- Email: [email protected]
Endothelin ETB receptor heterodimerization: beyond the ETA receptor
co m m e nt a r y
http://www.kidney-international.org © 2008 International Society of Nephrology
see original article on page 750
Endothelin ETB receptor heterodimerization: beyond the ETA receptor Erika I. Boesen1 The idea that endothelin ETA and ETB receptors may form homodimers and heterodimers has gained increasing interest in recent years. The existence of such interactions between endothelin receptors has the potential to explain some puzzling results from receptor binding and functional studies. Zeng and colleagues take ETB receptor heterodimerization beyond the ETA receptor, reporting renal endothelin ETB–dopamine D3 receptor interactions at the cellular level that appear to have functional consequences in vivo. Kidney International (2008) 74, 693–694. doi:10.1038/ki.2008.324
Over the years, a number of studies have reported results concerning the behavior of the two endothelin receptor subtypes, ETA and ETB, that do not fit the classical model of two G protein-coupled receptors acting independently of one another. For example, in the rat anterior pituitary gland, both ETA and ETB receptors are expressed, but competitive binding studies indicated that ETB receptor-selective ligands competed for binding with endothelin-1 (ET-1) only when an ETA receptor-selective ligand was present.1 Simultaneous blockade of both receptor subtypes was necessary to inhibit clearance of ET-1 by astrocytes, with antagonists of individual receptor subtypes having no effect when administered alone.2 A similar cooperative interaction between the two receptors, necessitating blockade of both subtypes in order to abolish responses to ET-1, has also been noted in a variety of preparations involving vascular or airway smooth muscle. Systematic experiments by Just and colleagues3 demonstrated that the contribu1Vascular Biology Center, Medical College of
Georgia, Augusta, Georgia, USA Correspondence: Erika I. Boesen, Vascular Biology Center, Medical College of Georgia, Room CB3330, 1459 Laney Walker Boulevard, Augusta, Georgia 30912, USA. E-mail: [email protected] Kidney International (2008) 74
tions of each receptor subtype to the renal vascular effects of ET-1 appear highly complex and are not merely additive. This may not be surprising given differences in receptor expression along the renal vascular tree combined with vasodilator and ET-1 clearance functions of endothelialcell ETB receptors. However, those factors do not adequately explain the observation that either an ETA receptor-selective or an ETB receptor-selective antagonist was able to abolish afferent arteriolar constrictor responses to low concentrations of ET-1.4 In recent years, there has been much interest within the endothelin community regarding the possibility that ETA and ETB receptors may form homoand heterodimers. Heterodimerization of endothelin receptors may provide an explanation for the results mentioned in the previous paragraph and might also account for other reports of atypical endothelin binding sites, although splice variants and post-translational modification of receptors should also be considered. It has been demonstrated with the aid of fluorescence resonance energy transfer analysis that, at least in transfected cells, ETA and ETB receptors can constitutively form heterodimers and homodimers.5,6 Furthermore, ETB receptors may internalize at a slower
rate when present as ETA–ETB hetero dimers.5 Consistent with this effect, HEK 293 cells transfected with both ETA and ETB receptors display a markedly prolonged (>4 minutes) increase in [Ca2+] in response to exposure to ET-1 or the selective ETB receptor antagonist sarafotoxin 6c, compared with more transient (1–2 minutes) responses of cells transfected with either receptor subtype alone.7 At least with endothelin receptors, most work to date has focused on heterod imerization of ETA and ETB receptors, rather than heterodimerization of endothelin receptors with receptors that bind other ligands. However, multiple combinations of receptor heterodimers have been described over the past 20 years, including