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Biomedical subjects

G Tigyi

Publications and source records attributed to G Tigyi.

At least 19 recordsLinked to original sources
PubMed2001

A single amino acid determines lysophospholipid specificity of the S1P1 (EDG1) and LPA1 (EDG2) phospholipid growth factor receptors.

The phospholipid growth factors sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are ligands for the related G protein-coupled receptors S1P(1)/EDG1 and LPA(1)/EDG2, respectively. We have developed a model of LPA(1) that predicts interactions between three polar residues and LPA. One of these, glutamine 125, which is conserved in the LPA receptor subfamily (LPA(1)/EDG2, LPA(2)/EDG4, and LPA(3)/EDG7), hydrogen bonds with the LPA hydroxyl group. Our previous S1P(1) study identified that the corresponding glutamate residue, conserved in all S1P receptors, ion pairs with the S1P ammonium. These two results predict that this residue might influence ligand recognition and specificity. Characterization of glutamate/glutamine interchange point mutants of S1P(1) and LPA(1) validated this prediction as the presence of glutamate was required for S1P recognition, whereas LPA recognition was possible with either glutamine or glutamate. The most likely explanation for this dual specificity behavior is a shift in the equilibrium between the acid and conjugate base forms of glutamic acid due to other amino acids surrounding that position in LPA(1), producing a mixture of receptors including those having an anionic glutamate that recognize S1P and others with a neutral glutamic acid that recognize LPA. Thus, computational modeling of these receptors provided valid information necessary for understanding the molecular pharmacology of these receptors.

Amino Acid Sequence↗
PubMed2001

Sphingosylphosphocholine is a naturally occurring lipid mediator in blood plasma: a possible role in regulating cardiac function via sphingolipid receptors.

Blood plasma and serum contain factors that activate inwardly rectifying GIRK1/GIRK4 K+ channels in atrial myocytes via one or more non-atropine-sensitive receptors coupled to pertussis-toxin-sensitive G-proteins. This channel is also the target of muscarinic M(2) receptors activated by the physiological release of acetylcholine from parasympathetic nerve endings. By using a combination of HPLC and TLC techniques with matrix-assisted laser desorption ionization-time-of-flight MS, we purified and identified sphingosine 1-phosphate (SPP) and sphingosylphosphocholine (SPC) as the plasma and serum factors responsible for activating the inwardly rectifying K+ channel (I(K)). With the use of MS the concentration of SPC was estimated at 50 nM in plasma and 130 nM in serum; those concentrations exceeded the 1.5 nM EC(50) measured in guinea-pig atrial myocytes. With the use of reverse-transcriptase-mediated PCR and/or Western blot analysis, we detected Edg1, Edg3, Edg5 and Edg8 as well as OGR1 sphingolipid receptor transcripts and/or proteins. In perfused guinea-pig hearts, SPC exerted a negative chronotropic effect with a threshold concentration of 1 microM. SPC was completely removed after perfusion through the coronary circulation at a concentration of 10 microM. On the basis of their constitutive presence in plasma, the expression of specific receptors, and a mechanism of ligand inactivation, we propose that SPP and SPC might have a physiologically relevant role in the regulation of the heart.

Animals↗
PubMed2001

Physiological responses to lysophosphatidic acid and related glycero-phospholipids.

1-Acyl-2-hydroxy(lyso)-sn-glycero-3-phosphate (lysophosphatidic acid, LPA) has attracted a lot of attention in recent years due to the wide range of its biological effects that span the phylogenetic tree from slime mold to human. LPA can be viewed as a pleiotropic phospholipid growth factor that utilizes the same signal transduction mechanisms as traditional polypeptide growth factors; however, LPA activates these mechanism via specific G protein-coupled receptors. The concentration of LPA in serum is in the high micromolar range, making it the most abundant mitogen/survival factor present in serum, one that is often unknowingly utilized in tissue culture. The present review gives a historical perspective and a critical analysis of the LPA literature with a special emphasis on the physiological implications of its effects.

