Antagonism of the ATP-gated P2X7 receptor: a potential therapeutic strategy for cancer

Purinergic Signalling - The P2X receptor 7 (P2X7R) is a plasma membrane receptor sensing extracellular ATP associated with a wide variety of cellular functions. It is most commonly expressed on...     

Internalization of the TXA2 receptor alpha and beta isoforms. Role of the differentially spliced cooh terminus in agonist-promoted receptor internalization

Thromboxane A2 (TXA2) potently stimulates platelet aggregation and smooth muscle constriction and is thought to play a role in myocardial infarction, atherosclerosis, and bronchial asthma. The TXA2 receptor (TXA2R) is a member of the G protein-coupled receptor family and is found as two alternatively spliced isoforms, alpha (343 residues) and beta (407 residues), which share the first 328 residues. In the present report, we demonstrate by enzyme-linked immunosorbent assay and immunofluorescence microscopy that the TXA2Rbeta, but not the TXA2Ralpha, undergoes agonist-induced internalization when expressed in HEK293 cells as well as several other cell types. Various dominant negative mutants were used to demonstrate that the internalization of the TXA2Rbeta is dynamin-, GRK-, and arrestin-dependent in HEK293 cells, suggesting the involvement of receptor phosphorylation and clathrin-coated pits in this process. Interestingly, the agonist-stimulated internalization of both the alpha and beta isoforms, but not of a mutant truncated after residue 328, can be promoted by overexpression of arrestin-3, identifying the C-tails of both receptors as necessary in arrestin-3 interaction. Simultaneous mutation of two dileucine motifs in the C-tail of TXA2Rbeta did not affect agonist-promoted internalization. Analysis of various C-tail deletion mutants revealed that a region between residues 355 and 366 of the TXA2Rbeta is essential for agonist-promoted internalization. These data demonstrate that alternative splicing of the TXA2R plays a critical role in regulating arrestin binding and subsequent receptor internalization.     

Heterogeneity of packing: structural approach.

Analysis of the heterogeneity of packing in proteins showed that different groups of the protein preferentially contribute to low- or high-density regions. Statistical distribution reveals the two preferable values for packing density in the form of two peaks. One peak occurs in the range of densities 0.55-0.65, the other occurs in the range 0.75-0.8. The high-density peak is originated primarily by high packing inside the hydrogen bonded backbone and to some extent by side chains. Polar/charged and apolar side chains both contribute to the low-density peak. The average packing density values of individual atomic groups significantly vary for backbone atoms as well as for side chain atoms. The carbonyl oxygen atoms of protein backbone and the end groups of side chains show lower packing density than the rest of the protein. The side-chain atomic groups of a secondary structure element when packed against the neighboring secondary structure element form stronger contacts with the side chains of this element than with its backbone. Analysis of the low-density regions around each buried peptide group was done for the set of proteins with different types of packing, including alpha-alpha, alpha-beta, and beta-beta packing. It was shown that cavities are regularly situated in the groove of secondary structure element packed against neighboring elements for all types of packing. Low density in the regions surrounding the peptide groups and the end groups of side chains can be explained by their positioning next to a cavity formed upon the association of secondary structure elements. The model proposed can be applied to the analysis of protein internal motions, mechanisms of cellular signal transduction, diffusion through protein matrix, and other events.     

Costimulation of the Gi-coupled ADP receptor and the Gq-coupled TXA2 receptor is required for ERK2 activation in collagen-induced platelet aggregation

