BBA - Molecular and Cell Biology of Lipids (v.1761, #9)

NF-κB independent inhibition of lipopolysaccharide-induced cyclooxygenase by a conjugated linoleic acid cognate, conjugated nonadecadienoic acid by Guangming Li; Baiyan Dong; Daniel E. Butz; Yeonhwa Park; Michael W. Pariza; Mark E. Cook (969-972).
10t, 12c-CLA was shown to inhibit COX-2 expression through the NF-κB pathway. In the current study, conjugated nonadecadienoic acid (CNA) was shown to decrease inducible COX-2 protein and mRNA and PGE2 release to the similar extent as 10t, 12c-CLA in Raw264.7 macrophage. However, unlike 10t, 12c-CLA, inhibition of COX-2 mRNA/protein by CNA was independent of the NF-κB pathway. The data indicate the regulation of COX-2 by select conjugated fatty acids and hence their anti-inflammatory actions could operate through different signal transduction pathways.
Keywords: Conjugated linoleic acid; Conjugated nonadecadienoic acid; Cyclooxygenase; NF-κB; Prostaglandin;

Peroxisomal multifunctional protein-2: The enzyme, the patients and the knockout mouse model by Steven Huyghe; Guy P. Mannaerts; Myriam Baes; Paul P. Van Veldhoven (973-994).
The mammalian multifunctional protein-2 (MFP-2, also called multifunctional enzyme 2, D-bifunctional enzyme or 17-β-estradiol dehydrogenase type IV) was identified by several groups about a decade ago. It plays a central role in peroxisomal β-oxidation as it handles most, if not all, peroxisomal β-oxidation substrates. Deficiency of this enzyme in man causes a severe developmental syndrome with abnormalities in several organs but in particular in the brain, leading to death within the first year of life. Accumulation of branched-long-chain fatty acids and very-long-chain fatty acids and a disturbed synthesis of bile acids were documented in these patients. A mouse model with MFP-2 deficiency only partly phenocopies the human disease. Although the expected metabolic abnormalities are present, no neurodevelopmental aberrations are observed. However, the survival of these mice into adulthood allowed to document the importance of this enzyme for the normal functioning of the brain, eyes and testis. In the present review, the identification and biochemical characteristics of MFP-2, and the consequences of MFP-2 dysfunction in humans and in mice will be discussed.
Keywords: multifunctional protein 2; hydroxysteroid dehydrogenase type 4; peroxisome; β-oxidation; patients; knockout mice;

Exploring the specific features of interfacial enzymology based on lipase studies by Ahmed Aloulou; Jorge A. Rodriguez; Sylvie Fernandez; Dirk van Oosterhout; Delphine Puccinelli; Frédéric Carrière (995-1013).
Many enzymes are active at interfaces in the living world (such as in the signaling processes at the surface of cell membranes, digestion of dietary lipids, starch and cellulose degradation, etc.), but fundamental enzymology remains largely focused on the interactions between enzymes and soluble substrates. The biochemical and kinetic characterization of lipolytic enzymes has opened up new paths of research in the field of interfacial enzymology. Lipases are water-soluble enzymes hydrolyzing insoluble triglyceride substrates, and studies on these enzymes have led to the development of specific interfacial kinetic models. Structure–function studies on lipases have thrown light on the interfacial recognition sites present in the molecular structure of these enzymes, the conformational changes occurring in the presence of lipids and amphiphiles, and the stability of the enzymes present at interfaces. The pH-dependent activity, substrate specificity and inhibition of these enzymes can all result from both “classical” interactions between a substrate or inhibitor and the active site, as well as from the adsorption of the enzymes at the surface of aggregated substrate particles such as oil drops, lipid bilayers or monomolecular lipid films. The adsorption step can provide an alternative target for improving substrate specificity and developing specific enzyme inhibitors. Several data obtained with gastric lipase, classical pancreatic lipase, pancreatic lipase-related protein 2 and phosphatidylserine-specific phospholipase A1 were chosen here to illustrate these specific features of interfacial enzymology.
Keywords: Lipase; Phospholipase; Colipase; Interfacial enzymology; Enzyme structure; Lipid-protein interaction; Protein adsorption; Lipase inhibitor; PS-PLA1; PLRP2; Gastric lipase; Pancreatic lipase;