Cell Cycle↗
PubMed2001

Topical application of the phospholipid growth factor lysophosphatidic acid promotes wound healing in vivo.

The lipid mediator lysophosphatidic acid (LPA) regulates cell proliferation and enhances cell motility in vitro, both of which are important events in wound healing. To evaluate the effects of LPA in vivo, it was applied to a full-thickness wound of rat skin. LPA in micromolar concentrations, or solvent, was applied daily. Animals were killed at 1, 3, 6, and 9 days after wounding and processed for histological evaluation, including hematoxylin-eosin staining and histochemical markers for macrophage-histiocytes, proliferating cells, and capillary endothelial cells. LPA treatment accelerated wound closing and increased neoepithelial thickness. Cytological evaluation showed no evidence for a secondary inflammation-mediated injury, infection, or increased keloid formation. Whereas LPA caused only a modest dose-dependent increase in proliferating cells, a marked increase in the immigration of histiocyte-macrophage cells was observed as early as day 1. The peaks of several cytological features and immunohistological markers preceded those of the untreated side. Our data suggest that exogenously applied LPA in this model promotes healing and that macrophage-histiocytes are the primary LPA-responsive cells in vivo.

Administration, Topical↗
PubMed2001

Short-chain phosphatidates are subtype-selective antagonists of lysophosphatidic acid receptors.

Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are members of the phospholipid growth factor family. A major limitation in the field to date has been a lack of receptor subtype-specific agonists and antagonists. Here, we report that dioctylglycerol pyrophosphate and dioctylphosphatidic acid are selective antagonists of the LPA(1) and LPA(3) receptors, but prefer LPA(3) by an order of magnitude. Neither molecule had an agonistic or antagonistic effect on LPA(2) receptor. Consistent with this receptor subtype selectivity, dioctylglycerol pyrophosphate inhibited cellular responses to LPA in NIH3T3 fibroblasts, HEY ovarian cancer cells, PC12 pheochromocytoma cells, and Xenopus laevis oocytes. Responses elicited by S1P in these cell lines that endogenously express S1P(1), S1P(2), S1P(3), and S1P(5) receptors were unaffected by dioctylglycerol pyrophosphate. Responses evoked by the G protein-coupled receptor ligands acetylcholine, serotonin, ATP, and thrombin receptor-activating peptide were similarly unaffected, suggesting that the short-chain phosphatidates are receptor subtype-specific lysophosphatidate antagonists.

3T3 Cells↗
PubMed2000

Identification of Edg1 receptor residues that recognize sphingosine 1-phosphate.

Originating from its DNA sequence, a computational model of the Edg1 receptor has been developed that predicts critical interactions with its ligand, sphingosine 1-phosphate. The basic amino acids Arg(120) and Arg(292) ion pair with the phosphate, whereas the acidic Glu(121) residue ion pairs with the ammonium moiety of sphingosine 1-phosphate. The requirement of these interactions for specific ligand recognition has been confirmed through examination of site-directed mutants by radioligand binding, ligand-induced [(35)S]GTPgammaS binding, and receptor internalization assays. These ion-pairing interactions explain the ligand specificity of the Edg1 receptor and provide insight into ligand specificity differences within the Edg receptor family. This computational map of the ligand binding pocket provides information necessary for understanding the molecular pharmacology of this receptor, thus underlining the potential of the computational method in predicting ligand-receptor interactions.

Amino Acid Sequence↗
PubMed2000

Sphingosine 1-phosphate as a major bioactive lysophospholipid that is released from platelets and interacts with endothelial cells.

