsR) is decreased due to a functional disturbance of InsR-mediated intracellular signaling. Insulin promotes the entry of glucose into the tissues and skeletal muscle is the most important tissue responsible for the insulin's action of decreasing blood glucose levels. Endothelin-1 (ET-1), a potent vasoconstrictor and pro-inflammatory peptide, induces insulin resistance through a direct action on skeletal muscle. However, the signaling pathways of ET-1-induced insulin resistance in skeletal muscle remain unclear. Here we show molecular mechanism underlying the inhibitory effect of ET-1 on insulin-stimulated Akt phosphorylation and glucose uptake in myotubes of rat L6 skeletal muscle cell line. mRNA expression levels of differentiation marker genes, MyoD and myogenin, were increased during L6 myoblasts differentiation into myotubes. Some of myotubes possessed the ability to spontaneously contract. In myotubes, insulin promoted Akt phosphorylation at Thr308 and Ser473, and [3H]-labelled 2-deoxy-D-glucose ([3H]2-DG) uptake. The insulin-facilitated Akt phosphorylation and [3H]2-DG uptake were inhibited by ET-1. The inhibitory effect of ET-1 was counteracted by blockade of ET type A receptor (ETAR), inhibition of Gq/11 protein, and siRNA knockdown of G protein-coupled receptor kinase 2 (GRK2). The exogenously overexpressed GRK2 directly bound to endogenous Akt and their association was facilitated by ET-1. In summary, activation of ETAR with ET-1 inhibits insulin-induced Akt phosphorylation and [3H]2-DG uptake in a Gq/11 protein- and GRK2-dependent manner in skeletal muscle. These findings indicate that ETAR and GRK2 are potential targets for insulin resistance.

Impact of the number of repeated inhalations and patient characteristics on the residual amount of inhaled laninamivir octanoate hydrate dry powder in pediatric patients with influenza.

BACKGROUND: A dry powder inhaled formulation is used for the anti-influenza drug laninamivir octanoate hydrate (laninamivir). Although two successive inhalations (puffs) are recommended to minimize residual amounts of active ingredients, previous reports suggest that pediatric patients with low peak inspiratory flow are unable to inhale the active ingredient adequately. In the present study, we prospectively investigated the appropriate number of repeated inhalations of laninamivir dry powder and factors influencing the residual amount of ingredients in pediatric patients with influenza. METHODS: The study enrolled 64 patients receiving laninamivir dry powder inhaler (Inavir®) between January and March 2016 at Tsu emergency medical center/pediatric clinic and dental clinic. All patients enrolled used a laninamivir dry powder inhaler in four repeated inhalations, as instructed by a pharmacist. The residual amount of laninamivir dry powder was calculated by measuring the device weight before and after each inhalation and a residual amount of >20% was defined as an unsuccessful inhalation. RESULTS: The inadequate inhalation rate after two successive inhalations was 45%, and it decreased as number of inhalation repeats increased, reaching 23% after four successive inhalations. Peak inspiratory flow in patients with inadequate inhalation was significantly lower than that in patients with adequate inhalation, for all numbers of inhalation repeats analyzed. Receiver operating characteristic analyses indicated peak inspiratory flow cut-off values of 140, 120, 100, and 100 L/min at 1-4 successive inhalations, respectively. CONCLUSIONS: The present findings suggest that a proportion of patients with low peak inspiratory flow were unable to inhale the active ingredient adequately when laninamivir dry powder inhaler was administered as two successive inhalations, as recommended in the instruction manual. Three or four repeated inhalations of laninamivir dry powder inhaler should be administered to pediatric patients with low peak inspiratory flow.

Using Phos-Tag in Western Blotting Analysis to Evaluate Protein Phosphorylation.

