Double Trouble for Type 1 Angiotensin Receptors in Atherosclerosis
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《新英格兰医药杂志》
G protein–coupled receptors represent the largest superfamily of receptors in the human genome. They are intricate sensors and transducers and are fundamental to physiology and disease. Drugs that target these receptors have therapeutic utility and potential and include beta-blockers and blockers of type 1 angiotensin (AT1) receptors. Traditionally, G protein–coupled receptors were thought to act as monomers, in which one ligand binds and activates a single receptor–G protein complex. Newer evidence,1 however, points to the existence of higher-order complexes (dimers or oligomers) with altered pharmacology, responsiveness, or both.2,3 A recent report by AbdAlla and colleagues4 not only underscores the critical nature of a dimer made up of AT1 receptors, but also describes a pivotal event in the formation of the dimer (Figure 1).
Figure 1. Monocytic Activation Leading to Atherosclerosis.
As reported by AbdAlla et al.,4 monocytes from patients with hypertension and dyslipidemic mice had increased adherence to the endothelium of blood vessels in an angiotensin II (AngII)–dependent manner. These monocytes are the precursors of subendothelial macrophages that acquire and store cholesterol and establish the inflammatory characteristics and complex lesions of atherosclerosis. The increased adherence is dependent on two properties of the activated monocytes: an increased capacity to generate angiotensin II by means of the angiotensin-converting enzyme (ACE) and — as shown by AbdAlla et al.4 — increased activity of activated factor XIII (factor XIIIa) transglutaminase; transglutaminase catalyzes the cross-linking of monomeric AT1 receptors into covalent dimers with enhanced signaling capacity. The target of factor XIIIa transglutaminase is glutamine at position 315 (Gln315) in the carboxy terminus of the 359-amino-acid AT1 receptor and presumably one or more of the many available lysine residues present in the intracellular domains of an adjacent receptor.
Blood pressure is governed in part by the angiotensin-converting enzyme (ACE), which generates the peptide hormone angiotensin II in blood and tissues. In turn, angiotensin II acts through the AT1 receptor to regulate arterial blood pressure and water–salt balance. Overactivity of the angiotensin II–AT1 axis leads to hypertension; cardiac, renal, and vascular remodeling; and substantial morbidity and mortality from conditions such as myocardial infarction, congestive heart failure, stroke, and type 2 diabetes. ACE inhibitors and AT1-receptor blockers alleviate these conditions.
The AT1 receptor is a G protein–coupled receptor that activates the Gq/11 protein and initiates a plethora of signals that affect cell function, including the liberation of inositol phosphates that mobilize intracellular calcium. Because this is one of the most comprehensively studied homeostatic systems, some might argue that further research into angiotensin II and AT1 receptors could yield only incremental advances. Thus, the study by AbdAlla et al. is remarkable in that it sheds light on another twist in the repertoire of the actions of angiotensin II, as well as on another mechanism of dimerization.
AbdAlla et al.4 first confirmed that monocytes from patients with essential hypertension were activated in an angiotensin II–dependent manner, responding to the peptide with enhanced cytokine release and adhesion to endothelial cells. The source of angiotensin II seemed likely to be the monocytes themselves, because their ACE activity was higher in patients with hypertension than in normotensive subjects. Although monocytes isolated from normotensive subjects and patients with hypertension expressed similar levels of AT1 receptors, immunoblotting of monocyte membranes with an AT1-receptor antibody demonstrated markedly different patterns of migration of the receptor protein on sodium dodecyl sulfate–polyacrylamide-gel electrophoresis. In normotensive subjects, the AT1 receptor resolved predominantly as a monomer, whereas in patients with hypertension, the receptor appeared to be in a dimeric form, which was stable under the reducing and denaturing conditions used. This finding suggested that the dimerized receptors were covalently bound.
The researchers then found that the levels and activity of activated factor XIII (factor XIIIa) transglutaminase were higher in the monocytes from hypertensive patients than in those from normotensive subjects. Transglutaminases are a family of calcium-dependent enzymes that catalyze the cross-linking of proteins, including membrane proteins, through the amino acids glutamine and lysine.5 Factor XIIIa is best known for cross-linking the fibrin network during blood coagulation. However, factor XIIIa appears to be acting within monocytes. As might be expected, increasing the levels of intracellular calcium by means of the calcium ionophore ionomycin stimulated the transglutaminase activity of factor XIIIa in monocytes. Remarkably, AbdAlla et al. found that treatment of monocytes with both angiotensin II and ionomycin resulted in the formation of covalent AT1-receptor dimers. Site-directed mutagenesis established that glutamine at position 315 (Gln315) within the carboxy terminus of the AT1 receptor is the probable target of factor XIIIa transglutaminase activity and cross-linking. Interestingly, dimerized wild-type AT1 receptor (but not the Gln315 mutant) was characterized by increases in coupling to Gq/11, generation of inositol phosphate, and mobilization of calcium, as compared with the monomeric form, indicating that dimeric AT1 receptors are hyperactive.