The serum-borne lysophospholipid mediators sphingosine 1-phosphate (Sph-1-P) and lysophosphatidic acid (LPA) have been shown to be released from activated platelets and to act on endothelial cells. In this study, we employed the repeated lipid extraction (under alkaline and acidic conditions), capable of detecting Sph-1-P, LPA, and possibly structurally similar lysophospholipids, whereby a marked formation of [(32)P]Sph-1-P, but not [(32)P]LPA, was observed in [(32)P]orthophosphate-labeled platelets. Platelet Sph-1-P release, possibly mediated by protein kinase C, was greatly enhanced in the presence of albumin, which formed a complex with Sph-1-P. This finding suggests that platelet Sph-1-P may become accessible to depletion by albumin when its transbilayer movement (flipping) across the plasma membrane is enhanced by protein kinase C. Although human umbilical vein endothelial cells expressed receptors for both Sph-1-P and LPA, Sph-1-P acted much more potently than LPA on the cells in terms of intracellular Ca(++) mobilization, cytoskeletal reorganization, and migration. The results suggest that Sph-1-P, rather than LPA, is a major bioactive lysophospholipid that is released from platelets and interacts with endothelial cells, under the conditions in which critical platelet-endothelial interactions (including thrombosis, angiogenesis, and atherosclerosis) occur. Furthermore, albumin-bound Sph-1-P may account for at least some of the serum biological activities on endothelial cells, which have been ascribed to the effects of albumin-bound LPA, based on the similarities between LPA and serum effects.

Biological Transport↗
PubMed2000

Lipid phosphate phosphatase-1 and Ca2+ control lysophosphatidate signaling through EDG-2 receptors.

The serum-derived phospholipid growth factor, lysophosphatidate (LPA), activates cells through the EDG family of G protein-coupled receptors. The present study investigated mechanisms by which dephosphorylation of exogenous LPA by lipid phosphate phosphatase-1 (LPP-1) controls cell signaling. Overexpressing LPP-1 decreased the net specific cell association of LPA with Rat2 fibroblasts by approximately 50% at 37 degrees C when less than 10% of LPA was dephosphorylated. This attenuated cell activation as indicated by diminished responses, including cAMP, Ca(2+), activation of phospholipase D and ERK, DNA synthesis, and cell division. Conversely, decreasing LPP-1 expression increased net LPA association, ERK stimulation, and DNA synthesis. Whereas changing LPP-1 expression did not alter the apparent K(d) and B(max) for LPA binding at 4 degrees C, increasing Ca(2+) from 0 to 50 micrometer increased the K(d) from 40 to 900 nm. Decreasing extracellular Ca(2+) from 1.8 mm to 10 micrometer increased LPA binding by 20-fold, shifting the threshold for ERK activation to the nanomolar range. Hence the Ca(2+) dependence of the apparent K(d) values explains the long-standing discrepancy of why micromolar LPA is often needed to activate cells at physiological Ca(2+) levels. In addition, the work demonstrates that LPP-1 can regulate specific LPA association with cells without significantly depleting bulk LPA concentrations in the extracellular medium. This identifies a novel mechanism for controlling EDG-2 receptor activation.

Animals↗
PubMed2000

Pharmacological characterization of phospholipid growth-factor receptors.

The phospholipid growth-factor (PLGE) terminology is proposed to describe a group of endogenous glycerol- and sphingolipid mediators that regulate cell proliferation through plasma membrane receptors. In addition to LPA and SPP, multiple PLGFs are present in blood plasma and serum. PLGF activity is regulated by its stimulus-coupled production and by endogenous inhibitors. In addition to LPA and SPP, alkenyl-glycerophosphate, cyclic-phosphatidic acid, and sphingosylphosphorylcholine were detected in biological fluids using mass spectrometry. Heterologous desensitization studies indicate the expression of multiple LPA-activated receptors in a variety of cell types, which are differentially activated by the different PLGFs. Northern blot and RT-PCR results reinforce the coexpression of PSP24 alpha and different members of the EDG1-7 receptors in the same cell. Stable heterologous expression of the PSP24 alpha, EDG2, and EDG4 receptors in HEK293 cells show distinct PLGF specificities and dose-response properties for each receptor subtype. Thus, both the controlled availability of the different agonists/inhibitors and the regulated expression of their receptors regulate the biological effects of PLGFs.

