Effects of peptide and non-peptide antagonists of angiotensin II receptors on drinking behavior in rats.

The effects of the non-peptide selective angiotensin II AT1 receptor antagonist DuP 753 and its metabolite EXP 3174, of the peptide ANGII analogues saralasin and sarmesin and of the newly synthesized imidazole compound (1-methyl-4,5-diphenylimidazole) on ANGII-induced drinking in rats were investigated. The effect of the AT2 selective antagonist PD 123319 on ANGII-induced drinking in rats was also studied. DuP 753, EXP 3174, saralasin and sarmesin (peptides and non-peptides) dose-dependently inhibited ANGII-induced water intake. The ID50 values of these drugs showed the following order of potency: EXP 3174 > saralasin > sarmesin > DuP 753 indicating their ability to block central AT1 receptors. The imidazole compound increased ANGII-induced water intake suggesting its AT1 receptor agonistic properties. PD 123319 inhibited ANGII-induced water intake at a higher dose (64 nmol), allowing to assume AT1 receptor agonistic properties.     

Angiotensin Receptor Type 1 Blockade in Astroglia Decreases Hypoxia-Induced Cell Damage and TNF Alpha Release

The present study investigated the role of angiotensin receptors (AT-R) in the survival and inflammatory response of astroglia upon hypoxic injury. Exposure of rat astroglial primary cultures (APC) to hypoxic conditions (HC) led to decreased viability of the cells and to a 3.5-fold increase in TNF-alpha release. AT-R type1 (AT1-R) antagonist losartan and its metabolite EXP3174 decrease the LDH release (by 36 ± 9%; 45 ± 6%) from APC under HC. Losartan diminished TNF-alpha release (by 40 ± 15%) and the number of TUNEL-cells by 204 ± 38% under HC, alone and together with angiotensin II (ATII), while EXP3174 was dependent on ATII for its effect on TNF-alpha. The AT2-R antagonist, PD123.319, did not influence the release of LDH and TNF-alpha under normoxic (NC) and HC. These data suggest that AT1-R may decrease the susceptibility of astrocytes to hypoxic injury and their propensity to release TNF-alpha. AT1-R antagonists may therefore be of therapeutic value during hypoxia-associated neurodegeneration.     

Molecular mechanism underlying inverse agonist of angiotensin II type 1 receptor

To delineate the molecular mechanism underlying the inverse agonist activity of olmesartan, a potent angiotensin II type 1 (AT1) receptor antagonist, we performed binding affinity studies and an inositol phosphate production assay. Binding affinity of olmesartan and its related compounds to wild-type and mutant AT1 receptors demonstrated that interactions between olmesartan and Tyr113, Lys199, His256, and Gln257 in the AT1 receptor were important. The inositol phosphate production assay of olmesartan and related compounds using mutant receptors indicated that the inverse agonist activity required two interactions, that between the hydroxyl group of olmesartan and Tyr113 in the receptor and that between the carboxyl group of olmesartan and Lys199 and His256 in the receptor. Gln257 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 AT1 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 Tyr113 in TM III and between the carboxyl group and His256 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 AT1 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 protein-coupled receptor antagonists with inverse agonist activity.     

How can the differences among AT1-receptor antagonists Be explained?

Over the last few years we have seen a new class of antihypertensive drug evolve, the angiotensin II subtype 1 receptor antagonists. Hypothetically, all substances in this class should have the same effect on blood pressure and on end-organ damage as they all block the AT1 receptor. However, there are distinctions between them that may explain the significant and clinically important differences that seem to exist within this class of drug. An explanation for the differences may be found in receptor-antagonist kinetics. The receptor-antagonist interaction may be fitted to a two-state, two-step model which determines how large a part of the binding that will be surmountable and how large a part that will be insurmountable. The proportion of surmountable/insurmountable binding fits nicely to the duration of binding of the antagonist to the receptor, which may be translated into efficacy for the antagonist as outlined in the following review.     

Inhibitory effects of DuP 753 and EXP3174 on responses to angiotensin II in pulmonary vascular bed of the cat.