Multiple lines of evidence indicate that the transglutaminase activity of factor XIIIa is responsible for covalently dimerizing AT1 receptors. First, dimers do not form on monocytes from patients with a congenital deficiency of factor XIIIa. Second, factor XIIIa–dependent dimerization could be induced in cells made to express the AT1 receptor and factor XIIIa but not in cells expressing a catalytically inactive form of factor XIII. Third, after three months of treatment with ACE inhibitors, hypertensive patients had primarily monomeric AT1 receptors, decreased levels of factor XIIIa, and diminished monocyte adhesion. Finally, in mice with an inactivated gene for apolipoprotein E — an established angiotensin II–dependent model of hypercholesterolemic atherosclerosis — inhibition of ACE or factor XIIIa eliminated AT1 receptor dimers, halted monocyte infiltration, and abolished the propensity for vascular lesions.
What makes the study by AbdAlla et al. provocative? First, transglutaminase-mediated cross-linking of G protein–coupled receptors is a novel finding and represents a paradigm shift from theories of dimerization that are based on disulfide bridging or interactions between transmembrane domains.1 As with any major advance, healthy skepticism is warranted until this observation has been replicated. Nevertheless, the apparent dimerization-selective activation and subsequent adhesion of monocytes expands the astounding array of actions ascribed to angiotensin II and AT1 receptors with respect to cardiovascular dysfunction. Given that the monocyte transglutaminase activity of factor XIIIa is intracellular and therefore sequestered from thrombin-dependent clot formation in blood, this discovery may provide the impetus for the development of monocyte-selective therapies in the form of agents that target factor XIIIa. Finally, determining why AT1-receptor homodimers are substantially more active than AT1-receptor monomers may provide new avenues for designing drugs that target G protein–coupled receptor function.
Source Information
From the Baker Heart Research Institute, Melbourne, Australia.
References
Milligan G. G protein-coupled receptor dimerization: function and ligand pharmacology. Mol Pharmacol 2004;66:1-7.
AbdAlla S, Lother H, Quitterer U. AT1-receptor heterodimers show enhanced G-protein activation and altered receptor sequestration. Nature 2000;407:94-98.
Barki-Harrington L, Luttrell LM, Rockman HA. Dual inhibition of beta-adrenergic and angiotensin II receptors by a single antagonist: a functional role for receptor-receptor interaction in vivo. Circulation 2003;108:1611-1618.
AbdAlla S, Lother H, Langer A, el Faramawy Y, Quitterer U. Factor XIIIA transglutaminase crosslinks AT1 receptor dimers of monocytes at the onset of atherosclerosis. Cell 2004;119:343-354.
Lorand L, Graham RM. Transglutaminases: crosslinking enzymes with pleiotropic functions. Nat Rev Mol Cell Biol 2003;4:140-156.(Walter G. Thomas, Ph.D.)
Figure 1. Monocytic Activation Leading to Atherosclerosis.
As reported by AbdAlla et al.,4 monocytes from patients with hypertension and dyslipidemic mice had increased adherence to the endothelium of blood vessels in an angiotensin II (AngII)–dependent manner. These monocytes are the precursors of subendothelial macrophages that acquire and store cholesterol and establish the inflammatory characteristics and complex lesions of atherosclerosis. The increased adherence is dependent on two properties of the activated monocytes: an increased capacity to generate angiotensin II by means of the angiotensin-converting enzyme (ACE) and — as shown by AbdAlla et al.4 — increased activity of activated factor XIII (factor XIIIa) transglutaminase; transglutaminase catalyzes the cross-linking of monomeric AT1 receptors into covalent dimers with enhanced signaling capacity. The target of factor XIIIa transglutaminase is glutamine at position 315 (Gln315) in the carboxy terminus of the 359-amino-acid AT1 receptor and presumably one or more of the many available lysine residues present in the intracellular domains of an adjacent receptor.
Blood pressure is governed in part by the angiotensin-converting enzyme (ACE), which generates the peptide hormone angiotensin II in blood and tissues. In turn, angiotensin II acts through the AT1 receptor to regulate arterial blood pressure and water–salt balance. Overactivity of the angiotensin II–AT1 axis leads to hypertension; cardiac, renal, and vascular remodeling; and substantial morbidity and mortality from conditions such as myocardial infarction, congestive heart failure, stroke, and type 2 diabetes. ACE inhibitors and AT1-receptor blockers alleviate these conditions.
The AT1 receptor is a G protein–coupled receptor that activates the Gq/11 protein and initiates a plethora of signals that affect cell function, including the liberation of inositol phosphates that mobilize intracellular calcium. Because this is one of the most comprehensively studied homeostatic systems, some might argue that further research into angiotensin II and AT1 receptors could yield only incremental advances. Thus, the study by AbdAlla et al. is remarkable in that it sheds light on another twist in the repertoire of the actions of angiotensin II, as well as on another mechanism of dimerization.
