Identification of Distinct Conformations of the Angiotensin-II Type 1 Receptor Associated with the Gq/11 Protein Pathway and the ß-Arrestin Pathway Using Molecular Dynamics Simulations.

Biased signaling represents the ability of G protein-coupled receptors to engage distinct pathways with various efficacies depending on the ligand used or on mutations in the receptor. The angiotensin-II type 1 (AT1) receptor, a prototypical class A G protein-coupled receptor, can activate various effectors upon stimulation with the endogenous ligand angiotensin-II (AngII), including the Gq/11 protein and ß-arrestins. It is believed that the activation of those two pathways can be associated with distinct conformations of the AT1 receptor. To verify this hypothesis, microseconds of molecular dynamics simulations were computed to explore the conformational landscape sampled by the WT-AT1 receptor, the N111G-AT1 receptor (constitutively active and biased for the Gq/11 pathway), and the D74N-AT1 receptor (biased for the ß-arrestin1 and -2 pathways) in their apo-forms and in complex with AngII. The molecular dynamics simulations of the AngII-WT-AT1, N111G-AT1, and AngII-N111G-AT1 receptors revealed specific structural rearrangements compared with the initial and ground state of the receptor. Simulations of the D74N-AT1 receptor revealed that the mutation stabilizes the receptor in the initial ground state. The presence of AngII further stabilized the ground state of the D74N-AT1 receptor. The biased agonist [Sar(1),Ile(8)]AngII also showed a preference for the ground state of the WT-AT1 receptor compared with AngII. These results suggest that activation of the Gq/11 pathway is associated with a specific conformational transition stabilized by the agonist, whereas the activation of the ß-arrestin pathway is linked to the stabilization of the ground state of the receptor.

Characterization of Angiotensin II Molecular Determinants Involved in AT1 Receptor Functional Selectivity.

The octapeptide angiotensin II (AngII) exerts a variety of cardiovascular effects through the activation of the AngII type 1 receptor (AT1), a G protein-coupled receptor. The AT1 receptor engages and activates several signaling pathways, including heterotrimeric G proteins Gq and G12, as well as the extracellular signal-regulated kinases (ERK) 1/2 pathway. Additionally, following stimulation, ßarrestin is recruited to the AT1 receptor, leading to receptor desensitization. It is increasingly recognized that specific ligands selectively bind and favor the activation of some signaling pathways over others, a concept termed ligand bias or functional selectivity. A better understanding of the molecular basis of functional selectivity may lead to the development of better therapeutics with fewer adverse effects. In the present study, we developed assays allowing the measurement of six different signaling modalities of the AT1 receptor. Using a series of AngII peptide analogs that were modified in positions 1, 4, and 8, we sought to better characterize the molecular determinants of AngII that underlie functional selectivity of the AT1 receptor in human embryonic kidney 293 cells. The results reveal that position 1 of AngII does not confer functional selectivity, whereas position 4 confers a bias toward ERK signaling over Gq signaling, and position 8 confers a bias toward ßarrestin recruitment over ERK activation and Gq signaling. Interestingly, the analogs modified in position 8 were also partial agonists of the protein kinase C (PKC)-dependent ERK pathway via atypical PKC isoforms PKCζ and PKCι.

Structure of the human angiotensin II type 1 (AT1) receptor bound to angiotensin II from multiple chemoselective photoprobe contacts reveals a unique peptide binding mode.

Breakthroughs in G protein-coupled receptor structure determination based on crystallography have been mainly obtained from receptors occupied in their transmembrane domain core by low molecular weight ligands, and we have only recently begun to elucidate how the extracellular surface of G protein-coupled receptors (GPCRs) allows for the binding of larger peptide molecules. In the present study, we used a unique chemoselective photoaffinity labeling strategy, the methionine proximity assay, to directly identify at physiological conditions a total of 38 discrete ligand/receptor contact residues that form the extracellular peptide-binding site of an activated GPCR, the angiotensin II type 1 receptor. This experimental data set was used in homology modeling to guide the positioning of the angiotensin II (AngII) peptide within several GPCR crystal structure templates. We found that the CXC chemokine receptor type 4 accommodated the results better than the other templates evaluated; ligand/receptor contact residues were spatially grouped into defined interaction clusters with AngII. In the resulting receptor structure, a ß-hairpin fold in extracellular loop 2 in conjunction with two extracellular disulfide bridges appeared to open and shape the entrance of the ligand-binding site. The bound AngII adopted a somewhat vertical binding mode, allowing concomitant contacts across the extracellular surface and deep within the transmembrane domain core of the receptor. We propose that such a dualistic nature of GPCR interaction could be well suited for diffusible linear peptide ligands and a common feature of other peptidergic class A GPCRs.