The effects of the non-peptide antagonist DuP 753 and its metabolite EXP3174 on responses to angiotensin II were investigated in the pulmonary vascular bed of the intact-chest cat. Under conditions of controlled blood flow and constant left atrial pressure, injections of angiotensin II into the perfused lobar artery caused dose-related increases in lobar arterial pressure. Responses to angiotensin II were reproducible and were not changed by meclofenamate or prazosin, indicating that prostaglandin or norepinephrine release does not mediate or modulate pulmonary vascular responses to the peptide. DuP 753 (1-5 mg/kg iv) decreased responses to angiotensin II in a competitive manner, and the duration of the blockade was related to dose of the antagonist. DuP 753 had no significant effect on responses to U-46619, norepinephrine, serotonin, endothelin-1, vasopressin, or BAY K 8644. EXP3174 also decreased responses to angiotensin II without altering responses to agents that act by a variety of mechanisms. The inhibitory effect of EXP3174 (1 mg/kg iv) was not overcome by angiotensin II in the range of doses studied, and the shift to the right of the dose-response curve was nonparallel, suggesting that the blockade was noncompetitive. The blockade was long in duration, and, when the dose of EXP3174 was decreased to 0.1 mg/kg iv, the blockade was surmounted and the shift to the right of the dose-response relationship was parallel. DuP 753 and EXP3174 had little effect on mean baseline pressures in the cat.(ABSTRACT TRUNCATED AT 250 WORDS)     

Agonist-induced phosphorylation of the angiotensin II (AT(1A)) receptor requires generation of a conformation that is distinct from the inositol phosphate-signaling state

G protein-coupled receptors are thought to isomerize between distinct inactive and active conformations, an idea supported by receptor mutations that induce constitutive (agonist-independent) activation. The agonist-promoted active state initiates signaling and, presumably, is then phosphorylated and internalized to terminate the signal. In this study, we examined the phosphorylation and internalization of wild type and constitutively active mutants (N111A and N111G) of the type 1 (AT(1A)) angiotensin II receptor. Cells expressing these receptors were stimulated with angiotensin II (AngII) and [Sar(1),Ile(4),Ile(8)]AngII, an analog that only activates signaling through the constitutive receptors. Wild type AT(1A) receptors displayed a basal level of phosphorylation, which was stimulated by AngII. Unexpectedly, the constitutively active AT(1A) receptors did not exhibit an increase in basal phosphorylation nor was phosphorylation enhanced by AngII stimulation. Phosphorylation of the constitutively active receptors was unaffected by pretreatment with the non-peptide AT(1) receptor inverse agonist, EXP3174, and was not stimulated by the selective ligand, [Sar(1),Ile(4),Ile(8)]AngII. Paradoxically, [Sar(1),Ile(4), Ile(8)]AngII produced a robust ( approximately 85% of AngII), dose-dependent phosphorylation of the wild type AT(1A) receptor at sites in the carboxyl terminus similar to those phosphorylated by AngII. Moreover, internalization of both wild type and constitutive receptors was induced by AngII, but not [Sar(1),Ile(4),Ile(8)]AngII, providing a differentiation between the phosphorylated and internalized states. These data suggest that the AT(1A) receptor can attain a conformation for phosphorylation without going through the conformation required for inositol phosphate signaling and provide evidence for a transition of the receptor through multiple states, each associated with separate stages of receptor activation and regulation. Separate transition states may be a common paradigm for G protein-coupled receptors.     

Slow Receptor Dissociation Kinetics Differentiate Macitentan from Other Endothelin Receptor Antagonists in Pulmonary Arterial Smooth Muscle Cells