Wide proinflammatory effect of electronegative low-density lipoprotein on human endothelial cells assayed by a protein array by Sònia Benítez; Mercedes Camacho; Cristina Bancells; Luis Vila; José Luis Sánchez-Quesada; Jordi Ordóñez-Llanos (1014-1021).
Electronegative low-density lipoprotein (LDL(−)) is a modified subfraction of LDL present in plasma able to induce the release of interleukin 8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) by human umbilical vein endothelial cells (HUVEC). To ascertain whether further inflammation mediator release could be induced by LDL(−), a protein array system was used to measure 42 cytokines and related compounds. Native LDL and LDL(−) isolated from normolipemic subjects were incubated for 24 h with HUVEC and culture supernatants were used to measure inflammation mediator release. The protein array revealed that IL-6, granulocyte/monocyte colony-stimulating factor (GM-CSF) and growth-related oncogene (GRO) release were increased by cultured HUVEC in response to LDL(−). LDL(−) enhanced production of IL-6 (4-fold vs. LDL(+)), GM-CSF (4-fold), GROβ (2-fold) and GROγ (7-fold) was confirmed by ELISA. Time-course experiments revealed that IL-6 was released earlier than the other inflammation mediators, suggesting a first-wave cytokine action. However, the addition of IL-6 alone did not stimulate the production of IL-8, MCP-1 or GM-CSF. Moreover, IL-8, MCP-1 or GM-CSF alone did not promote the release of the other inflammatory molecules. Modification of LDL(+) by phospholipase A2-mediated lipolysis or by loading with non-esterified fatty acids (NEFA) reproduced the action of LDL(−), thereby suggesting the involvement of NEFA and/or lysophosphatidylcholine in the release of these molecules. Our results indicate that LDL(−) promotes a proinflammatory phenotype in endothelial cells through the production of cytokines, chemokines and growth factors.
Keywords: Electronegative LDL; Interleukin 6; Granulocyte/monocyte colony-stimulating factor; GRO; Inflammation;

Unique molecular signatures of glycerophospholipid species in different rat tissues analyzed by tandem mass spectrometry by Amy M. Hicks; Cynthia J. DeLong; Michael J. Thomas; Michael Samuel; Zheng Cui (1022-1029).
Glycerophospholipids (GPL) in animal tissues are composed of a large array of molecular species that mainly differ in the fatty acyl composition. In order to further understand the roles of GPL at the molecular level, it is necessary to have comprehensive, accurate accounts of the molecular makeup for these molecules in animal tissues. However, this task was difficult simply because the conventional technologies of profiling GPL species depended heavily on technical skill for accuracy and reliability and were extremely labor-intensive. In recent years, tandem mass spectrometry (MS/MS) proved to be a highly reliable and sensitive technology for profiling small molecules, including GPL, in biological samples. In this study, we used this technology to perform simultaneous comparative analyses for phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI) in the same lipid preparations of liver, lung, kidney, heart, pancreas, stomach, small intestine, spleen, skeleton muscle and brain of an adult rat. We produced molecular profiles of these 4 GPL classes in these 10 different tissues that are highly reproducible between different scans of the same sample and between samples from different animals. It is intriguing that each tissue was found to possess a unique signature of GPL profile that may be used to identify unknown tissues. More importantly, these profiles may also set reference points for studying changes of GPL metabolism in different physiological and pathological conditions.
Keywords: Rat tissue; Phospholipid molecular specie; Tandem mass spectrometry; Phospholipid profiling;