Critical hydrogen bond formation for activation of the angiotensin II type 1 receptor.

G protein-coupled receptors contain selectively important residues that play central roles in the conformational changes that occur during receptor activation. Asparagine 111 (N111(3.35)) is such a residue within the angiotensin II type 1 (AT(1)) receptor. Substitution of N111(3.35) for glycine leads to a constitutively active receptor, whereas substitution for tryptophan leads to an inactivable receptor. Here, we analyzed the AT(1) receptor and two mutants (N111G and N111W) by molecular dynamics simulations, which revealed a novel molecular switch involving the strictly conserved residue D74(2.50). Indeed, D74(2.50) forms a stable hydrogen bond (H-bond) with the residue in position 111(3.35) in the wild-type and the inactivable receptor. However, in the constitutively active mutant N111G-AT(1) receptor, residue D74 is reoriented to form a new H-bond with another strictly conserved residue, N46(1.50). When expressed in HEK293 cells, the mutant N46G-AT(1) receptor was poorly activable, although it retained a high binding affinity. Interestingly, the mutant N46G/N111G-AT(1) receptor was also inactivable. Molecular dynamics simulations also revealed the presence of a cluster of hydrophobic residues from transmembrane domains 2, 3, and 7 that appears to stabilize the inactive form of the receptor. Whereas this hydrophobic cluster and the H-bond between D74(2.50) and W111(3.35) are more stable in the inactivable N111W-AT(1) receptor, the mutant N111W/F77A-AT(1) receptor, designed to weaken the hydrophobic core, showed significant agonist-induced signaling. These results support the potential for the formation of an H-bond between residues D74(2.50) and N46(1.50) in the activation of the AT(1) receptor.

STIM1 participates in the contractile rhythmicity of HL-1 cells by moderating T-type Ca(2+) channel activity.

STIM1 plays a crucial role in Ca(2+) homeostasis, particularly in replenishing the intracellular Ca(2+) store following its depletion. In cardiomyocytes, the Ca(2+) content of the sarcoplasmic reticulum must be tightly controlled to sustain contractile activity. The presence of STIM1 in cardiomyocytes suggests that it may play a role in regulating the contraction of cardiomyocytes. The aim of the present study was to determine how STIM1 participates in the regulation of cardiac contractility. Atomic force microscopy revealed that knocking down STIM1 disrupts the contractility of cardiomyocyte-derived HL-1 cells. Ca(2+) imaging also revealed that knocking down STIM1 causes irregular spontaneous Ca(2+) oscillations in HL-1 cells. Action potential recordings further showed that knocking down STIM1 induces early and delayed afterdepolarizations. Knocking down STIM1 increased the peak amplitude and current density of T-type voltage-dependent Ca(2+) channels (T-VDCC) and shifted the activation curve toward more negative membrane potentials in HL-1 cells. Biotinylation assays revealed that knocking down STIM1 increased T-VDCC surface expression and co-immunoprecipitation assays suggested that STIM1 directly regulates T-VDCC activity. Thus, STIM1 is a negative regulator of T-VDCC activity and maintains a constant cardiac rhythm by preventing a Ca(2+) overload that elicits arrhythmogenic events.

Type 1 inositol-1,4,5-trisphosphate receptor is a late substrate of caspases during apoptosis.