Two endothelin receptor antagonists (ERAs), bosentan and ambrisentan, are currently approved for the treatment of pulmonary arterial hypertension (PAH), a devastating disease involving an activated endothelin system and aberrant contraction and proliferation of pulmonary arterial smooth muscle cells (PASMC). The novel ERA macitentan has recently concluded testing in a Phase III morbidity/mortality clinical trial in PAH patients. Since the association and dissociation rates of G protein-coupled receptor antagonists can influence their pharmacological activity in vivo, we used human PASMC to characterize inhibitory potency and receptor inhibition kinetics of macitentan, ambrisentan and bosentan using calcium release and inositol-1-phosphate (IP₁) assays. In calcium release assays macitentan, ambrisentan and bosentan were highly potent ERAs with K_b values of 0.14 nM, 0.12 nM and 1.1 nM, respectively. Macitentan, but not ambrisentan and bosentan, displayed slow apparent receptor association kinetics as evidenced by increased antagonistic potency upon prolongation of antagonist pre-incubation times. In compound washout experiments, macitentan displayed a significantly lower receptor dissociation rate and longer receptor occupancy half-life (ROt_1/2) compared to bosentan and ambrisentan (ROt_1/2∶17 minutes versus 70 seconds and 40 seconds, respectively). Because of its lower dissociation rate macitentan behaved as an insurmountable antagonist in calcium release and IP₁ assays, and unlike bosentan and ambrisentan it blocked endothelin receptor activation across a wide range of endothelin-1 (ET-1) concentrations. However, prolongation of the ET-1 stimulation time beyond ROt_1/2 rendered macitentan a surmountable antagonist, revealing its competitive binding mode. Bosentan and ambrisentan behaved as surmountable antagonists irrespective of the assay duration and they lacked inhibitory activity at high ET-1 concentrations. Thus, macitentan is a competitive ERA with significantly slower receptor dissociation kinetics than the currently approved ERAs. Slow dissociation caused insurmountable antagonism in functional PASMC-based assays and this could contribute to an enhanced pharmacological activity of macitentan in ET-1-dependent pathologies.     

Differential effects of losartan and candesartan on vasoconstrictor responses in the rat

Losartan has been reported to have inhibitory effects on thromboxane (TP) receptor-mediated responses. In the present study, the effects of 2 nonpeptide angiotensin II (AT1) receptor antagonists, losartan and candesartan, on responses to angiotensin II, the thromboxane A2 mimic, U46619, and norepinephrine were investigated and compared in the pulmonary and systemic vascular beds of the intact-chest rat. In this study, intravenous injections of angiotensin II, U46619, and norepinephrine produced dose-related increases in pulmonary and systemic arterial pressure. Losartan and candesartan, in the doses studied, decreased or abolished responses to angiotensin II. Losartan, but not candesartan, and only in a higher dose, produced small, but statistically significant, reductions in pressor responses to U46619 and to norepinephrine in the pulmonary and systemic vascular beds. Furthermore, losartan significantly reduced arachidonic acid-induced platelet aggregation, whereas candesartan had no effect. Pressor responses to angiotensin II were not changed by thromboxane and alpha-adrenergic receptor antagonists, or by cyclooxygenase and NO synthase inhibitors. These results show that losartan and candesartan are potent selective AT1 receptor antagonists in the pulmonary and systemic vascular beds and that losartan can attenuate thromboxane and alpha-adrenergic responses when administered at a high dose, whereas candesartan in the highest dose studied had no effect on responses to U46619 or to norepinephrine. The present data show that the effects of losartan and candesartan on vasoconstrictor responses are different and that pulmonary and systemic pressor responses to angiotensin II are not modulated or mediated by the release of cyclooxygenase products, activation of TP receptors, or the release of NO in the anesthetized rat.     

Site-specific cleavage of G protein-coupled receptor-engaged beta-arrestin. Influence of the AT1 receptor conformation on scissile site selection