The stimulation of platelets by low doses of collagen induces extracellular signal-regulated kinase 2 (ERK2) activation. In this report, we demonstrate that collagen-induced ERK2 activation depends on thromboxane A(2) (TXA(2)) formation and ADP release. The collagen-induced ERK2 activation was inhibited by indomethacin (88%) and by AR-C69931MX (70%), a specific antagonist of P2Y12, a Gi-coupled ADP receptor. AR-C69931MX (10 microM) inhibition was overcome by epinephrine (1 microM), an agonist of the Gi-coupled alpha(2A)-adrenergic receptor, suggesting that the Gi-coupled receptor was necessary for ERK2 activation by collagen. By contrast, MRS 2179 (10 microM), a specific antagonist of P2Y1, a Gq-coupled ADP receptor, did not affect collagen-induced ERK2 activation. Little or no ERK2 activation was observed with ADP alone (10 microM). By contrast, U46619 (10 microM), a stable analog of TXA(2), induced ERK2 activation in an ADP-dependent manner, via the P2Y12 receptor. These results suggest that the Gi-dependent signaling pathway, stimulated by ADP or epinephrine, was not the only pathway required for ERK2 activation by collagen. Costimulation of the specific G(12/13)-coupled TXA(2) receptor with a low dose of U46619 (10 nM) and of Gi- and Gq-coupled ADP receptor (10 microM) induced very low levels of ERK2 activation, similar to those observed with ADP alone, suggesting that G(12/13) is not involved or not sufficient to induce the additional pathway necessary for ERK2 activation. The Gq-coupled TXA(2) receptor was required for ERK2 activation by U46619 (10 microM) and low doses of collagen, clearly showing that a coordinated pathway through both Gq from TXA(2) and Gi from ADP was necessary for ERK2 activation. Finally, we demonstrate that ERK2 activation is involved in collagen-induced aggregation and secretion.     

Effect of thromboxane A2 (TXA2) synthase inhibitor and TXA2 receptor antagonist alone and in combination on antigen-induced bronchoconstriction in guinea pigs

Thromboxane A2 (TXA2) causes bronchoconstriction and bronchial hyperresponsiveness. Two types of TXA2 modifiers, one synthase inhibitor and one receptor antagonist, are widely used for the treatment of asthma in Japan. Although the target of TXA2 modifiers is to inhibit bioactivity of TXA2, the pharmacological properties are somewhat different between these drugs. We studied the inhibitory effects of the TXA2 synthase inhibitor CS-518 and the TXA2 receptor antagonist S-1452 alone and in combination on antigen-induced bronchoconstriction in passively sensitized guinea pigs treated with diphenhydramine. Both CS-518 and S-1452 inhibited the antigen-induced bronchoconstriction dose-dependently with the plateau. The combination of these drugs at the maximal inhibitory doses did not have any more effect compared with each single dosing. The combination at the submaximal doses tended to show an additive effect, but the effect was not significant. These findings suggest that other prostanoids such as PGE2, PGI2, PGD2 and PGF2alpha may not take an important role in the antiasthmatic effects of TXA2 modifiers.     

17β Estradiol-induced increase in brain dopamine D-2 receptor: Antagonism by MIF-1

Animal behavioral and neurochemical studies implicate dopaminergic systems in the neurological sequelae induced by estrogen. In the present study, we demonstrated for the first time that MIF-1, a neuropeptide unrelated to classical dopamine agonists, when given prior to, concurrently with, and after 17β-estradiol, antagonized significantly the estrogen-induced increase in the density of dopamine D-2 receptor both in the striatum and the mesolimbic area of male rat brain. The current findings have implications for the prophylactic and therapeutic potential for MIF-1 in extrapyramidal motor disorders caused by estrogen imbalance in humans.     

Modulation of TXA2 generation of platelets by human lipoproteins

The lipoprotein (LP) fractions VLDL, LDL, HDL2 and HDL3 were prepared by ultracentrifugation of plasma from healthy volunteers and from patients with coronary heart disease (CHD). We investigated the capacity of platelets from healthy volunteers and patients with atherosclerosis to generate thromboxane A2 (TXA2) during spontaneous clotting of whole blood under the influence of the lipoprotein fractions. In our experiments the serum concentration of TXB2, reflecting the capacity of platelets to generate TXA2 during clotting, depends on several factors: the type of LP fraction used, the blood used for generation of TXA2, and for the same LP fraction whether it was taken from plasma of healthy volunteers or patients with CHD. VLDL prepared from plasma of healthy volunteers inhibited but VLDL prepared from plasma of patients with CHD enhanced the TXA2 formation of platelets from healthy volunteers (p less than 0.05, resp.). LDL from CHD patients inhibited the TXA2 formation of platelets from atherosclerotic patients (p less than 0.01). The HDL subfractions HDL2 and HDL3 from healthy volunteers inhibited TXA2 formation by platelets from healthy volunteers as well as those from atherosclerotic patients (p less than 0.05; p less than 0.01, respectively). HDL2 from patients with CHD inhibited only the TXA2 formation of platelets from healthy volunteers (p less than 0.01), whereas HDL3 from CHD patients inhibited only the TXA2 formation of platelets from atherosclerotic patients (p less than 0.01).     