dimers that bind the same ligand (for example, β2-adrenoceptor–α2-adrenoceptor dimers) or different ligands (for example, angiotensin AT1–bradykinin B2 receptor dimers).7 Heterodimerization of receptors alters their ligand-binding properties and in some cases appears essential for activation of downstream signaling.7 Zeng and colleagues8 (this issue) now present results consistent with the possibility that endothelin ETB receptors and dopamine D3 receptors form heterodimers in renal proximal tubule cells from Wistar–Kyoto (WKY) rats. Dopamine receptors have previously been shown to heterodimerize with adenosine receptors in neurons, allowing adenosine agonists and antagonists to modulate the binding affinity and signaling activity of dopamine receptors. With both D3 and ETB receptors being expressed by the proximal tubule, it seems plausible that heterodimerization of ETB receptors with D3 receptors could also occur. Zeng and colleagues8 report that in a proximal tubular cell line derived from WKY rats, D3 receptors colocalize with ETB receptors in the plasma membrane. In contrast, this interaction appears to be disrupted in the corresponding cell line derived from spontaneously hypertensive rats (SHRs), with ETB receptors primarily localized in the cytoplasm and D3 receptors present in both the plasma membrane and cytoplasm. This difference 693
co m m e nt a r y
may be functionally important, as the investigators reported that WKY rats produced a natriuretic response to intrarenal infusion of a selective D3 agonist, whereas SHRs, at least under conditions of high salt intake, did not. Moreover, the natriuretic effect of the D3 agonist in WKY rats could be attenuated by treatment with an ETB receptor antagonist, arguing in favor of some kind of functional cross-talk existing between ETB and D3 receptors in WKY rats. A question not addressed in the study is whether the relationship goes both ways—do D3 receptor antagonists impair the natriuretic effects of tubular ETB receptors? Also, what are the effects of agonists of D3 and ETB receptors on the actions mediated by one another? This study appears to provide an additional example, albeit an unanticipated one, of the importance of renal ETB receptor function in the normal control of blood pressure and salt and water homeostasis. ETB receptor-deficient rats, collecting duct-specific ETB receptor knockout mice, and rats chronically treated with an ETB receptor antagonist all display an elevation of arterial pressure that is exaggerated when the animals are placed on a high-salt diet. Although these effects may be at least partially attributable to loss of ETB receptor-mediated NO production, the work of Zeng and colleagues8 would suggest that impairment of D3 receptor-induced natriuresis might also contribute to the hypertension observed in those animals. Since D3 and ETB receptors are both expressed by medullary collecting duct cells, it would be interesting to know whether collecting duct-specific ETB receptor knockout mice display reduced diuretic and natriuretic responses to D3 receptor agonists. Although further experiments are needed to determine definitively whether D3–ETB receptor heterodimers exist, for example, by using fluorescence resonance energy transfer analysis or coimmunoprecipitation of receptors, the results of the study by Zeng and colleagues8 appear to indicate that D3–ETB receptor interactions in the renal tubules facilitate D3 agonist-induced natriuresis. Zeng and colleagues have previously reported that ETB and AT1 receptors can be co-immunoprecipitated from 694
Endothelin receptor dimers Evidence supporting dimer formation
ETB ETB
ETB ETA
ETA ETA
FRET analysis, confocal microscopic colocalization, altered ligand binding and receptor trafficking, growing functional evidence
ETB AT1
ETB D3
ETA
α1
Confocal microscopic Confocal Not yet colocalizaton, comicroscopic determined. immunoprecipitation colocalization, Functional some functional interactions evidence only
Figure 1 | Summary of proposed endothelin receptor dimer combinations and the evidence in support of their existence. FRET, fluorescence resonance energy transfer.