Apoptosis is characterized by the proteolytic cleavage of hundreds of proteins. One of them, the type 1 inositol-1,4,5-trisphosphate receptor (IP(3) R-1), a multimeric receptor located on the endoplasmic reticulum (ER) membrane that is critical to calcium homeostasis, was reported to be cleaved during staurosporine (STS) induced-apoptosis in Jurkat cells. Because the reported cleavage site separates the IP(3) binding site from the channel moiety, its cleavage would shut down a critical signaling pathway that is common to several cellular processes. Here we show that IP(3) R-1 is not cleaved in 293 cells treated with STS, TNFα, Trail, or ultra-violet (UV) irradiation. Further, it is not cleaved in Hela or Jurkat cells induced to undergo apoptosis with Trail, TNFα, or UV. In accordance with previous reports, we demonstrate that it is cleaved in a Jurkat cell line treated with STS. However its cleavage occurs only after poly(ADP-ribose) polymerase (PARP), which cleavage is a hallmark of apoptosis, and p23, a poor caspase-7 substrate, are completely cleaved, suggesting that IP(3) R-1 is a relatively late substrate of caspases. Nevertheless, the receptor is fully accessible to proteolysis in cellulo by ectopically overexpressed caspase-7 or by the tobacco etch virus (TEV) protease. Finally, using recombinant caspase-3 and microsomal fractions enriched in IP(3) R-1, we show that the receptor is a poor caspase-3 substrate. Consequently, we conclude that IP(3) R-1 is not a key death substrate.

Analysis of transmembrane domains 1 and 4 of the human angiotensin II AT1 receptor by cysteine-scanning mutagenesis.

The octapeptide hormone angiotensin II (AngII) exerts a wide variety of cardiovascular effects through the activation of the AT(1) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein-coupled receptors, the AT(1) receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. Here, we investigated the role of the first and fourth transmembrane domains (TMDs) in the formation of the binding pocket of the human AT(1) receptor using the substituted-cysteine accessibility method. Each residue within the Phe-28((1.32))-Ile-53((1.57)) fragment of TMD1 and Leu-143((4.40))-Phe-170((4.67)) fragment of TMD4 was mutated, one at a time, to a cysteine. The resulting mutant receptors were expressed in COS-7 cells, which were subsequently treated with the charged sulfhydryl-specific alkylating agent methanethiosulfonate ethylammonium (MTSEA). This treatment led to a significant reduction in the binding affinity of TMD1 mutants M30C((1.34))-AT(1) and T33C((1.37))-AT(1) and TMD4 mutant V169C((4.66))-AT(1). Although this reduction in binding of the TMD1 mutants was maintained when examined in a constitutively active receptor (N111G-AT(1)) background, we found that V169C((4.66))-AT(1) remained unaffected when treated with MTSEA compared with untreated in this context. Moreover, the complete loss of binding observed for R167C((4.64))-AT(1) was restored upon treatment with MTSEA. Our results suggest that the extracellular portion of TMD1, particularly residues Met-30((1.34)) and Thr-33((1.37)), as well as residues Arg-167((4.64)) and Val-169((4.66)) at the junction of TMD4 and the second extracellular loop, are important binding determinants within the AT(1) receptor binding pocket but that these TMDs undergo very little movement, if at all, during the activation process.

Positive regulation of inositol 1,4,5-trisphosphate-induced Ca2+ release by mammalian target of rapamycin (mTOR) in RINm5F cells.

The inositol 1,4,5-trisphosphate receptor (IP(3)R), a ligand-gated Ca(2+) channel, is the main regulator of intracellular Ca(2+) mobilization in non-excitable cells. An emerging body of evidence suggests that specific regulatory control of the Ca(2+) signaling pathway is modulated by the activation of additional signaling pathways. In the present study, we investigated the influence of the PI3-kinase/mammalian target of rapamycin (mTOR) pathway on the activity of the IP(3)R/Ca(2+) signaling pathway in RINm5F cells. We used a co-immunoprecipitation approach to show that mTOR physically interacts with IP(3)R-3 in an mTOR activity-dependent manner. We also showed that IP(3)R is phosphorylated by mTOR in cellulo. All the conditions known to modulate mTOR activity (IGF-1, wortmannin, rapamycin, PP242, and nutrient starvation) were shown to modify carbachol-induced Ca(2+) signaling in RINm5F cells. Lastly, we used an assay that directly measures the activity of IP(3)R, to show that mTOR increases the apparent affinity of IP(3)R. Given that mTOR controls cell proliferation and cell homeostasis, and that Ca(2+) plays a key role in these two phenomena, it follows that mTOR facilitates IP(3)R-mediated Ca(2+) release when the nutritional status of cells requires it.

