Endothelin A Receptor (ETAR)
Endothelin A Receptor (ETAR), Table 1 Agonists and antagonists acting at ETAR
ETAR is expressed in many tissues and organs. It is the principal receptor subtype in the cardiovascular system mediating vasoconstriction, vascular cell proliferation, and the hypertrophic effects and positive inotropic effects of ET-1 in cardiac myocytes. In contrast, ETBR is the predominant receptor on endothelial cells and its activation opposes many of the effects mediated by ETAR stimulation; thus ETBR stimulation results in vasodilation (NO and prostacyclin release from endothelial cells), natriuresis, and clearance of ET-1 from the circulation. Endothelin binds irreversibly to ETAR and, depending on cell type, couples to multiple G proteins (Gq/11, Gi, G12/13) activating a network of signaling pathways. Activation of ETAR is important in wound healing and is implicated in many of the pathophysiological effects of ET-1, including cardiovascular disease, pulmonary and systemic hypertension, atherosclerosis, fibrotic diseases, renal disease, diabetes, cancer, inflammation, pain, and hyperalgesia (Schneider et al. 2007; Barton and Yanagisawa 2008; Khimji and Rockey 2010; Bagnato et al. 2011).
Localization of the ETA Receptor
ETAR is the predominant subtype expressed in vascular smooth muscle cells and cardiac myocytes, but is absent in endothelial cells. ETAR is present in cells and tissues of the lungs, kidneys, liver, CNS, adrenal glands, eyes, and ovaries. Confocal microscopy and immunohistochemical analysis demonstrate that ETAR is largely localized on cell surface on plasma membranes, and the cytoplasmic tail of ETAR is important for proper localization. In cardiac myocytes, ETAR is present on the sarcolemma, transverse-tubules and, when internalized, is located in the nuclear envelope of ventricular myocytes (Kuc et al. 2006; Bkaily et al. 2011; Hilal-Dandan and Brunton 2010).
ETAR-Activated Signaling Pathways
ET-1 stimulation of ETAR may induce a variety of physiological responses, depending on the differentiated properties of the target cells. ETAR can couple to multiple G proteins, principally Gq, Gi, and likely G12 to modulate a large array of signaling pathways (Hilal-Dandan et al. 1997; Takigawa et al. 1995; Ivey et al. 2008). Takigawa et al. (1995) co-expressed ETAR with different G-protein a subunits in COS-7 cells and found that ETAR coupled to Gq/11, G12/13, and Gs. There are a few reports of physiological coupling of ETAR activation to elevation of cyclic AMP, but this is likely not direct but mediated by paracrine mechanisms such as ET-stimulated eicosanoid production (Ivey et al. 2008).
Ligand Binding, Internalization, and Regulation of Activity
Human ETAR is located on chromosome 4, is 427 amino acids long, and shares ∼60% sequence homology with ETBR. The intracellular third loop and carboxyl-terminal domains are required for binding and signaling. Based on chimeric experiments between human ETAR and ETBR, the transmembrane (TM) helices 4–6 seem to determine isoform selectivity, whereas portions of TM 1–3 and 7 plus adjacent extracellular loop regions bind the ETAR-selective antagonist BQ-123 (reviewed by Davenport 2002; Hilal-Dandan and Brunton 2010). The selectivity of ETAR binding to ET isoforms and the snake venoms S6b and S6c pharmacologically distinguishes ETAR from the nonselective ETBR. The order of potency for ETAR binding and activation is ET-1 = ET-2 > S6b > ET-3; S6c is inactive (Hilal-Dandan et al. 1997). Numerous ETAR-selective and nonselective antagonists have been developed, including both peptide and non-peptide ligands (see Table 1).
The binding of ET-1 to ETAR is quasi-irreversible and the primary effects on Gq and Gi pathways are sustained and cannot by removed by washing cells exposed to ET-1 (Hilal-Dandan et al. 1997). Thus, attempts to reverse ET-1 action at ETAR using competitive inhibitors are not successful, whereas simultaneous addition of receptor antagonists and ET-1 is effective at reducing ET-1’s subsequent effects. Following ligand binding, conformational changes induce receptor internalization and recycling. Ligand-bound ETAR remains intact for up to 2 h following internalization, and maintains G-protein-coupled signaling. Internalization of ligand-bound ETAR follows the ß-arrestin–dynamin–caveolae and clathrin-coated pit pathways. The internalized receptors co-localize with transferrin and are recycled to the surface. Internalized ET-1/ETAR complexes can localize in perinuclear structures. The cytoplasmic C-terminal tail of ETAR determines its intracellular trafficking through a pericentriolar pathway for recycling. Truncation of the C-terminal tail causes ETAR to be directed toward lysosomal degradation (Paasche et al. 2005; Bkaily et al. 2011; see review Hilal-Dandan and Brunton 2010).