Gangliosides of the stroma layer participate in the interferon-gamma receptor-dependent controls of myelopoiesis by Claudia M.B. Andrade; Ana L. Ziulkoski; Aline X.S. dos Santos; Leonardo C. Boldrini; Vera M.T. Trindade; Radovan Borojevic; Fátima C.R. Guma (1030-1040).
Stroma-mediated myelopoiesis depends upon growth-factors and an appropriate intercellular microenvironment, whose polarity is relevant for granulocyte-macrophage colony stimulating factor (GM-CSF) mediated myeloid cell proliferation. Here we have studied qualitative and quantitative aspects of ganglioside participation in controls of the microenvironment required to sustain myelopoiesis. We analysed ganglioside synthesis, expression and shedding by two primary liver stromal cell cultures isolated from wild type and interferon-gamma (IFNγ) receptor knockout mice. The latter one has a higher capacity to sustain myelopoiesis. FDC-P1 myeloid growth factor-dependent cell line was used as the reporter system, monitoring the cell survival and proliferation that reflect the bio-availability and the activity of GM-CSF. Although the two stromal cells synthesised the same gangliosides their relative content was quite different. FDC-P1 proliferation decreased in cultures in which ganglioside synthesis was inhibited in the stroma, as well as in presence of stroma cell supernatants in which GM3 was neutralised by the anti-GM3 monoclonal antibody. Addition of exogenous GM3 reverted the inhibition and sustained proliferation of FDC-P1 cells. FDC-P1 cells do not accumulate GM3, but they are able to take up the stroma-produced sphingolipids. Thus, stroma has a double role in sustaining myelopoiesis, providing both growth factor(s) and ganglioside(s) required for the optimal stimulation of the myeloid cell proliferation, and the IFNγ mediated stroma-dependent controls of myelopoiesis are determinant for this cell interaction.
Keywords: Ganglioside; GM-CSF; Haemopoiesis; Myelopoiesis;

Inhibition of ATP binding to the carboxyl terminus of Kir6.2 by epoxyeicosatrienoic acids by Xiao-Li Wang; Tong Lu; Sheng Cao; Vijay H. Shah; Hon-Chi Lee (1041-1049).
Epoxyeicosatrienoic acids (EETs), the cytochrome P450 metabolites of arachidonic acid (AA), are potent and stereospecific activators of cardiac ATP-sensitive K+(KATP) channels. EETs activate KATP channels by reducing channel sensitivity to ATP. In this study, we determined the direct effects of EETs on the binding of ATP to KATP channel protein. A fluorescent ATP analog, 2,4,6-trinitrophenyl (TNP)-ATP, which increases its fluorescence emission significantly upon binding with proteins, was used for binding studies with glutathione-S-transferase (GST) Kir6.2 fusion proteins. TNP-ATP bound to GST fusion protein containing the C-terminus of Kir6.2 (GST-Kir6.2C), but not to the N-terminus of Kir6.2, or to GST alone. 11,12-EET (5 μM) did not change TNP-ATP binding K D to GST-Kir6.2C, but B max was reduced by half. The effect of 11,12-EET was dose-dependent, and 8,9- and 14,15-EETs were as effective as 11,12-EET in inhibiting TNP-ATP binding to GST-Kir6.2C. AA and 11,12-dihydroxyeicosatrienoic acid (11,12-DHET), the parent compound and metabolite of 11,12-EET, respectively, were not effective inhibitors of TNP-ATP binding to GST-Kir6.2C, whereas the methyl ester of 11,12-EET was. These findings suggest that the epoxide group in EETs is important for modulation of ATP binding to Kir6.2. We conclude that EETs bind to the C-terminus of KATP channels, inhibiting binding of ATP to the channel.
Keywords: Epoxyeicosatrienoic acid; ATP-sensitive potassium channel; Kir 6.2; ATP; TNP-ATP; binding;

Brain elongation of linoleic acid is a negligible source of the arachidonate in brain phospholipids of adult rats by James C. DeMar; Ho-Joo Lee; Kaizong Ma; Lisa Chang; Jane M. Bell; Stanley I. Rapoport; Richard P. Bazinet (1050-1059).
The extent to which the adult brain can derive some of its arachidonic acid (AA) through internalized synthesis from linoleic acid (LA) is uncertain. Thus, we determined for plasma-derived LA in vivo rates for brain incorporation, β-oxidation, and conversion to AA. Adult male unanesthetized rats, reared on a diet enriched in LA but low in AA, were infused intravenously for 5 min with [1-14C]LA. Timed arterial samples were collected until the animals were killed at 5 min and the brain was removed after microwaving. Within plasma lipids, > 96% of radioactivity was in the form of unchanged [1-14C]LA, but [14C]AA was insignificant (< 0.2%). Eighty-six percent of brain radioactivity at 5 min was present as β-oxidation products, whereas the remainder was mainly in ‘stable’ phospholipid or triglyceride as LA or AA (11 and < 1%, respectively). Unesterified unlabeled LA rapidly enters brain from plasma, but its incorporation into brain total phospholipid and triglyceride, in the form of synthesized AA, is < 1% of the amount that enters the brain. Thus, in rats fed even a diet containing low amounts of AA, the LA that enters brain is largely β-oxidized, and is not a major source of AA in brain.
Keywords: Arachidonic; Brain; Incorporation; Linoleic; Phospholipid; Rat;