The activation state of the inositol 1,4,5-trisphosphate receptor regulates the velocity of intracellular Ca2+ waves in bovine aortic endothelial cells.

Ca(2+) is a highly versatile second messenger that plays a key role in the regulation of many cell processes. This versatility resides in the fact that different signals can be encoded spatio-temporally by varying the frequency and amplitude of the Ca(2+) response. A typical example of an organized Ca(2+) signal is a Ca(2+) wave initiated in a given area of a cell that propagates throughout the entire cell or within a specific subcellular region. In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP(3) R) is responsible for the release of Ca(2+) from the endoplasmic reticulum. IP(3) R activity can be directly modulated in many ways, including by interacting molecules, proteins, and kinases such as PKA, PKC, and mTOR. In the present study, we used a videomicroscopic approach to measure the velocity of Ca(2+) waves in bovine aortic endothelial cells under various conditions that affect IP(3) R function. The velocity of the Ca(2+) waves increased with the intensity of the stimulus while extracellular Ca(2+) had no significant impact on wave velocity. Forskolin increased the velocity of IP(3) R-dependent Ca(2+) waves whereas PMA and rapamycin decreased the velocity. We used scatter plots and Pearson's correlation test to visualize and quantify the relationship between the Ca(2+) peak amplitude and the velocity of Ca(2+) waves. The velocity of IP(3) R-dependent Ca(2+) waves poorly correlated with the amplitude of the Ca(2+) response elicited by agonists in all the conditions evaluated, indicating that the velocity depended on the activation state of IP(3) R, which can be modulated in many ways.

Dopamine 5 receptor mediates Ang II type 1 receptor degradation via a ubiquitin-proteasome pathway in mice and human cells.

Hypertension is a multigenic disorder in which abnormal counterregulation between dopamine and Ang II plays a role. Recent studies suggest that this counterregulation results, at least in part, from regulation of the expression of both the antihypertensive dopamine 5 receptor (D5R) and the prohypertensive Ang II type 1 receptor (AT1R). In this report, we investigated the in vivo and in vitro interaction between these GPCRs. Disruption of the gene encoding D5R in mice increased both blood pressure and AT1R protein expression, and the increase in blood pressure was reversed by AT1R blockade. Activation of D5R increased the degradation of glycosylated AT1R in proteasomes in HEK cells and human renal proximal tubule cells heterologously and endogenously expressing human AT1R and D5R. Confocal microscopy, Förster/fluorescence resonance energy transfer microscopy, and fluorescence lifetime imaging microscopy revealed that activation of D5R initiated ubiquitination of the glycosylated AT1R at the plasma membrane. The regulated degradation of AT1R via a ubiquitin/proteasome pathway by activation of D5R provides what we believe to be a novel mechanism whereby blood pressure can be regulated by the interaction of 2 counterregulatory GPCRs. Our results therefore suggest that treatments for hypertension might be optimized by designing compounds that can target the AT1R and the D5R.

Activation induces structural changes in the liganded angiotensin II type 1 receptor.

The octapeptide hormone angiotensin II (AngII) binds to and activates the human angiotensin II type 1 receptor (hAT(1)) of the G protein-coupled receptor class A family. Several activation mechanisms have been proposed for this family, but they have not yet been experimentally validated. We previously used the methionine proximity assay to show that 11 residues in transmembrane domain (TMD) III, VI, and VII of the hAT(1) receptor reside in close proximity to the C-terminal residue of AngII. With the exception of a single change in TMD VI, the same contacts are present on N111G-hAT(1), a constitutively active mutant; this N111G-hAT(1) is a model for the active form of the receptor. In this study, two series of 53 individual methionine mutations were constructed in TMD I, II, IV, and V on both receptor forms. The mutants were photolabeled with a neutral antagonist, (125)I-[Sar(1),p-benzoyl-L-Phe(8)]AngII, and the resulting complexes were digested with cyanogen bromide. Although no new contacts were found for the hAT(1) mutants, two were found in the constitutively active mutants, Phe-77 in TMD II and Asn-200 in TMD V. To our knowledge, this is the first time that a direct ligand contact with TMD II and TMD V has been reported. These contact point differences were used to identify the structural changes between the WT-hAT(1) and N111G-hAT(1) complexes through homology-based modeling and restrained molecular dynamics. The model generated revealed an important structural rearrangement of several TMDs from the basal to the activated form in the WT-hAT(1) receptor.