Binding of a thromboxane A2 (TXA2)/prostaglandin H2 (PGH2) receptor antagonist to rabbit and pig heart membrane protein.

This study aimed at testing the hypothesis that the binding sites for TXA2/PGH2 are present and different in the heart as compared to platelets and blood vessels. Kinetic studies on the thromboxane binding to protein of membrane preparations from rabbit and pig hearts were carried out using [125I]-PTA-OH, a potent specific thromboxane receptor antagonist. The following points are stressed: 1. the binding sites to 125I-PTA-OH were shown to be present in the heart membrane whatever the adopted experimental conditions were i.e. the temperature (4 or 30 degrees C) used for incubation the protein fractions under study: either the 105,000 g supernatant or the 200,000 g supernatant of the solubilized pellet (105,000 g) the animal species: rabbit and pig 2. the radioligand binding was rapid, saturable and reversible 3. the kinetically determined Kd's were in the picomolar range--11 and 14 pM for the rabbit and pig heart membrane preparation respectively--instead of the nanomolar range found in other tissues of the nanomolar range found in other tissues--27-39 nM and 2 nM for human platelets and bovine artery endothelial cells respectively- using the same thromboxane receptor antagonist.     

Protein structural homology: a metric approach

The flexibility of the polypeptide fold of proteins is essentially due to the rotational freedom about the main chain bonds involving C alpha atoms. The polypeptide fold can therefore be represented by virtual bonds joining consecutive C alpha atoms. The ordered sequence of virtual torsion and bond angles involving these bonds can be used to specify the fold. Such representations can then be compared to reveal structural similarities using the Needleman & Wünsch algorithm, which has been developed for comparison of amino acid sequences. Such an approach is presented and illustrated with examples. The method is suitable for detecting structural similarities that extend over 7 or more residues.     

Metabolic control theory: a structural approach

In the general framework of metabolic control theory, we describe a method of mathematical modelling that provides a way of analysing the sensitivity of a metabolic system to perturbations of the environment or of the internal state of this system. The method can be applied to any metabolic system, involving for instance conservation relationships, non-specific external parameters, etc., and leads in particular to a characterization of the control matrices and to a generalization of the summation and connectivity theorems. In this paper, we emphasize the structural characterizations and properties of the systems which depend only on the structure of the metabolic network, and not on the reaction kinetics. The advantage of this approach lies of course in the fact that the structure of the metabolic network is an invariant of the system which depends neither on the environment nor on the internal state of this system. The aim of this paper is to show the efficiency of such a structural approach.     

The PGI2-analogue iloprost and the TXA2-receptor antagonist sulotroban synergistically inhibit TXA2-dependent platelet activation

The stable PGI2-analogue iloprost and the TXA2-receptor antagonist sulotroban (BM 13177) were investigated for possible synergistic effects on platelet aggregation in human platelet rich plasma in vitro. Iloprost and sulotroban synergistically inhibited U 46619, collagen, and the second wave of ADP-induced platelet aggregation. Iloprost and sulotroban at concentrations showing little or no inhibition alone resulted, in combination, in marked or complete inhibition of U 46619 or collagen induced aggregation. Combination of iloprost 10(-10) M, which had no effect on the concentration-response curve (CRC) to U 46619, with sulotroban 5 x 10(-6) M, which shifted the CRC to U 46619 by a factor of 3 to the right, resulted in a rightward shift of the U 46619 CRC by a factor of 4.5. To attain a 4.5-fold shift with either compound alone, a concentration of 5 x 10(-10) M iloprost or 10(-5) M sulotroban was required. A similar mutual enhancement of inhibitory effects was seen for combinations of the PGI2-analogue cicaprost (ZK 96.480) with sulotroban or the TXA2-receptor antagonist SQ 29548 with iloprost. When the TXA2-dependent part of collagen-induced aggregation was fully inhibited by sulotroban, the concentrations of iloprost necessary for 90% inhibition were reduced by a factor of 2.5 - 3. In the presence of acetylsalicylic acid, the synergistic action of sulotroban and iloprost was reduced and merely additive effects against U 46619-induced platelet aggregation were found, suggesting that the release of endogenous TXA2 plays an important role for the synergistic effect of the two compounds. The combination of a PGI2-analogue and a TXA2-antagonist may lead to a safer and more effective control of platelet activation than with either compound alone.     