SHR and WKY renal proximal tubular cells.9,10 Although Zeng and colleagues reported that stimulation of either the ETB or the AT 1 receptor resulted in changes in expression and phosphorylation of the other receptor,9,10 the physiological consequences of these ETB–AT1 receptor interactions were not directly addressed. An intriguing possibility is that endothelin receptor–AT1 receptor heterodimerization could be one of the mechanisms underlying the reported ability of endothelin receptor antagonists to attenuate or abolish acute and chronic effects of angiotensin II (see, for example, Riggleman et al.11). Several studies suggest that ETA receptor activation may impair α1-adrenoceptor-mediated responses.12,13 However, this appears to occur, at least in fibroblasts, via phosphorylation of the α1-adrenoceptor,14 and, as ET-1 did not stimulate internalization of the α1-adrenoceptor, this effect may not involve the existence of ETA receptor–α1adrenoceptor heterodimers. A pictorial summary of the endothelin receptor dimers proposed to date is given in Figure 1. Whether endothelin receptors form heterodimers with any other receptors, and the functional consequences of any such interactions, remains an open field of future inquiry. ACKNOWLEDGMENTS The author wishes to thank David Pollock for his helpful advice on this Commentary. DISCLOSURE The author declared no competing interests.
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References
1.
2.
Harada N, Himeno A, Shigematsu K et al. Endothelin-1 binding to endothelin receptors in the rat anterior pituitary gland: possible formation of an ETA-ETB receptor heterodimer. Cell Mol Neurobiol 2002; 22: 207–226. Hasselblatt M, Kamrowski-Kruck H, Jensen N et al. ETA and ETB receptor antagonists
14.
synergistically increase extracellular endothelin-1 levels in primary rat astrocyte cultures. Brain Res 1998; 785: 253–261. Just A, Olson AJ, Arendshorst WJ. Dual constrictor and dilator actions of ET(B) receptors in the rat renal microcirculation: interactions with ET(A) receptors. Am J Physiol Renal Physiol 2004; 286: F660–F668. Inscho EW, Imig JD, Cook AK et al. ETA and ETB receptors differentially modulate afferent and efferent arteriolar responses to endothelin. Br J Pharmacol 2005; 146: 1019–1026. Gregan B, Jürgensen J, Papsdorf G et al. Liganddependent differences in the internalization of endothelin A and endothelin B receptor heterodimers. J Biol Chem 2004; 279: 27679– 27687. Gregan B, Schaefer M, Rosenthal W et al. Fluorescence resonance energy transfer analysis reveals the existence of endothelin-A and endothelin-B receptor homodimers. J Cardiovasc Pharmacol 2004; 44: S30–S33. Gomes I, Jordan BA, Gupta A et al. G protein coupled receptor dimerization: implications in modulating receptor function. J Mol Med 2001; 79: 226–242. Zeng C, Asico LD, Yu C et al. Renal D3 dopamine receptor stimulation induces natriuresis by endothelin B receptor interactions. Kidney Int 2008; 74: 750–759. Zeng C, Hopfer U, Asico LD et al. Altered AT1 receptor regulation of ETB receptors in renal proximal tubule cells of spontaneously hypertensive rats. Hypertension 2005; 46: 926– 931. Zeng C, Wang Z, Asico LD et al. Aberrant ETB receptor regulation of AT1 receptors in immortalized renal proximal tubule cells of spontaneously hypertensive rats. Kidney Int 2005; 68: 623–631. Riggleman A, Harvey J, Baylis C. Endothelin mediates some of the renal actions of acutely administered angiotensin II. Hypertension 2001; 38: 105–109. D’Angelo G, Pollock JS, Pollock DM. In vivo evidence for endothelin-1-mediated attenuation of α1-adrenergic stimulation. Am J Physiol Heart Circ Physiol 2006; 290: H1251–H1258. Bender SB, Klabunde RE. Altered role of smooth muscle endothelin receptors in coronary endothelin-1 and α1-adrenoceptor-mediated vasoconstriction in type 2 diabetes. Am J Physiol Heart Circ Physiol 2007; 293: H2281–H2288. Vazquez-Prado J, Medina LC, Garcia-Sainz JA. Activation of endothelin ETA receptors induces phosphorylation of α1b-adrenoceptors in rat-1 fibroblasts. J Biol Chem 1997; 272: 27330–27337. Kidney International (2008) 74