Modifications of ETAR include glycosylation, phosphorylation, and palmitoylation. Palmitoylation of the cysteine residues in the cytoplasmic tail modulates ETAR’s activity in coupling to Gq signaling, ERK activation, and Gi signaling, but has no effect on ligand binding. Homologous desensitization of ETAR can be induced by GRK2- and GRK3-mediated phosphorylation in vascular smooth muscle cells and cardiac myocytes; the specific residues that are phosphorylated have not been identified (for review see Hilal-Dandan and Brunton 2010).
ETAR can form homodimers or heterodimers with ETBR but the functional significance is not clear. ETAR–ETBR heterodimers in certain cell types have been reported to modify ligand binding and transmembrane signaling, which may explain nontypical receptor behavior. Pharmacologically, selective antagonists to both ETAR and ETBR may be required to inhibit the function of heterodimers (Evans and Walker 2008). For example, there is an increased sensitivity to the contractile actions of ET-1 in rat and human ischemic cerebral arteries following subarachnoid hemorrhage (Edvinsson and Povlsen 2011). In these ischemic arteries, ETBR but not ETAR is upregulated. While the ETBR agonist S6c does not elicit any contractile responses in these arteries, antagonists to ETBR attenuate ET-1-induced contraction; these results suggest possible interaction or dimerization between ETAR and ETBR that enhances sensitivity to ET-1 (Edvinsson and Povlsen 2011). ETAR can form a complex with ß-arrestin that interacts with c-Src to stimulate transactivation of EGFR (Rosano et al. 2009).
The human ETAR gene has eight exons and seven introns. Alternative mRNA transcripts of ETAR translate into truncated and nonfunctional receptors. Splice variants of ETAR isolated from human tissue include two alternative transcripts designated ETAR?4 and ETAR?3,4 (with deletions corresponding to exon 4 and exons 3 and 4, respectively) that result in nonfunctional receptors; a third mRNA transcript designated ETAR?3 (deletion corresponding to exon 3), identified in human placental tissue, results in a truncated and nonfunctional ETAR. The function of these splice variants is not clear and may involve regulation of ETAR expression and function (reviewed by Davenport 2002; Hilal-Dandan and Brunton 2010).
Genetically engineered homozygous ETAR knockout mice are not viable and resemble ET-1-knockout mice. ETAR−/− mice have defects in cephalic neural crest cell derivatives. Abnormalities include craniofacial deformities, a poorly formed mandible, abnormal development of the middle ear structures, and tracheal narrowing. Cardiovascular defects include a defective aortic septum and defects in cardiac outflow tract with abnormalities in the aortic arch and great vessels. Signaling through ETAR is important for proper development of pharyngeal arches and aortic arch patterning. ET-1/ETAR signaling is responsible for a choice of morphogenetic program that determines maxillary and mandibular structures. The loss of ET-1/ETAR signaling affects homeotic genes, causing transformation of lower jaw into upper jaw structures, whereas constitutive activation of ETAR has the opposite effect, conversion of maxillary structures into mandibular structures (Sato et al. 2008).
ETAR in Disease
Signaling through ETAR is anti-apoptotic and induces cell growth, hypertrophy and proliferation, cell invasiveness and migration, fibrosis, and inflammation; thus, endothelin and the activation of ETAR are implicated in numerous pathological conditions.
Downregulation of ETAR may also occur as a consequence of elevated ET-1 plasma levels. Downregulation of ETAR in the heart, aorta, and pulmonary arteries is reported in hypertension induced by adenovirus-mediated gene transfer of ET-1, and in ETBR−/− deficient mice that have elevated circulating ET-1 levels due to reduced clearance by ETBR (Kuc et al. 2006). About 20–50% downregulation in ETAR levels has been observed in the aorta and pulmonary arteries of some human diseased hearts, and this has been suggested to be a compensation for elevated plasma ET-1 levels (Kuc et al. 2006).