Simplified modeling approach suggests structural mechanisms for constitutive activation of the C5a receptor

Molecular modeling of conformational changes occurring in the transmembrane region of the complement factor 5a receptor (C5aR) during receptor activation was performed by comparing two constitutively active mutants (CAMs) of C5aR, NQ (I124N/L127Q), and F251A, to those of the wild-type C5aR and NQ-N296A (I124N/L127Q/N296A), which have the wild-type phenotype. Modeling involved comprehensive sampling of various rotations of TM helices aligned to the crystal template of the dark-adapted rhodopsin along their long axes. By assuming that the relative energies of the spontaneously activated states of CAMs should be lower or at least comparable to energies characteristic for the ground states, we selected the plausible models for the conformational states associated with constitutive activation in C5aR. The modeling revealed that the hydrogen bonds between the side chains of D82-N119, S85-N119, and S131-C221 characteristic for the ground state were replaced by the hydrogen bonds D82-N296, N296-Y300, and S131-R134, respectively, in the activated states. Also, conformational transitions that occurred upon activation were hindered by contacts between the side chains of L127 and F251. The results rationalize the available data of mutagenesis in C5aR and offer the first specific molecular mechanism for the loss of constitutive activity in NQ-N296A. Our results also contributed to understanding the general structural mechanisms of activation in G-protein-coupled receptors lacking the "ionic lock", R(3.50) and E/D(6.30). Importantly, these results were obtained by modeling approaches that deliberately simplify many elements in order to explore potential conformations of GPCRs involving large-scale molecular movements.     

The structural evolution of a P2Y-like G-protein-coupled receptor

Based on the now available crystallographic data of the G-protein-coupled receptor (GPCR) prototype rhodopsin, many studies have been undertaken to build or verify models of other GPCRs. Here, we mined evolution as an additional source of structural information that may guide GPCR model generation as well as mutagenesis studies. The sequence information of 61 cloned orthologs of a P2Y-like receptor (GPR34) enabled us to identify motifs and residues that are important for maintaining the receptor function. The sequence data were compared with available sequences of 77 rhodopsin orthologs. Under a negative selection mode, only 17% of amino acid residues were preserved during 450 million years of GPR34 evolution. On the contrary, in rhodopsin evolution approximately 43% residues were absolutely conserved between fish and mammals. Despite major differences in their structural conservation, a comparison of structural data suggests that the global arrangement of the transmembrane core of GPR34 orthologs is similar to rhodopsin. The evolutionary approach was further applied to functionally analyze the relevance of common scaffold residues and motifs found in most of the rhodopsin-like GPCRs. Our analysis indicates that, in contrast to other GPCRs, maintaining the unique function of rhodopsin requires a more stringent network of relevant intramolecular constrains.     

Synthesis of [17,18-3H] trans-13-azaprostanoic acid. A labeled probe for the PGH2/TXA2 receptor

Because of its highly unstable nature, TXA2, produced by platelet metabolism of arachidonic acid, does not lend itself to use as a receptor probe for its own receptor. As such, the stable TXA2/PGH2 antagonist, trans-13-azaprostanoic acid (trans-13-APA, 12b), was prepared as the [17, 18 3H] derivative [( 3H] trans-13-APA, 12c) to study this receptor and to better evaluate the mechanism of action of these azaprostanoids. Tritiated trans-13-APA, 12c, was prepared in nearly theoretical specific activity (57 Ci/mmole) from (17Z)-trans-13-azaprost-17-enoic acid (11b) by catalytic tritiation. The unsaturated 11b was prepared by condensation of cis-7-amino-3-heptene (8) with 2-(6-carboxyhexyl) cyclopentanone (9), NaBH4 reduction, chromatography, and hydrolysis of the trans isomer so isolated. The olefins 11a and b were also of biochemical interest because of the unsaturation in the lower side chain. The presence of similar unsaturation in PGH3(4) and TXA3 (3) renders these prostaglandins inactive as proaggregatory agents. Evaluation of the antiaggregatory activity of 11a and b indicated it to be about the same potency in inhibiting human platelet aggregation as the parent cis and trans-13-APAs, suggesting that introduction of a double bond at the 17 position in platelet prostaglandin antagonists is unlikely to result in enhanced antiplatelet activity.     