Protein phosphorylation has traditionally been detected by radioisotope phosphate labeling of proteins with radioactive ATP. Several nonradioactive assays with phosphorylation site-specific antibodies are now available for the analysis of phosphorylation status at target sites. However, due to their high specificity, these antibodies they cannot be used to detect unidentified phosphorylation sites. Recently, Phos-tag technology has been developed to overcome the disadvantages and limitations of phosphospecific antibodies. Phos-tag and its derivatives conjugated to biotin, acrylamide, or agarose, form alkoxide-bridged dinuclear metal complexes, which can capture phosphate monoester dianions bound to serine, threonine, and tyrosine residues, in an amino acid sequence-independent manner. Here, we describe our method, which is based on in vitro kinase assay and Western blotting analysis using biotinylated Phos-tag and horseradish peroxidase-conjugated streptavidin, to determine the sites of TRPC6 (transient receptor potential canonical 6) channel phosphorylated by protein kinase A.

Endothelin-1 suppresses insulin-stimulated Akt phosphorylation and glucose uptake via GPCR kinase 2 in skeletal muscle cells.

BACKGROUND AND PURPOSE: Endothelin-1 (ET-1) reduces insulin-stimulated glucose uptake in skeletal muscle, inducing insulin resistance. Here, we have determined the molecular mechanisms underlying negative regulation by ET-1 of insulin signalling. EXPERIMENTAL APPROACH: We used the rat L6 skeletal muscle cells fully differentiated into myotubes. Changes in the phosphorylation of Akt was assessed by Western blotting. Effects of ET-1 on insulin-stimulated glucose uptake was assessed with [(3) H]-2-deoxy-d-glucose ([(3) H]2-DG). The C-terminus region of GPCR kinase 2 (GRK2-ct), a dominant negative GRK2, was overexpressed in L6 cells using adenovirus-mediated gene transfer. GRK2 expression was suppressed by transfection of the corresponding short-interfering RNA (siRNA). KEY RESULTS: In L6 myotubes, insulin elicited sustained Akt phosphorylation at Thr(308) and Ser(473) , which was suppressed by ET-1. The inhibitory effects of ET-1 were prevented by treatment with a selective ETA receptor antagonist and a Gq protein inhibitor, overexpression of GRK2-ct and knockdown of GRK2. Insulin increased [(3) H]2-DG uptake rate in a concentration-dependent manner. ET-1 noncompetitively antagonized insulin-stimulated [(3) H]2-DG uptake. Blockade of ETA receptors, overexpression of GRK2-ct and knockdown of GRK2 prevented the ET-1-induced suppression of insulin-stimulated [(3) H]2-DG uptake. In L6 myotubes overexpressing FLAG-tagged GRK2, ET-1 facilitated the interaction of endogenous Akt with FLAG-GRK2. CONCLUSIONS AND IMPLICATIONS: Activation of ETA receptors with ET-1 suppressed insulin-induced Akt phosphorylation at Thr(308) and Ser(473) and [(3) H]2-DG uptake in a GRK2-dependent manner in skeletal muscle cells. These findings suggest that ETA receptors and GRK2 are potential targets for overcoming insulin resistance.

Agonist-promoted ubiquitination differentially regulates receptor trafficking of endothelin type A and type B receptors.

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.

A simple and rapid method for standard preparation of gas phase extract of cigarette smoke.

Cigarette smoke consists of tar and gas phase: the latter is toxicologically important because it can pass through lung alveolar epithelium to enter the circulation. Here we attempt to establish a standard method for preparation of gas phase extract of cigarette smoke (CSE). CSE was prepared by continuously sucking cigarette smoke through a Cambridge filter to remove tar, followed by bubbling it into phosphate-buffered saline (PBS). An increase in dry weight of the filter was defined as tar weight. Characteristically, concentrations of CSEs were represented as virtual tar concentrations, assuming that tar on the filter was dissolved in PBS. CSEs prepared from smaller numbers of cigarettes (original tar concentrations ≤ 15 mg/ml) showed similar concentration-response curves for cytotoxicity versus virtual tar concentrations, but with CSEs from larger numbers (tar ≥ 20 mg/ml), the curves were shifted rightward. Accordingly, the cytotoxic activity was detected in PBS of the second reservoir downstream of the first one with larger numbers of cigarettes. CSEs prepared from various cigarette brands showed comparable concentration-response curves for cytotoxicity. Two types of CSEs prepared by continuous and puff smoking protocols were similar regarding concentration-response curves for cytotoxicity, pharmacology of their cytotoxicity, and concentrations of cytotoxic compounds. These data show that concentrations of CSEs expressed by virtual tar concentrations can be a reference value to normalize their cytotoxicity, irrespective of numbers of combusted cigarettes, cigarette brands and smoking protocols, if original tar concentrations are ≤15 mg/ml.