The fifth transmembrane domain of angiotensin II Type 1 receptor participates in the formation of the ligand-binding pocket and undergoes a counterclockwise rotation upon receptor activation.

The octapeptide hormone angiotensin II exerts a wide variety of cardiovascular effects through the activation of the angiotensin II Type 1 (AT(1)) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein- coupled receptors, the AT(1) receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. The role of the fifth transmembrane domain (TMD5) was investigated using the substituted cysteine accessibility method. All of the residues within Thr-190 to Leu-217 region were mutated one at a time to cysteine, and after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of L197C-AT(1), N200C-AT(1), I201C-AT(1), G203C-AT(1), and F204C-AT(1) mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT(1) receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD5 reporter cysteines engineered in a constitutively active N111G-AT(1) receptor background. Indeed, mutant I201C-N111G-AT(1) became more sensitive to MTSEA, whereas mutant G203C-N111G-AT(1) lost some sensitivity. Our results suggest that constitutive activation of AT(1) receptor causes an apparent counterclockwise rotation of TMD5 as viewed from the extracellular side.

A single-nucleotide polymorphism of alanine to threonine at position 163 of the human angiotensin II type 1 receptor impairs Losartan affinity.

BACKGROUND AND OBJECTIVE: AT1 is the principal receptor for angiotensin II (AngII), which regulates blood pressure and osmotic homeostasis. Earlier studies have shown that position 163 interacts with the antihypertensive nonpeptide antagonist, Losartan. A recently discovered polymorphism found in humans (rs12721226) coding for residue 163 led us to determine whether this polymorphism would affect Losartan antihypertensive therapies. The pharmacological properties of the A163T hAT1 variant are described. METHOD AND RESULTS: The A163T hAT1 mutation was evaluated by testing its affinity by dose displacement of AngII analogs in COS-7 cells expressing either wild-type hAT1 or the A163T hAT1. The expressions of the receptors were evaluated by saturation binding and the efficacies were assessed by measuring the 3H-inositol phosphate production. The results showed that the A163T hAT1 receptor is comparable with the affinity, expression, and efficacy of native hAT1 towards peptide ligands. The affinities were also tested with nonpeptide antagonists Losartan, L-158 809, valsartan, telmisartan, irbesartan, candesartan, and EXP3174. Losartan and EXP3174 displayed a 7-fold loss in affinity towards A163T hAT1. The ability of Losartan to inhibit AngII-induced inositol triphosphate production also confirmed a loss in efficacy. Molecular modeling showed a higher steric and hydrophilic hindrance of the A163T hAT1-Losartan complex. CONCLUSION: The polymorphism that codes for the A163T hAT1 variant results in a receptor with normal physiological properties toward the endogenous hormone. However, the significant reduction in affinity to Losartan and its active metabolite, EXP3174, could significantly impair the clinical effectiveness of an antihypertensive therapy using Losartan with patients bearing the A163T polymorphism.

The transcription factors NFAT and CREB have different susceptibilities to the reduced Ca2+ responses caused by the knock down of inositol trisphosphate receptor in HEK 293A cells.