Both ET-1 levels and ETAR are elevated in chronic kidney disease (CKD) and acute renal failure (Kohan 2010). The ET system is involved in regulation of renal blood flow, reabsorption of Na+ and water, cell proliferation, and extracellular matrix accumulation. Preclinical studies indicate that selective blockade of the ETAR increases renal blood flow and reduces proteinuria, blood pressure, and arterial stiffness in CKD (Dhaun et al. 2011). Increased ET-1 levels are reported in dysfunctional podocytes that will cause an increase in kidney damage and protein filtration. Blockade of ETAR reverses glomerulosclerosis and inhibits release of ET-1 and the shedding of podocyte-specific protein nephrin that is elevated in CKD. Clinical studies are currently assessing the long-term effects of ETAR blockade on morbidity and mortality of patients with CKD and diabetic nephropathy (Kohan 2010).
Upregulation of the ET system and activation of ETAR are associated with proliferation of VSMC. The mitogenic effects of ET-1 are amplified by synergistic interactions with growth factors such as epidermal growth factor (EFG), insulin, transforming growth factor-ß (TGF-ß), basic fibroblast growth factor (b- FGF), platelet-derived growth factor (PDGF), and release of cytokines, which contribute to the development of atherosclerotic plaques (Schneider et al. 2007; Ivey et al. 2008; Khimji and Rockey 2010). In animal models, ETAR blockade inhibits formation of atherosclerotic plaques and improves NO-mediated vasodilation (Barton and Yanagisawa 2008).
In many cancers, the ET system is upregulated and the levels correlate with the severity and progression of the disease (Bagnato et al. 2011). Increased expression of ET-1 and ETAR is associated with tumor growth and progression, neovascularizaion, and metastasis in several cancers including ovarian, prostate, breast, cervical, colon, and lung cancers (Bagnato et al. 2011). The mitogenic effects, mediated through ETAR, are enhanced by transactivation of the EGF receptor (EGFR) through activation of matrix-metalloproteinases (MMPs) that release precursor ligands of EGFR such as the heparin binding EGF-like growth factor (HB-EGF), or through direct phosphorylation by c-Src. Transactivation of EGFR potentiates cell proliferation, cell survival, and tumorigenesis by activating protein tyrosine kinases, PI3-kinase-dependent Akt activation, and Ras–MAPK pathways (Ivey et al. 2008; Rosano et al. 2009). ZD4054, an ETAR-specific antagonist, reportedly inhibits EGFR transactivation and metastasis and enhances tumor regression in ovarian cancer cell xenografts (Rosano et al. 2009). Also, silencing of ß-arrestins 1 and 2 prevents ETAR-induced Src activation, transactivation of EFGR, and metastasis (Rosano et al. 2009). Neovascularization and tumor progression correlate with vascular endothelial growth factor (VEGF) levels. ETAR stimulation enhances VEGF levels by increasing the protein levels of hypoxia-inducible factor 1a (HIF-1a) and increasing expression of cyclooxygenase (COX)-1 and COX-2 and the production of PGE2 (Bagnato et al. 2011). Clinical trials using the ETAR antagonist ZD4054 in patients with prostate cancer have reported improved overall survival (Bagnato et al. 2011). More studies are under way to evaluate the therapeutic value of ETAR antagonism in cancer treatment.
Injection of ET-1 in both human and animals induces spontaneous pain and hyperalgesia (increased sensitivity to pain). The nociceptive and hyperalgesic effects of ET-1 are predominantly mediated through ETAR activation, while ETBR may elicit either pain responses or analgesic effects through release of endorphins (Khodorova et al. 2009). The ETAR is localized on the cell bodies of small diameter sensory neurons of the dorsal root ganglia (DRG) which are associated with pain impulses. ETAR antagonists can ameliorate ET-1-induced acute pain in humans and animal models (Khodorova et al. 2009). Antagonists of ETAR are being tested for their potential analgesic effects in cancer-related pain (Bagnato et al. 2011).
Endothelin works through ETAR and ETBR to regulate multiple physiological events such as vascular tone, renal function, and cell growth and proliferation. Most of the studied pathophysiological effects of ET-1 are mediated through ETAR. Activation of ETAR is implicated in cardiac hypertrophy and congestive heart failure, ischemic heart disease, pulmonary and systemic hypertension, atherosclerosis, and renal disease. ETAR antagonists are being clinically evaluated in a number of diseases. Both ETAR-selective and nonselective blockers have proved to be beneficial and are currently in use for treatment of pulmonary hypertension. Given the significant pathophysiological role that has emerged of ET-1/ETAR activation in nociception, inflammation, and the progression and metastasis of many cancers, ETAR-selective blockade may provide a promising target of intervention in analgesia and cancer therapy.
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