The oxidized phospholipids POVPC and PGPC inhibit growth and induce apoptosis in vascular smooth muscle cells by Gilbert O. Fruhwirth; Alexandra Moumtzi; Alexandra Loidl; Elisabeth Ingolic; Albin Hermetter (1060-1069).
Oxidized phospholipids, including 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) are typically present in oxidatively modified low density lipoprotein (oxLDL) and have been found in atherosclerotic lesions. These compounds are gaining increasing importance as inducers of different cellular responses like inflammation, proliferation, or cell death. The aim of this study was to elicit the type and outcome of the cellular response of vascular smooth muscle cells (VSMC) upon treatment with POVPC and PGPC. Both oxidized phospholipids led to inhibition of cell proliferation and showed cytotoxic effects in VSMC. Several morphological criteria, the presence of typical DNA fragments, and a phosphatidylserine shift towards the outer leaflet of the cell membrane revealed that apoptosis was the predominant mode of cell death. In all experiments, POVPC was found to be a more potent inducer of apoptosis than PGPC. Interestingly, in the presence of high levels of serum in the growth media the proapoptotic but not the antiproliferative effects of both oxidized phospholipids were abolished. Thus, we conclude that under low serum conditions both intact POVPC and PGPC are proapoptotic mediators. Under high serum conditions, these lipids are hydrolyzed and the resultant lipid mixture containing the degradation products is no longer apoptotic but antiproliferative. Thus, the direct and indirect effects of POVPC and PGPC on cell viability may account for the detrimental actions of oxLDL on VSMC.
Keywords: Oxidized phospholipid; Cell proliferation; Apoptosis; Cell morphology;

Sex differences in atherosclerosis in mice with elevated phospholipid transfer protein activity are related to decreased plasma high density lipoproteins and not to increased production of triglycerides by Jessica Lie; Matthijs Moerland; Teus van Gent; Rien van Haperen; Leo Scheek; Farah Sadeghi-Niaraki; Rini de Crom; Arie van Tol (1070-1077).
Plasma phospholipid transfer protein (PLTP) has atherogenic properties in genetically modified mice. PLTP stimulates hepatic triglyceride secretion and reduces plasma levels of high density lipoproteins (HDL). The present study was performed to relate the increased atherosclerosis in PLTP transgenic mice to one of these atherogenic effects. A humanized mouse model was used which had decreased LDL receptor expression and was transgenic for human cholesterylester transfer protein (CETP) in order to obtain a better resemblance to the plasma lipoprotein profile present in humans. It is well known that female mice are more susceptible to atherosclerosis than male mice. Therefore, we compared male and female mice expressing human PLTP. The animals were fed an atherogenic diet and the effects on plasma lipids and lipoproteins, triglyceride secretion and the development of atherosclerosis were measured. The development of atherosclerosis was sex-dependent. This effect was stronger in PLTP transgenic mice, while PLTP activity levels were virtually identical. Also, the rates of hepatic secretion of triglycerides were similar. In contrast, plasma levels of HDL were about 2-fold lower in female mice than in male mice after feeding an atherogenic diet. We conclude that increased atherosclerosis caused by overexpression of PLTP is related to a decrease in HDL, rather than to elevated hepatic secretion of triglycerides.
Keywords: Atherosclerosis; Lipid metabolism; Lipoprotein; Transgenic animal model;