Animals↗
PubMed1999

Localization of the PAK1-, WASP-, and IQGAP1-specifying regions of Cdc42.

The Rho family small GTPase Cdc42 transmits divergent intracellular signals through multiple effector proteins to elicit cellular responses such as cytoskeletal reorganization. Potential effectors of Cdc42 implicated in mediating its cytoskeletal effect in mammalian cells include PAK1, WASP, and IQGAP1. To investigate the determinants of Cdc42-effector specificity, we utilized recombinant Cdc42 mutants and chimeras made between Cdc42 and RhoA to map the regions of Cdc42 contributing to specific effector p21-binding domain (PBD) interaction. Site-directed mutants of the switch I domain and neighboring regions of Cdc42 demonstrated differential binding patterns toward the PBDs of PAK1, WASP, and IQGAP1, suggesting that switch I provides essential determinants for the effector binding, but recognition of each effector by Cdc42 involves a distinct mechanism. Differing from Rac1, the switch I domain and the surrounding region (amino acids 29 to 55) of Cdc42 appeared to be sufficient for specific binding to PAK1, whereas determinants outside the switch I domain, residues 157-191 and 84-120 in particular, were necessary and sufficient to confer specificity to WASP and IQGAP1, respectively. In addition, IQGAP1, but not PAK1 nor WASP, required the unique "insert region," residues 122-134, of Cdc42 to achieve high affinity binding. Microinjection of the constitutively active Cdc42/RhoA chimeras into serum-starved Swiss 3T3 cells showed that although preserving PAK1- and WASP-binding activity could retain the peripheral actin microspike (PAM)-inducing activity of Cdc42, interaction with PAK1 or WASP was not required for this activity. Moreover, IQGAP1-binding alone by Cdc42 was insufficient for PAM-induction. Thus, Cdc42 utilizes multiple distinct structural determinants to specify different effector recognition and to elicit PAM-inducing effect.

3T3 Cells↗
PubMed1999

Structurally similar oxidized phospholipids differentially regulate endothelial binding of monocytes and neutrophils.

We previously have demonstrated that oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC), a component of minimally modified low density lipoprotein (MM-LDL), activates endothelial cells to bind monocytes. 1-Palmitoyl-2- (5-oxovaleroyl)-sn-glycero-3-phosphorylcholine (POVPC) and 1- palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC), which are present in OxPAPC, MM-LDL, and atherosclerotic lesions, were shown to have a major role in the activation of endothelial cells. We now demonstrate that these two highly similar molecules have dramatically different effects on leukocyte endothelial interactions. POVPC is a potent regulator of monocyte-specific endothelial interactions. Treatment of endothelial cells with POVPC increased monocyte binding by inducing the surface expression of the connecting segment 1 domain of fibronectin; no increase in neutrophil binding was observed. In addition, POVPC strongly inhibited lipopolysaccharide-mediated induction of neutrophil binding and expression of E-selectin protein and mRNA. This inhibition was mediated by a protein kinase A-dependent pathway, resulting in down-regulation of NF-kappaB-dependent transcription. In contrast, PGPC induced both monocyte and neutrophil binding and expression of E-selectin and vascular cell adhesion molecule 1. We present evidence to suggest that the two phospholipids act by different novel receptors present in Xenopus laevis oocytes and that POVPC, but not PGPC, stimulates a cAMP-mediated pathway. At concentrations equal to that present in MM-LDL, the effect of POVPC dominates and inhibits PGPC-induced neutrophil binding and E-selectin expression in endothelial cells. In summary, our data provide evidence that both POVPC and PGPC are important regulators of leukocyte-endothelial interactions and that POVPC may play a dominant role in a number of chronic inflammatory processes where oxidized phospholipids are known to be present.

Aorta↗