http://www.kidney-international.org © 2008 International Society of Nephrology
see original article on page 750
Endothelin ETB receptor heterodimerization: beyond the ETA receptor Erika I. Boesen1 The idea that endothelin ETA and ETB receptors may form homodimers and heterodimers has gained increasing interest in recent years. The existence of such interactions between endothelin receptors has the potential to explain some puzzling results from receptor binding and functional studies. Zeng and colleagues take ETB receptor heterodimerization beyond the ETA receptor, reporting renal endothelin ETB–dopamine D3 receptor interactions at the cellular level that appear to have functional consequences in vivo. Kidney International (2008) 74, 693–694. doi:10.1038/ki.2008.324
Over the years, a number of studies have reported results concerning the behavior of the two endothelin receptor subtypes, ETA and ETB, that do not fit the classical model of two G protein-coupled receptors acting independently of one another. For example, in the rat anterior pituitary gland, both ETA and ETB receptors are expressed, but competitive binding studies indicated that ETB receptor-selective ligands competed for binding with endothelin-1 (ET-1) only when an ETA receptor-selective ligand was present.1 Simultaneous blockade of both receptor subtypes was necessary to inhibit clearance of ET-1 by astrocytes, with antagonists of individual receptor subtypes having no effect when administered alone.2 A similar cooperative interaction between the two receptors, necessitating blockade of both subtypes in order to abolish responses to ET-1, has also been noted in a variety of preparations involving vascular or airway smooth muscle. Systematic experiments by Just and colleagues3 demonstrated that the contribu1Vascular Biology Center, Medical College of
Georgia, Augusta, Georgia, USA Correspondence: Erika I. Boesen, Vascular Biology Center, Medical College of Georgia, Room CB3330, 1459 Laney Walker Boulevard, Augusta, Georgia 30912, USA. E-mail: [email protected] Kidney International (2008) 74
tions of each receptor subtype to the renal vascular effects of ET-1 appear highly complex and are not merely additive. This may not be surprising given differences in receptor expression along the renal vascular tree combined with vasodilator and ET-1 clearance functions of endothelialcell ETB receptors. However, those factors do not adequately explain the observation that either an ETA receptor-selective or an ETB receptor-selective antagonist was able to abolish afferent arteriolar constrictor responses to low concentrations of ET-1.4 In recent years, there has been much interest within the endothelin community regarding the possibility that ETA and ETB receptors may form homoand heterodimers. Heterodimerization of endothelin receptors may provide an explanation for the results mentioned in the previous paragraph and might also account for other reports of atypical endothelin binding sites, although splice variants and post-translational modification of receptors should also be considered. It has been demonstrated with the aid of fluorescence resonance energy transfer analysis that, at least in transfected cells, ETA and ETB receptors can constitutively form heterodimers and homodimers.5,6 Furthermore, ETB receptors may internalize at a slower
rate when present as ETA–ETB hetero dimers.5 Consistent with this effect, HEK 293 cells transfected with both ETA and ETB receptors display a markedly prolonged (>4 minutes) increase in [Ca2+] in response to exposure to ET-1 or the selective ETB receptor antagonist sarafotoxin 6c, compared with more transient (1–2 minutes) responses of cells transfected with either receptor subtype alone.7 At least with endothelin receptors, most work to date has focused on heterod imerization of ETA and ETB receptors, rather than heterodimerization of endothelin receptors with receptors that bind other ligands. However, multiple combinations of receptor heterodimers have been described over the past 20 years, including dimers that bind the same ligand (for