The yeast alpha-factor receptor: structural properties deduced from the sequence of the STE2 gene.

The STE2 gene of the yeast Saccharomyces cerevisiae encodes a component of the receptor for the oligopeptide pheromone alpha-factor. We have cloned and determined the nucleotide sequence of the STE2 gene. A sequence involved in the control of cell-type expression of the STE2 gene was found 5' of an open reading frame that could encode a protein of 431 amino acids. The predicted STE2 protein contains seven hydrophobic segments, suggesting that the alpha-factor receptor is an integral membrane protein. No extensive homology at the primary sequence level was detected between the predicted STE2 protein and other available protein sequences.     

Ecological determinants of divorce: a structural approach to the explanation of Japanese divorce.

This paper examines the ecological determinants of contemporary Japanese divorce rates on the prefectural level. LISREL and computer-generated graphics are the analytic methods used. The aggregate level of analysis demands the use of the ecological model which posits that demographic changes, economic activities, migration patterns, and the level of urbanization are significant predictors of divorce rate. Our analysis demonstrates that sex ratio, female labor force participation, female in-migration patterns, population increase, and net household income all play a significant role in affecting the divorce rate. Our findings also confirm the well-supported hypothesis that both population density and modernization positively influence modern Japan's divorce rates. The residual analysis also points out that in order to account for the large proportion of the unexplained variance of Japanese divorce, behavioral-related variables and island- or prefecture-specific dimensions need to be included in the ecological model of divorce.     

Design of a minimal protein oligomerization domain by a structural approach

Because of the simplicity and regularity of the alpha-helical coiled coil relative to other structural motifs, it can be conveniently used to clarify the molecular interactions responsible for protein folding and stability. Here we describe the de novo design and characterization of a two heptad-repeat peptide stabilized by a complex network of inter- and intrahelical salt bridges. Circular dichroism spectroscopy and analytical ultracentrifugation show that this peptide is highly alpha-helical and 100% dimeric tinder physiological buffer conditions. Interestingly, the peptide was shown to switch its oligomerization state from a dimer to a trimer upon increasing ionic strength. The correctness of the rational design principles used here is supported by details of the atomic structure of the peptide deduced from X-ray crystallography. The structure of the peptide shows that it is not a molten globule but assumes a unique, native-like conformation. This de novo peptide thus represents an attractive model system for the design of a molecular recognition system.     

Effect of thromboxane A(2) (TXA(2)) synthase inhibitor and TXA(2) receptor antagonist alone and in combination on antigen-induced bronchoconstriction in guinea pigs

Thromboxane A(2) (TXA(2)) causes bronchoconstriction and bronchial hyperresponsiveness. Two types of TXA(2) modifiers, one synthase inhibitor and one receptor antagonist, are widely used for the treatment of asthma in Japan. Although the target of TXA(2) modifiers is to inhibit bioactivity of TXA(2), the pharmacological properties are somewhat different between these drugs. We studied the inhibitory effects of the TXA(2) synthase inhibitor CS-518 and the TXA(2) receptor antagonist S-1452 alone and in combination on antigen-induced bronchoconstriction in passively sensitized guinea pigs treated with diphenhydramine.Both CS-518 and S-1452 inhibited the antigen-induced bronchoconstriction dose-dependently with the plateau. The combination of these drugs at the maximal inhibitory doses did not have any more effect compared with each single dosing. The combination at the submaximal doses tended to show an additive effect, but the effect was not significant.These findings suggest that other prostanoids such as PGE(2), PGI(2), PGD(2) and PGF(2alpha) may not take an important role in the antiasthmatic effects of TXA(2) modifiers.