The discovery of beta-arrestin-related approximately 46-kDa polypeptide in transfected cells and mouse hearts led us to examine angiotensin II type 1 receptor (AT(1)R)-dependent proteolytic cleavage of beta-arrestin(s). Receptor-ligand induced proteolysis of beta-arrestin(s) is novel, especially in the endocrine system, since proteolytic and/or splice variants of nonvisual arrestins are unknown. We used a strategy to retrieve AT(1)R-engaged isoforms of beta-arrestin 1 to confirm direct interaction of fragments with this G protein-coupled receptor and determine cleavage sites. Here we show that the angiotensin II-AT(1)R complex is associated with full-length and approximately 46-kDa beta-arrestin forms. Mass spectrometric analysis of the AT(1)R-associated short form suggested a scissile site located within the Arg(363)-Arg(393) region in the bovine beta-arrestin 1. Edman degradation analysis of a beta-arrestin 1 C-terminal fragment fused to enhanced green fluorescent protein confirmed the major cleavage to be after Phe(388) and a minor cleavage after Asn(375). Rather unexpectedly, the inverse agonist EXP3174-bound AT(1)R generated different fragmentation of bovine beta-arrestin 1, at Pro(276). The angiotensin II-induced cleavage is independent of inositol 1,4,5-trisphosphate- and Ca(2+)-mediated signaling pathways. The proteolysis of beta-arrestin 2 occurs, but the pattern is more complex. Our findings suggest that beta-arrestin cleavage upon AT(1)R stimulation is a part of the unraveling beta-arrestin-mediated G protein-coupled receptor signaling diversity.     

Angiotensin II induces formation of the early growth response gene-1 protein in rat vascular smooth muscle cells.

The effect of angiotensin II (Ang II) on the early growth response gene-1 (Egr-1) mRNA, on the Egr-1 protein and on the phosphoinositide PI turnover signalling system was investigated in the presence and absence of EXP3174, a potent non-peptide Ang II receptor antagonist. Ang II induced an accumulation of 3.4 kb Egr-1 mRNA and the 80 kDa Egr-1 protein, with a maximum at 30 min and 60 min, respectively. EXP3174 blocked the Ang II-induced increase of inositol phosphates, Egr-1 mRNA and the Egr-1 protein, suggesting the involvement of the PI signalling system by the expression of the Egr-1 gene.     

Effect of angiotensin II type 1 receptor antagonist on tumor growth and angiogenesis in a xenograft model of human bladder cancer

Several authors have investigated the antitumor activity of angiotensin II type 1 receptor (AT1R) antagonists, which are widely used as antihypertensive drugs. In this study, we evaluated the efficacy of the AT1R antagonist candesartan against bladder cancer. For the study in vitro, human bladder cancer cells (KU-19-19) were cultured with and without angiotensin II (A II) and candesartan, and cell viability and vascular endothelial growth factor (VEGF) secretion were analyzed. Also for the study in vivo, a tumor xenograft model was prepared in nude mice using KU-19-19 cells. Mice were administered candesartan daily by oral gavage, and paclitaxel via intravenous infusion. Microvessel density, VEGF expression, and apoptosis were investigated. Candesartan did not induce direct toxicity in KU-19-19 cells, but VEGF was significantly lower in candesartan-treated cells than in the A II-treated control cells. In mice, candesartan, paclitaxel and candesartan-paclitaxel significantly suppressed tumor growth to 46.0%, 35.8% and 17.3%, respectively, of the tumor volume in the control group, showing that combined treatment significantly inhibited tumor growth compared to the candesartan group. Microvessel density and VEGF were significantly decreased in the candesartan and candesartan-paclitaxel groups compared to the control group. The apoptotic index was significantly increased in the paclitaxel and candesartan-paclitaxel groups compared to the control and candesartan groups. In our experimental model, candesartan prevented bladder cancer growth by inhibiting angiogenesis. Furthermore, combined treatment with candesartan and paclitaxel enhanced paclitaxel-induced cytotoxicity. These results suggest that the AT1R antagonist candesartan may be a candidate for innovative therapy for bladder cancer.     

The constitutively active N111G-AT1 receptor for angiotensin II modifies the morphology and cytoskeletal organization of HEK-293 cells