Endothelin-1 activates extracellular signal-regulated kinases 1/2 via transactivation of platelet-derived growth factor receptor in rat L6 myoblasts.

AIMS: Endothelin (ET) system plays a critical role in the development of insulin resistance and type 2 diabetes. In skeletal muscle, differentiation of myoblasts to myotubes is accompanied by the development of insulin sensitivity. Activation of extracellular signal-regulated kinase (ERK) 1/2 inhibits the differentiation of myoblasts, leading to insulin resistance. Although ET receptor (ETR) stimulation generally activates ERK1/2, the mechanism for ETR-mediated ERK1/2 activation in skeletal muscle is unknown. The purpose of this study was to determine the signal transduction pathway involved in ET-1-stimulated ERK1/2 phosphorylation in L6 myoblasts derived from rat skeletal muscle. MAIN METHODS: Changes in phosphorylation levels of ERK1/2 following stimulation with ET-1 were analyzed by Western blot in L6 myoblasts. To inhibit receptor internalization, dominant-negative dynamin (K44A) was overexpressed in L6 myoblasts using adenovirus-mediated gene transfer. KEY FINDINGS: ET-1 induced phosphorylation of ERK1/2 in L6 myoblasts. The ERK1/2 phosphorylation was abolished by BQ123 (a selective ET type A receptor (ETAR) antagonist), YM-254890 (a Gαq/11 protein inhibitor), and AG370 (a platelet-derived growth factor receptor (PDGFR) kinase inhibitor), while U-73122 (a phospholipase C (PLC) inhibitor) was less potent. The ERK1/2 phosphorylation was inhibited by overexpression of dominant-negative dynamin (K44A). These results suggest that ETAR stimulation induces ERK1/2 phosphorylation in L6 myoblasts through Gq/11 protein-dependent, PLC-independent PDGFR transactivation which requires dynamin-dependent ETAR internalization. SIGNIFICANCE: Because activation of ERK1/2 is considered to inhibit differentiation of myoblasts with the development of insulin sensitivity, the ETAR-mediated PDGFR transactivation and subsequent ERK1/2 activation play an important role in ET-1-induced insulin resistance.

Filamin-interacting proteins, Cfm1 and Cfm2, are essential for the formation of cartilaginous skeletal elements.

Mutations of Filamin genes, which encode actin-binding proteins, cause a wide range of congenital developmental malformations in humans, mainly skeletal abnormalities. However, the molecular mechanisms underlying Filamin functions in skeletal system formation remain elusive. In our screen to identify skeletal development molecules, we found that Cfm (Fam101) genes, Cfm1 (Fam101b) and Cfm2 (Fam101a), are predominantly co-expressed in developing cartilage and intervertebral discs (IVDs). To investigate the functional role of Cfm genes in skeletal development, we generated single knockout mice for Cfm1 and Cfm2, as well as Cfm1/Cfm2 double-knockout (Cfm DKO) mice, by targeted gene disruption. Mice with loss of a single Cfm gene displayed no overt phenotype, whereas Cfm DKO mice showed skeletal malformations including spinal curvatures, vertebral fusions and impairment of bone growth, showing that the phenotypes of Cfm DKO mice resemble those of Filamin B (Flnb)-deficient mice. The number of cartilaginous cells in IVDs is remarkably reduced, and chondrocytes are moderately reduced in Cfm DKO mice. We observed increased apoptosis and decreased proliferation in Cfm DKO cartilaginous cells. In addition to direct interaction between Cfm and Filamin proteins in developing chondrocytes, we showed that Cfm is required for the interaction between Flnb and Smad3, which was reported to regulate Runx2 expression. Furthermore, we found that Cfm DKO primary chondrocytes showed decreased cellular size and fewer actin bundles compared with those of wild-type chondrocytes. These results suggest that Cfms are essential partner molecules of Flnb in regulating differentiation and proliferation of chondryocytes and actin dynamics.

Endothelin receptor signaling: new insight into its regulatory mechanisms.

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.