Arachidonic acid can function as a signaling modulator by activating the TRPM5 cation channel in taste receptor cells by Hideaki Oike; Minoru Wakamori; Yasuo Mori; Hiroki Nakanishi; Ryo Taguchi; Takumi Misaka; Ichiro Matsumoto; Keiko Abe (1078-1084).
Vertebrate sensory cells such as vomeronasal neurons and Drosophila photoreceptor cells use TRP channels to respond to exogenous stimuli. In mammalian taste cells, bitter and sweet substances as well as some amino acids are received by G protein-coupled receptors (T2Rs or T1Rs). As a result of activation of G protein and phospholipase Cβ2, the TRPM5 channel is activated. Intracellular Ca2+ is known to be a TRPM5 activator, but the participation of lipid activators remains unreported. To clarify the effect of arachidonic acid on TRPM5 in taste cells, we investigated the expression profile of a series of enzymes involved in controlling the intracellular free arachidonic acid level, with the result that in a subset of taste bud cells, monoglyceride lipase (MGL) and cyclooxygenase-2 (COX-2) are expressed as well as the previously reported group IIA phospholipase A2 (PLA2-IIA). Double-labeling analysis revealed that MGL, COX-2 and PLA2-IIA are co-expressed in some cells that express TRPM5. We then investigated whether arachidonic acid activates TRPM5 via a heterologous expression system in HEK293 cells, and found that its activation occurred at 10 μM arachidonic acid. These results strongly suggest the possibility that arachidonic acid acts as a modulator of TRPM5 in taste signaling pathways.
Keywords: Taste signaling; Arachidonic acid; TRPM5; Monoglyceride lipase; Phospholipase A2; Cyclooxygenase;

The al-2 cDNA from Neurospora crassa was cloned, expressed and functionally characterized. The enzyme comprised the two catalytic activities of a phytoene synthase and a lycopene cyclase. In contrast to most other lycopene cyclases, single cyclizations were preferentially catalyzed. This N. crassa enzyme is the first CrtYB-type monocyclic-acting lycopene cyclase. Therefore, this cDNA has been evaluated for the heterologous synthesis of monocyclic hydroxy-keto carotenoids by combination with other carotenogenic genes in Escherichia coli. Depending on the degree of desaturation, 4-keto derivatives of γ-carotene and torulene with additional 2-hydroxy, 3-hydroxy and/or 1′-HO groups were generated and the following asymmetrical carotenoids identified and quantitated: 3-HO-4-keto-γ-carotene, 2-HO-4-keto-γ-carotene, 4-keto-1′-HO-γ-carotene, 3,1′-(HO)2-4-keto-γ-carotene, 3-HO-4-keto-torulene and 2-HO-4-keto-torulene. Among them all the monocyclic γ-carotene derivatives with 9 conjugated double bonds were not found naturally before. Furthermore, 2-HO-4-keto-torulene with 10 conjugated double bonds is another novel carotenoid.
Keywords: Gene al-2; Hydroxylated 4-keto-γ-carotene derivatives; Hydroxylated 4-keto-torulene derivatives; Lycopene cyclase CrtYB; Phytoene synthase; Phytoene synthase cDNA;

The proinflammatory mediator Platelet Activating Factor is an effective substrate for human group X secreted phospholipase A2 by Sarah Gora; Gerard Lambeau; James G. Bollinger; Michael Gelb; Ewa Ninio; Sonia-Athina Karabina (1093-1099).
Platelet Activating Factor (PAF) is a potent mediator of inflammation whose biological activity depends on the acetyl group esterified at the sn-2 position of the molecule. PAF-acetylhydrolase (PAF-AH), a secreted calcium-independent phospholipase A2, is known to inactivate PAF by formation of lyso-PAF and acetate. However, PAF-AH deficient patients are not susceptible to the biological effects of inhaled PAF in airway inflammation, suggesting that other enzymes may regulate extracellular levels of PAF. We therefore examined the hydrolytic activity of the recently described human group X secreted phospholipase A2 (hGX sPLA2) towards PAF. Among different sPLA2s, hGX sPLA2 has the highest affinity towards phosphatidylcholine (PC), the major phospholipid of cellular membranes and plasma lipoproteins. Our results show that unlike group IIA, group V, and the pancreatic group IB sPLA2, recombinant hGX sPLA2 can efficiently hydrolyze PAF. The hydrolysis of PAF by hGX sPLA2 rises abruptly when the concentration of PAF passes through its critical micelle concentration suggesting that the enzyme undergoes interfacial binding and activation to PAF. In conclusion, our study shows that hGX sPLA2 may be a novel player in PAF regulation during inflammatory processes.
Keywords: PAF; Inflammation; PAF-AH; Phospholipase A2 group X; Lp-PLA2; LDL;