The expression of a constitutively active G protein-coupled receptor is expected to trigger diverse cellular changes ranging from normal to adaptive responses. We report that confluent HEK-293 cells stably expressing the constitutively active mutant N111G-AT1 receptor for angiotensin II spontaneously exhibited dramatic morphological changes and cytoskeletal reorganization. Phase-contrast microscopy revealed that these cells formed a dense monolayer, whereas cells expressing the WT-AT1 receptor displayed large intercellular spaces and numerous filopodia. Confocal microscopy revealed an elaborate web of polymerized actin at the apical and basolateral surfaces of cells expressing the N111G-AT1 receptor. Interestingly, these phenotypic changes were prevented by culturing the cells in the presence of the inverse agonist EXP3174. Similar morphologic rearrangements and de novo polymerized actin structures were found in Ang II-stimulated cells expressing the WT-AT1 receptor. We further showed that AT1 receptor-induced cell-cell contact formation did not require an increase in intracellular Ca2+ concentration or the activity of protein kinase C. However, pretreatment with Y-27632 revealed that Rho-kinase activity was required for cell-cell contact formation upon AT1 receptor activation. These observations demonstrate that the expression of the constitutively active mutant N111G-AT1 receptor had a significant impact on the morphology and cytoskeletal organization of HEK-293 cells, possibly via a mechanism involving the activity of Rho-kinase.     

Conformational switch of angiotensin II type 1 receptor underlying mechanical stress-induced activation

The angiotensin II type 1 (AT(1)) receptor is a G protein-coupled receptor that has a crucial role in the development of load-induced cardiac hypertrophy. Here, we show that cell stretch leads to activation of the AT(1) receptor, which undergoes an anticlockwise rotation and a shift of transmembrane (TM) 7 into the ligand-binding pocket. As an inverse agonist, candesartan suppressed the stretch-induced helical movement of TM7 through the bindings of the carboxyl group of candesartan to the specific residues of the receptor. A molecular model proposes that the tight binding of candesartan to the AT(1) receptor stabilizes the receptor in the inactive conformation, preventing its shift to the active conformation. Our results show that the AT(1) receptor undergoes a conformational switch that couples mechanical stress-induced activation and inverse agonist-induced inactivation.     

Mechanical stress activates angiotensin II type 1 receptor without the involvement of angiotensin II

The angiotensin II type 1 (AT1) receptor has a crucial role in load-induced cardiac hypertrophy. Here we show that the AT1 receptor can be activated by mechanical stress through an angiotensin-II-independent mechanism. Without the involvement of angiotensin II, mechanical stress not only activates extracellular-signal-regulated kinases and increases phosphoinositide production in vitro, but also induces cardiac hypertrophy in vivo. Mechanical stretch induces association of the AT1 receptor with Janus kinase 2, and translocation of G proteins into the cytosol. All of these events are inhibited by the AT1 receptor blocker candesartan. Thus, mechanical stress activates AT1 receptor independently of angiotensin II, and this activation can be inhibited by an inverse agonist of the AT1 receptor.     

Jak2 acts as both a STAT1 kinase and as a molecular bridge linking STAT1 to the angiotensin II AT1 receptor

Angiotensin II activates the Jak-STAT pathway via the AT(1) receptor. We studied two mutant AT(1) receptors, termed M5 and M6, that contain Y to F substitutions for the tyrosine residues naturally found in the third intracellular loop and the carboxyl terminus. After binding ligand, both the M5 and M6 AT(1) receptors trigger STAT1 tyrosine phosphorylation equivalent to that observed with the wild type receptor, indicating that angiotensin II-mediated phosphorylation of STAT1 is independent of these receptor tyrosine residues. In response to angiotensin II, Jak2 autophosphorylates on tyrosine, and Jak2 and STAT1 physically associate, a process that depends on the SH2 domain of STAT1 in vitro. Evaluation of the wild type, M5, and M6 AT(1) receptors showed that angiotensin II-dependent AT(1) receptor-Jak2-STAT1 complex formation is dependent on catalytically active Jak2, not on the receptor tyrosine residues in the third intracellular loop and carboxyl tail. Immunodepletion of Jak2 virtually eliminated the ligand-dependent binding of STAT1 to the AT(1) receptor. These data indicate that the association of STAT1 with the AT(1) receptor is not strictly bimolecular; it requires Jak2 as both a STAT1 kinase and as a molecular bridge linking STAT1 to the AT(1) receptor.     