AbdAlla et al.4 first confirmed that monocytes from patients with essential hypertension were activated in an angiotensin II–dependent manner, responding to the peptide with enhanced cytokine release and adhesion to endothelial cells. The source of angiotensin II seemed likely to be the monocytes themselves, because their ACE activity was higher in patients with hypertension than in normotensive subjects. Although monocytes isolated from normotensive subjects and patients with hypertension expressed similar levels of AT1 receptors, immunoblotting of monocyte membranes with an AT1-receptor antibody demonstrated markedly different patterns of migration of the receptor protein on sodium dodecyl sulfate–polyacrylamide-gel electrophoresis. In normotensive subjects, the AT1 receptor resolved predominantly as a monomer, whereas in patients with hypertension, the receptor appeared to be in a dimeric form, which was stable under the reducing and denaturing conditions used. This finding suggested that the dimerized receptors were covalently bound.
The researchers then found that the levels and activity of activated factor XIII (factor XIIIa) transglutaminase were higher in the monocytes from hypertensive patients than in those from normotensive subjects. Transglutaminases are a family of calcium-dependent enzymes that catalyze the cross-linking of proteins, including membrane proteins, through the amino acids glutamine and lysine.5 Factor XIIIa is best known for cross-linking the fibrin network during blood coagulation. However, factor XIIIa appears to be acting within monocytes. As might be expected, increasing the levels of intracellular calcium by means of the calcium ionophore ionomycin stimulated the transglutaminase activity of factor XIIIa in monocytes. Remarkably, AbdAlla et al. found that treatment of monocytes with both angiotensin II and ionomycin resulted in the formation of covalent AT1-receptor dimers. Site-directed mutagenesis established that glutamine at position 315 (Gln315) within the carboxy terminus of the AT1 receptor is the probable target of factor XIIIa transglutaminase activity and cross-linking. Interestingly, dimerized wild-type AT1 receptor (but not the Gln315 mutant) was characterized by increases in coupling to Gq/11, generation of inositol phosphate, and mobilization of calcium, as compared with the monomeric form, indicating that dimeric AT1 receptors are hyperactive.
Multiple lines of evidence indicate that the transglutaminase activity of factor XIIIa is responsible for covalently dimerizing AT1 receptors. First, dimers do not form on monocytes from patients with a congenital deficiency of factor XIIIa. Second, factor XIIIa–dependent dimerization could be induced in cells made to express the AT1 receptor and factor XIIIa but not in cells expressing a catalytically inactive form of factor XIII. Third, after three months of treatment with ACE inhibitors, hypertensive patients had primarily monomeric AT1 receptors, decreased levels of factor XIIIa, and diminished monocyte adhesion. Finally, in mice with an inactivated gene for apolipoprotein E — an established angiotensin II–dependent model of hypercholesterolemic atherosclerosis — inhibition of ACE or factor XIIIa eliminated AT1 receptor dimers, halted monocyte infiltration, and abolished the propensity for vascular lesions.
What makes the study by AbdAlla et al. provocative? First, transglutaminase-mediated cross-linking of G protein–coupled receptors is a novel finding and represents a paradigm shift from theories of dimerization that are based on disulfide bridging or interactions between transmembrane domains.1 As with any major advance, healthy skepticism is warranted until this observation has been replicated. Nevertheless, the apparent dimerization-selective activation and subsequent adhesion of monocytes expands the astounding array of actions ascribed to angiotensin II and AT1 receptors with respect to cardiovascular dysfunction. Given that the monocyte transglutaminase activity of factor XIIIa is intracellular and therefore sequestered from thrombin-dependent clot formation in blood, this discovery may provide the impetus for the development of monocyte-selective therapies in the form of agents that target factor XIIIa. Finally, determining why AT1-receptor homodimers are substantially more active than AT1-receptor monomers may provide new avenues for designing drugs that target G protein–coupled receptor function.
Source Information
From the Baker Heart Research Institute, Melbourne, Australia.
References
Milligan G. G protein-coupled receptor dimerization: function and ligand pharmacology. Mol Pharmacol 2004;66:1-7.
AbdAlla S, Lother H, Quitterer U. AT1-receptor heterodimers show enhanced G-protein activation and altered receptor sequestration. Nature 2000;407:94-98.
Barki-Harrington L, Luttrell LM, Rockman HA. Dual inhibition of beta-adrenergic and angiotensin II receptors by a single antagonist: a functional role for receptor-receptor interaction in vivo. Circulation 2003;108:1611-1618.
AbdAlla S, Lother H, Langer A, el Faramawy Y, Quitterer U. Factor XIIIA transglutaminase crosslinks AT1 receptor dimers of monocytes at the onset of atherosclerosis. Cell 2004;119:343-354.
Lorand L, Graham RM. Transglutaminases: crosslinking enzymes with pleiotropic functions. Nat Rev Mol Cell Biol 2003;4:140-156.(Walter G. Thomas, Ph.D.)