example, β2-adrenoceptor–α2-adrenoceptor dimers) or different ligands (for example, angiotensin AT1–bradykinin B2 receptor dimers).7 Heterodimerization of receptors alters their ligand-binding properties and in some cases appears essential for activation of downstream signaling.7 Zeng and colleagues8 (this issue) now present results consistent with the possibility that endothelin ETB receptors and dopamine D3 receptors form heterodimers in renal proximal tubule cells from Wistar–Kyoto (WKY) rats. Dopamine receptors have previously been shown to heterodimerize with adenosine receptors in neurons, allowing adenosine agonists and antagonists to modulate the binding affinity and signaling activity of dopamine receptors. With both D3 and ETB receptors being expressed by the proximal tubule, it seems plausible that heterodimerization of ETB receptors with D3 receptors could also occur. Zeng and colleagues8 report that in a proximal tubular cell line derived from WKY rats, D3 receptors colocalize with ETB receptors in the plasma membrane. In contrast, this interaction appears to be disrupted in the corresponding cell line derived from spontaneously hypertensive rats (SHRs), with ETB receptors primarily localized in the cytoplasm and D3 receptors present in both the plasma membrane and cytoplasm. This difference 693
co m m e nt a r y
may be functionally important, as the investigators reported that WKY rats produced a natriuretic response to intrarenal infusion of a selective D3 agonist, whereas SHRs, at least under conditions of high salt intake, did not. Moreover, the natriuretic effect of the D3 agonist in WKY rats could be attenuated by treatment with an ETB receptor antagonist, arguing in favor of some kind of functional cross-talk existing between ETB and D3 receptors in WKY rats. A question not addressed in the study is whether the relationship goes both ways—do D3 receptor antagonists impair the natriuretic effects of tubular ETB receptors? Also, what are the effects of agonists of D3 and ETB receptors on the actions mediated by one another? This study appears to provide an additional example, albeit an unanticipated one, of the importance of renal ETB receptor function in the normal control of blood pressure and salt and water homeostasis. ETB receptor-deficient rats, collecting duct-specific ETB receptor knockout mice, and rats chronically treated with an ETB receptor antagonist all display an elevation of arterial pressure that is exaggerated when the animals are placed on a high-salt diet. Although these effects may be at least partially attributable to loss of ETB receptor-mediated NO production, the work of Zeng and colleagues8 would suggest that impairment of D3 receptor-induced natriuresis might also contribute to the hypertension observed in those animals. Since D3 and ETB receptors are both expressed by medullary collecting duct cells, it would be interesting to know whether collecting duct-specific ETB receptor knockout mice display reduced diuretic and natriuretic responses to D3 receptor agonists. Although further experiments are needed to determine definitively whether D3–ETB receptor heterodimers exist, for example, by using fluorescence resonance energy transfer analysis or coimmunoprecipitation of receptors, the results of the study by Zeng and colleagues8 appear to indicate that D3–ETB receptor interactions in the renal tubules facilitate D3 agonist-induced natriuresis. Zeng and colleagues have previously reported that ETB and AT1 receptors can be co-immunoprecipitated from 694
Endothelin receptor dimers Evidence supporting dimer formation
ETB ETB
ETB ETA
ETA ETA
FRET analysis, confocal microscopic colocalization, altered ligand binding and receptor trafficking, growing functional evidence
ETB AT1
ETB D3
ETA
α1
Confocal microscopic Confocal Not yet colocalizaton, comicroscopic determined. immunoprecipitation colocalization, Functional some functional interactions evidence only
Figure 1 | Summary of proposed endothelin receptor dimer combinations and the evidence in support of their existence. FRET, fluorescence resonance energy transfer.