The AT1A receptor "gain-of-function" mutant N111S/delta329 is both constitutively active and hyperreactive to angiotensin II

The renin-angiotensin-aldosterone system (RAAS) is central to cardiovascular and renal physiology. However, there is no animal model in which the activation of the RAAS only reflects the activation of the angiotensin II (ANG II) AT1 receptor. As a first step to developing such a model, we characterized a gain-of-function mutant of the mouse AT1A receptor. This mutant carries two mutations: N111S predicted to activate the receptor constitutively and a COOH-terminal deletion, delta329, expected to reduce receptor internalization and desensitization. We expressed this double mutant (AT1A-N111S/delta329) in heterologous cells. It showed a pharmacological profile consistent with that of other constitutively active mutants. Furthermore, it increased basal production of inositol phosphates, as well as basal cytosolic and nuclear ERK activities. Basal proliferation of cells expressing the mutant was also greater than that of the wild type. The double mutant was poorly internalized and failed to recruit beta-arrestin 2 in the presence of ANG II. It also showed hypersensitive and hyperreactive responses to ANG II for both inositol phosphate production and ERK activation. The additivity of the phenotypes of the two mutations makes this mutant an appropriate candidate to test the physiological consequences of the AT1A receptor activation itself in transgenic animal models.     

Reassessment of the Unique Mode of Binding between Angiotensin II Type 1 Receptor and Their Blockers

While the molecular structures of angiotensin II (Ang II) type 1 (AT₁) receptor blockers (ARBs) are very similar, they are also slightly different. Although each ARB has been shown to exhibit a unique mode of binding to AT₁ receptor, different positions of the AT₁ receptor have been analyzed and computational modeling has been performed using different crystal structures for the receptor as a template and different kinds of software. Therefore, we systematically analyzed the critical positions of the AT₁ receptor, Tyr¹¹³, Tyr¹⁸⁴, Lys¹⁹⁹, His²⁵⁶ and Gln²⁵⁷ using a mutagenesis study, and subsequently performed computational modeling of the binding of ARBs to AT₁ receptor using CXCR4 receptor as a new template and a single version of software. The interactions between Tyr¹¹³ in the AT₁ receptor and the hydroxyl group of olmesartan, between Lys¹⁹⁹ and carboxyl or tetrazole groups, and between His²⁵⁶ or Gln²⁵⁷ and the tetrazole group were studied. The common structure, a tetrazole group, of most ARBs similarly bind to Lys¹⁹⁹, His²⁵⁶ and Gln²⁵⁷ of AT₁ receptor. Lys¹⁹⁹ in the AT₁ receptor binds to the carboxyl group of EXP3174, candesartan and azilsartan, whereas oxygen in the amidecarbonyl group of valsartan may bind to Lys¹⁹⁹. The benzimidazole portion of telmisartan may bind to a lipophilic pocket that includes Tyr¹¹³. On the other hand, the n-butyl group of irbesartan may bind to Tyr¹¹³. In conclusion, we confirmed that the slightly different structures of ARBs may be critical for binding to AT₁ receptor and for the formation of unique modes of binding.     

Role of Gln1029 in the photoactivation processes of the LOV2 domain in adiantum phytochrome3

Phototropin (phot) is a blue-light receptor in plants. The molecule has two FMN (flavin mononucleotide)-binding domains named the LOV (light-oxygen-voltage) domain, that is a subset of a PAS (per-arnt-sim) superfamily. Illumination of phot-LOV domains produces a covalent C(4a) flavin-cysteinyl adduct, which is called the S390 intermediate state. According to the crystal structures of the LOV2 domain of Adiantum phytochrome3 (phy3), a fusion protein of phot containing the phytochrome chromophoric domain, in the unphotolyzed and S390 states, and the side chain of Gln1029 switches hydrogen bonds with the FMN chromophore. Gln1029 is the hydrogen-bonding donor of the C(4)=O group of FMN in the unphotolyzed state, whereas Gln1029 is the hydrogen-bonding acceptor of the N(5)-H group of FMN in S390. In this paper, we measured the light-induced structural changes in the Q1029L mutant protein of phy3-LOV2 by means of low-temperature FTIR spectroscopy, and the obtained spectra are compared with those of the wild type. Low-temperature UV-visible spectroscopy of Q1029L detected only one intermediate state, S390, at 77-295 K, as well as the wild type. The C(4)=O stretch of FMN at 1710 cm(-1) is shifted to 1723 cm(-1) in Q1029L, presumably because of the lack of hydrogen bonds between Gln1029 and FMN. Upon formation of S390, the C(4)=O group hydrogen bond is weakened in both wild type and Q1029L. These observations are fully consistent with the X-ray crystal structures of the unphotolyzed and S390 states. On the other hand, the C(4)=O stretch of FMN and amide-I vibrations are temperature-independent in Q1029L, in contrast to wild type, in which highly temperature-dependent FTIR spectra are detected. Amide-I vibrations of Q1029L at room temperature are similar to those of the wild type at 77-150 K but not at room temperature. These facts imply that the Q1029L mutant protein lacks progressive protein structural changes following flavin-cysteinyl adduct formation in the wild type, which eventually alter structures of beta sheet and alpha helix in the protein moiety. Hydrogen-bonding interaction of Gln1029 with the FMN chromophore likely plays an important role in the protein structural changes of phy3-LOV2.     