BACKGROUND/AIMS: The inositol 1,4,5-trisphosphate receptor (IP(3)R), a ligand-gated Ca(2+) channel, plays an important role in the control of intracellular Ca(2+). Three isoforms of IP(3)R have been identified and most cell types express different proportions of these isoforms. The purpose of this study was to investigate how IP(3)R signalling is involved in the activation of the Ca(2+)-sensitive transcription factors NFAT and CREB. METHODS: Each IP(3)R isoform expressed in HEK 293A cells was knocked down using selective siRNA. Free intracellular Ca(2+) was monitored spectrofluometrically. NFAT and CREB activities were measured with luciferase reporter constructs. RESULTS: IP(3)R-2-knocked down HEK 293A cells showed a deficient CCh-induced Ca(2+) response that could be rescued by co-stimulation with VIP, a cAMP increasing agonist. NFAT transcriptional activity, but not CREB transcriptional activity, was significantly reduced in IP(3)R-2-knocked down HEK 293A cells. Overexpression of IP(3)R-1 could fully compensate for IP(3)R-2 knock down to mobilize Ca(2+) and to activate NFAT. CONCLUSION: Our results show that the knock down of IP(3)R-2 significantly reduced the intracellular Ca(2+) response of HEK 293 cells. This reduced Ca(2+) response did not affect the activation of CREB but significantly decreased the activation of NFAT, suggesting that the Ca(2+) signals required for the activation of NFAT are stronger than those required for the activation of CREB.

Real-time monitoring of angiotensin II-induced contractile response and cytoskeleton remodeling in individual cells by atomic force microscopy.

Physiological processes, occurring as a result of specific receptor stimulation, are generally assessed via molecular biology techniques and microscopic approaches with the involvement of specific molecular markers. The recent progress in experimental approaches, allowing the mechanical characterization of individual biological entities, now makes it possible to address cellular processes occurring in individual cells as a result of their stimulation by hormones. Here, we demonstrate that the atomic force microscope (AFM) can be used to mechanically probe individual cells following the activation of the angiotensin-1 receptor, a receptor well known for its role in cell homeostasis regulation. Our goal is to demonstrate that the measurement of cantilever deflection can be used to quantify in real time the mechanical and morphological cell activity associated with the activation of the receptor. By combining the AFM with time-lapse sequences of phase-contrast and confocal micrographs, we show that the angiotensin-1 receptor stimulation with 100 nM angiotensin II produces an actin-dependent contractile response with an amplitude of 262 +/- 52 nm. We validated the mechanical origin of the responses by measuring the elastic modulus of the cell from indentation experiments performed at 30-s intervals. Additionally, nanoscaled height fluctuations of the cell membrane occurring after the initial contraction response could be attributed to an increased actin cytoskeleton activity and remodeling detected by confocal microscopy. Finally, by using inhibitors for specific elements of the angiotensin-1 receptor signaling pathways, we demonstrate that AFM real-time height monitoring allows a read out of the molecular processes responsible for the cell mechanical response.

Microfilament and microtubule assembly is required for the propagation of inositol trisphosphate receptor-induced Ca2+ waves in bovine aortic endothelial cells.

Ca2+ is a highly versatile second messenger that plays a key role in the regulation of numerous cell processes. One-way cells ensure the specificity and reliability of Ca2+ signals is by organizing them spatially in the form of waves that propagate throughout the cell or within a specific subcellular region. In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP3R) is responsible for the release of Ca2+ from the endoplasmic reticulum. The spatial aspect of the Ca2+ signal depends on the organization of various elements of the Ca2+ signaling toolkit and varies from tissue to tissue. Ca2+ is implicated in many of endothelium functions that thus depend on the versatility of Ca2+ signaling. In the present study, we showed that the disruption of caveolae microdomains in bovine aortic endothelial cells (BAEC) with methyl-beta-cyclodextrin was not sufficient to disorganize the propagation of Ca2+ waves when the cells were stimulated with ATP or bradykinin. However, disorganizing microfilaments with latrunculin B and microtubules with colchicine both prevented the formation of Ca2+ waves. These results suggest that the organization of the Ca2+ waves mediated by IP3R channels does not depend on the integrity of caveolae in BAEC, but that microtubule and microfilament cytoskeleton assembly is crucial.

Mutational analysis of the conserved Asp2.50 and ERY motif reveals signaling bias of the urotensin II receptor.