SHR and WKY renal proximal tubular cells.9,10 Although Zeng and colleagues reported that stimulation of either the ETB or the AT 1 receptor resulted in changes in expression and phosphorylation of the other receptor,9,10 the physiological consequences of these ETB–AT1 receptor interactions were not directly addressed. An intriguing possibility is that endothelin receptor–AT1 receptor heterodimerization could be one of the mechanisms underlying the reported ability of endothelin receptor antagonists to attenuate or abolish acute and chronic effects of angiotensin II (see, for example, Riggleman et al.11). Several studies suggest that ETA receptor activation may impair α1-adrenoceptor-mediated responses.12,13 However, this appears to occur, at least in fibroblasts, via phosphorylation of the α1-adrenoceptor,14 and, as ET-1 did not stimulate internalization of the α1-adrenoceptor, this effect may not involve the existence of ETA receptor–α1adrenoceptor heterodimers. A pictorial summary of the endothelin receptor dimers proposed to date is given in Figure 1. Whether endothelin receptors form heterodimers with any other receptors, and the functional consequences of any such interactions, remains an open field of future inquiry. ACKNOWLEDGMENTS The author wishes to thank David Pollock for his helpful advice on this Commentary. DISCLOSURE The author declared no competing interests.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
References
1.
2.
Harada N, Himeno A, Shigematsu K et al. Endothelin-1 binding to endothelin receptors in the rat anterior pituitary gland: possible formation of an ETA-ETB receptor heterodimer. Cell Mol Neurobiol 2002; 22: 207–226. Hasselblatt M, Kamrowski-Kruck H, Jensen N et al. ETA and ETB receptor antagonists
14.
synergistically increase extracellular endothelin-1 levels in primary rat astrocyte cultures. Brain Res 1998; 785: 253–261. Just A, Olson AJ, Arendshorst WJ. Dual constrictor and dilator actions of ET(B) receptors in the rat renal microcirculation: interactions with ET(A) receptors. Am J Physiol Renal Physiol 2004; 286: F660–F668. Inscho EW, Imig JD, Cook AK et al. ETA and ETB receptors differentially modulate afferent and efferent arteriolar responses to endothelin. Br J Pharmacol 2005; 146: 1019–1026. Gregan B, Jürgensen J, Papsdorf G et al. Liganddependent differences in the internalization of endothelin A and endothelin B receptor heterodimers. J Biol Chem 2004; 279: 27679– 27687. Gregan B, Schaefer M, Rosenthal W et al. Fluorescence resonance energy transfer analysis reveals the existence of endothelin-A and endothelin-B receptor homodimers. J Cardiovasc Pharmacol 2004; 44: S30–S33. Gomes I, Jordan BA, Gupta A et al. G protein coupled receptor dimerization: implications in modulating receptor function. J Mol Med 2001; 79: 226–242. Zeng C, Asico LD, Yu C et al. Renal D3 dopamine receptor stimulation induces natriuresis by endothelin B receptor interactions. Kidney Int 2008; 74: 750–759. Zeng C, Hopfer U, Asico LD et al. Altered AT1 receptor regulation of ETB receptors in renal proximal tubule cells of spontaneously hypertensive rats. Hypertension 2005; 46: 926– 931. Zeng C, Wang Z, Asico LD et al. Aberrant ETB receptor regulation of AT1 receptors in immortalized renal proximal tubule cells of spontaneously hypertensive rats. Kidney Int 2005; 68: 623–631. Riggleman A, Harvey J, Baylis C. Endothelin mediates some of the renal actions of acutely administered angiotensin II. Hypertension 2001; 38: 105–109. D’Angelo G, Pollock JS, Pollock DM. In vivo evidence for endothelin-1-mediated attenuation of α1-adrenergic stimulation. Am J Physiol Heart Circ Physiol 2006; 290: H1251–H1258. Bender SB, Klabunde RE. Altered role of smooth muscle endothelin receptors in coronary endothelin-1 and α1-adrenoceptor-mediated vasoconstriction in type 2 diabetes. Am J Physiol Heart Circ Physiol 2007; 293: H2281–H2288. Vazquez-Prado J, Medina LC, Garcia-Sainz JA. Activation of endothelin ETA receptors induces phosphorylation of α1b-adrenoceptors in rat-1 fibroblasts. J Biol Chem 1997; 272: 27330–27337. Kidney International (2008) 74