Dual roles of Gln137 of actin revealed by recombinant human cardiac muscle alpha-actin mutants

The actin filament is quite dynamic in the cell. To determine the relationship between the structure and the dynamic properties of the actin filament, experiments using actin mutants are indispensable. We focused on Gln(137) to understand the relationships between two activities: the conformational changes relevant to the G- to F-actin transition and the activation of actin ATPase upon actin polymerization. To elucidate the function of Gln(137) in these activities, we characterized Gln(137) mutants of human cardiac muscle alpha-actin. Although all of the single mutants, Q137E, Q137K, Q137P, and Q137A, as well as the wild type were expressed by a baculovirus-based system, only Q137A and the wild type were purified to high homogeneity. The CD spectrum of Q137A was similar to that of the wild type, and Q137A showed the typical morphology of negatively stained Q137A F-actin images. However, Q137A had an extremely low critical concentration for polymerization. Furthermore, we found that Q137A polymerized 4-fold faster, cleaved the gamma-phosphate group of bound ATP 4-fold slower, and depolymerized 5-fold slower, as compared with the wild-type rates. These results suggest that Gln(137) plays dual roles in actin polymerization, in both the conformational transition of the actin molecule and the mechanism of ATP hydrolysis.     

Distinct ETA Receptor Binding Mode of Macitentan As Determined by Site Directed Mutagenesis

The competitive endothelin receptor antagonists (ERA) bosentan and ambrisentan, which have long been approved for the treatment of pulmonary arterial hypertension, are characterized by very short (1 min) occupancy half-lives at the ET_A receptor. The novel ERA macitentan, displays a 20-fold increased receptor occupancy half-life, causing insurmountable antagonism of ET-1-induced signaling in pulmonary arterial smooth muscle cells. We show here that the slow ET_A receptor dissociation rate of macitentan was shared with a set of structural analogs, whereas compounds structurally related to bosentan displayed fast dissociation kinetics. NMR analysis showed that macitentan adopts a compact structure in aqueous solution and molecular modeling suggests that this conformation tightly fits into a well-defined ET_A receptor binding pocket. In contrast the structurally different and negatively charged bosentan-type molecules only partially filled this pocket and expanded into an extended endothelin binding site. To further investigate these different ET_A receptor-antagonist interaction modes, we performed functional studies using ET_A receptor variants harboring amino acid point mutations in the presumed ERA interaction site. Three ET_A receptor residues significantly and differentially affected ERA activity: Mutation R326Q did not affect the antagonist activity of macitentan, however the potencies of bosentan and ambrisentan were significantly reduced; mutation L322A rendered macitentan less potent, whereas bosentan and ambrisentan were unaffected; mutation I355A significantly reduced bosentan potency, but not ambrisentan and macitentan potencies. This suggests that – in contrast to bosentan and ambrisentan - macitentan-ET_A receptor binding is not dependent on strong charge-charge interactions, but depends predominantly on hydrophobic interactions. This different binding mode could be the reason for macitentan''s sustained target occupancy and insurmountable antagonism.