Class A (rhodopsin-like) G protein-coupled receptors possess conserved residues and motifs that are important for their specific activity. In the present study, we examined the role of residue Asp97(2.50) as well as residues Glu147(3.49), Arg148(3.50), and Tyr149(3.51) of the ERY motif on the functionality of the urotensin II receptor (UT). Mutations D97(2.50)A, R148(3.50)A, and R148(3.50)H abolished the ability of UT to activate phospholipase C, whereas mutations E147(3.49)A and Y149(3.51)A reduced the ability to activate PLC by 50%. None of the mutants exhibited constitutive activity. However, R148(3.50)A and R148(3.50)H promoted ERK1/2 activation, which was abolished by 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478), an inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase activity. Both these mutants were capable of directly activating EGFR, which confirmed that they activated the mitogen-activated protein kinase (MAPK) pathway by a Galpha(q/11)-independent transactivation of EGFR. The D97(2.50)A, R148(3.50)A, and R148(3.50)H mutants did not readily internalize and did not promote translocation or colocalize with beta-arrestin2-GFP. Finally, the agonist-induced internalization of the E147(3.49)A mutant receptor was significantly increased compared with wild-type receptor. This study highlights the major contribution of the conserved Asp(2.50) residue to the functionality of the UT receptor. The Arg residue in the ERY motif of UT is an important structural element in signaling crossroads that determine whether Galpha(q/11)-dependent and -independent events can occur.

Memantine potentiates agonist-induced Ca2+ responses in HEK 293 cells.

BACKGROUND/AIMS: The Alzheimer drug memantine (1-amino-3,5-dimethyl-adamantane) blocks the pore channel of the NMDA receptor. Since memantine also blocks the 5-HT(3) receptor, neuronal nicotinic receptor, and voltage-activated Na(+) channels, the purpose of our study was to verify whether memantine could influence other types of channels involved in the regulation of Ca(2+). METHODS: Free intracellular Ca(2+) concentrations in whole cells and in saponin-permeabilized cells were monitored spectrofluorometrically in HEK-293 cells stably expressing TRPC6. RESULTS: Memantine decreased the basal level of intracellular Ca(2+), increased the content of the intracellular Ca(2+) store, which in turn increased the agonist-induced intracellular Ca(2+) release, and increased the store-operated Ca(2+) entry. CONCLUSION: In addition to blocking the NMDA receptor, memantine also decreases the basal level of intracellular Ca(2+) and increases the sensitivity of cells to extracellular stimuli. All these effects may be of benefit in the treatment of Alzheimer's disease.

Ascorbic acid decreases the binding affinity of the AT1 receptor for angiotensin II.

BACKGROUND: Ascorbic acid is an essential vitamin and a powerful antioxidant. Many studies have highlighted the benefits of ascorbic acid for chronic cardiovascular diseases such as hypertension in which angiotensin II (Ang II) plays an significant role. We therefore hypothesized that ascorbic acid could modify the pharmacological properties of the AT(1) receptor for Ang II. METHODS: Binding studies and Ca(2+) mobilization studies were performed with HEK293 cells stably expressing the AT(1) receptor for Ang II. Smooth muscle contraction studies were performed with rabbit aorta strips that endogenously express the AT(1) receptor. RESULTS: Scatchard analysis revealed that ascorbic acid decreased the binding affinity of the AT(1) receptor without modifying its maximal binding capacity. Ascorbic acid did not modify the binding affinity of the AT(2) receptor for Ang II or of the UT receptor for urotensin II. In single-cell Ca(2+) imaging assays, ascorbic acid reduced the frequency of intracellular Ca(2+) oscillations induced by a low dose of Ang II. In functional assays, ascorbic acid significantly diminished the contraction of rabbit aorta pre-contracted with Ang II but not those pre-contracted with urotensin II. CONCLUSIONS: Ascorbic acid decreases the binding affinity of the AT(1) receptor. These results offer a mechanistic explanation for the reported blood pressure lowering effect of ascorbic acid.

Biological properties and functional determinants of the urotensin II receptor.

The urotensin II receptor (UT) is a member of the G protein-coupled receptor (GPCR) family and binds the cyclic undecapeptide urotensin II (U-II) as well as the octapeptide urotensin II-related peptide (URP). The active UT mediates pleiotropic effects through various signal transduction pathways, including coupling to G proteins and activating the mitogen-activated protein kinase pathway. Several highly conserved residues and motifs of class A GPCRs that are important for activity are found in UT. This review highlights some of the putative roles of these motifs in the binding, activation and desensitization of UT.