Molecular Mechanism Underlying Inverse Agonist of Angiotensin II Type 1 Receptor

Abstract

To delineate the molecular mechanism underlying the inverse agonist activity of olmesartan, a potent angiotensin II type 1 (AT 1 ) receptor antagonist, we performed binding affinity studies and an inositol phosphate production assay. 257 was found to be important for the interaction with olmesartan but not for the inverse agonist activity. Based on these results, we constructed a model for the interaction between olmesartan and the AT 1 receptor. Although the activation of G protein-coupled receptors is initiated by anti-clockwise rotation of transmembrane (TM) III and TM VI followed by changes in the conformation of the receptor, in this model, cooperative interactions between the hydroxyl group and Tyr 113 in TM III and between the carboxyl group and His 256 in TM VI were essential for the potent inverse agonist activity of olmesartan. We speculate that the specific interaction of olmesartan with these two TMs is essential for stabilizing the AT 1 receptor in an inactive conformation. A better understanding of the molecular mechanisms of the inverse agonism could be useful for the development of new G proteincoupled receptor antagonists with inverse agonist activity.Angiotensin II (Ang II) 3 receptor antagonists (ARBs) are highly selective for the Ang II type 1 (AT 1 ) receptor, which is a member of the G protein-coupled receptor (GPCR) superfamily, and block the diverse effects of Ang II. In addition to their blood pressure-lowering effects in hypertensive patients, ARBs have been shown to promote regression of left ventricular hypertrophy and decrease cardiovascular morbidity and mortality in patients with heart failure or hypertensive diabetic nephropathy with proteinuria (1). Many ARBs are available for clinical use. Because not all ARBs have the same effects, some benefits conferred by ARBs may not be class effects (2). This notion represents an exciting new area in ARB-based therapy, which holds the promise of reducing the incidence of cardiovascular disease.Inverse agonists, such as the opioid receptor ligand ICI174864 (3), block agonist-independent signal transduction by GPCRs. Many clinically important medications have been shown to behave as inverse agonists when tested against either wild-type (WT) or mutated GPCRs; e.g. olanzapine in the 5-hydroxytryptamine 2C receptors (4) and metoprolol in the ␤-adrenoreceptor (5). Spontaneous receptor mutations leading to constitutive activity have been implicated in some human diseases (6, 7). However, such spontaneous mutations have not been reported for the AT 1 receptor, and the WT AT 1 receptor shows slight constitutive activity (2). A recent study demonstrates that the WT AT 1 receptor is activated by mechanical stretching of cultured rat myocytes without the involvement of Ang II, and this was suppressed by an inverse agonist (8). The same study also demonstrates that cardiac hypertrophy induced by constricting the transverse aorta in angiotensinogen knock-out mice was attenuated by an inverse agonist, suggesting that the WT AT 1 receptor is activated ind...

Multivalent ligand–receptor interactions elicit inverse agonist activity of AT1 receptor blockers against stretch-induced AT1 receptor activation

“…9 We also found that basal c-fos promoter activity was suppressed by olmesartan in HEK293 cells expressing AT 1 -N111G (Figure 2). The inhibitory effect of olmesartan on basal c-fos promoter activity was significantly stronger than that of losartan ( Figure 3).…”

“…9 The tetrazole group of olmesartan also interacts with Gln 257 of the AT 1 receptor, but its binding is not required to reduce the basal activity level of the AT 1 receptor. 9 We first examined the effect of olmesartan on stretch-induced ERK activation in HEK293 cells overexpressing AT 1 -WT or an AT 1 mutant receptor harboring one of the following mutations: Y113F, K199Q, H256A or Q257A. As shown in Figure 6a, mechanical stretch-induced phosphorylation of ERKs occurred in AT 1 -Y113F, AT 1 -K199Q, AT 1 -H256A and AT 1 -Q257A cells in degrees equivalent to AT 1 -WT cells.…”

“…As the tetrazole ring of olmesartan interacts with Gln 257 of the AT 1 receptor, 9 we next examined the inhibitory effect that R-88145 (in which the tetrazole group was replaced with a carboxyl group, Figure 6b) had on stretch-induced ERK activation in HEK293 cells overexpressing AT 1 -WT. Although 10 À7 M R-88145 did not inhibit ERK activation induced by 10 À7 M AngII, 10 À5 M R-88145 could inhibit ERK activation to an extent equivalent to 10 À7 M olmesartan (Figure 6c).…”

“…6 Cardiomyocytes and HEK293 cells were cultured in DMEM supplemented with 10% fetal bovine serum and nutrient-starved under serum-free conditions for 48 h before AngII or stretch stimulation. The expression vector for AT 1 -WT and AT 1 -mutant receptors 9 was transfected using FuGENE 6 Transfection Reagent (Roche Diagnostics, Basel, Switzerland) according to the manufacturer's instructions. 7 …”

“…For example, the ternary interactions between the hydroxyl group of the imidazole ring and Tyr 113 of the AT 1 receptor and between the carboxyl group and Lys 199 and His 256 of the AT 1 receptor were required for the inverse agonist activity that olmesartan exerts on GTPase-stimulating activity in a constitutively active AT 1 -N111G mutant containing an Asn 111 to Gly mutation. 9 However, studies using substituted cysteine accessibility mapping (SCAM) showed that conformation of the AT 1 p-ERKs during stretch-induced activation is quite different from that of the AT 1 -N111G receptor. 7,10 Transmembrane domain 7 (TM7) of the AT 1 receptor undergoes a counterclockwise rotation and a shift toward the ligand-binding pocket in response to mechanical stretch, 7 but it shifts away from the ligand-binding pocket in the AT 1 -N111G receptor.…”