The N terminal domain of human apolipoprotein E3 (apoE3-NT) functions as a ligand for members of the low-density lipoprotein receptor (LDLR) family. Whereas lipid-free apoE3-NT adopts a stable four-helix bundle conformation, a lipid binding induced conformational change is required for LDLR recognition. To investigate the role of a leucine zipper motif identified in the helix bundle on lipid binding activity, three leucine residues in helix 2 (Leu63, Leu71 and Leu78) were replaced by alanine. Recombinant “leucine to alanine” (LA) apoE3-NT was produced in E. coli, isolated and characterized. Stability studies revealed a transition midpoint of guanidine hydrochloride induced denaturation of 2.7 M and 2.1 M for wild type (WT) and LA apoE3-NT, respectively. Results from fluorescent dye binding assays revealed that, compared to WT apoE3-NT, LA apoE3-NT has an increased content of solvent exposed hydrophobic surfaces. In phospholipid vesicle solubilization assays, LA apoE3-NT was more effective than WT apoE3-NT at inducing a time-dependent decrease in dimyristoylphosphatidylglycerol vesicle light scattering intensity. Likewise, in lipoprotein binding assays, LA apoE3-NT protected human low-density lipoprotein from phospholipase C induced aggregation to a greater extent than WT apoE3-NT. On the other hand, LA apoE3-NT and WT apoE3-NT were equivalent in terms of their ability to bind a soluble LDLR fragment. The results suggest that the leucine zipper motif confers stability to the apoE3-NT helix bundle state and may serve to modulate lipid binding activity of this domain and, thereby, influence the conformational transition associated with manifestation of LDLR binding activity.
Keywords: Leucine zipper; ApoE3; Lipid;

DnaJA4 is a SREBP-regulated chaperone involved in the cholesterol biosynthesis pathway by Céline Robichon; Mathilde Varret; Xavier Le Liepvre; Françoise Lasnier; Eric Hajduch; Pascal Ferré; Isabelle Dugail (1107-1113).
Using subtractive hybridization technique in 3T3-L1 adipocytes overexpressing constitutively active SREBP2, we have identified a DnaJ/Hsp40 chaperone, DnaJA4, as a new SREBP-responsive gene. SREBP2 regulation was demonstrated by changes in DnaJA4 mRNA under conditions of altered sterol status that were strictly parallel to that of well-characterized SREBP targets (LDL receptor and HMG-CoA reductase). The role of SREBP2 was further established using adenoviral overexpression of a dominant negative SREBP2, which abolished cholesterol-regulated changes in DnaJA4 expression. To determine the functional significance of this regulation, DnaJA4 was overexpressed in COS cells, which induced a specific increase in the synthesis of cholesterol from acetate. We also observed that DnaJA4 overexpression increased the activity and the protein content of HMG-CoA reductase, the rate limiting enzyme in this pathway. At the molecular level, DnaJA4 overexpression did not alter HMG-CoA reductase stability or mRNA levels, suggesting a co-translational effect of the chaperone. In the DnaJ/Hsp40 family, DnaJA4 uniquely exhibited SREBP-regulated expression, and also responded to heat shock. Through its responsiveness to SREBP, and its stimulatory effect on cholesterol synthesis, the DnaJA4 chaperone can be viewed as a new player in cholesterol synthesis. These data suggest a link between molecular chaperones, heat stress and cholesterol synthesis.
Keywords: Cholesterol synthesis; SREBP; HMG-CoA reductase; Chaperone; Heat shock;

Thromboxane (TX) A2 is a potent stimulator of platelet activation/aggregation and smooth muscle contraction and contributes to a variety of pathologies within the vasculature. In this study, we investigated the mechanism whereby the cellular responses to TXA2 mediated through the TPβ isoform of the human TXA2 receptor (TP) are dynamically regulated by examining the mechanism of agonist-induced desensitization of intracellular signalling and second messenger generation by TPβ. It was established that TPβ is subject to profound agonist-induced homologous desensitization of signalling (intracellular calcium mobilization and inositol 1,3,5 trisphosphate generation) in response to stimulation with the TXA2 mimetic U46619 and this occurs through two key mechanisms: TPβ undergoes partial agonist-induced desensitization that occurs through a GF 109203X-sensitive, protein kinase (PK)C mechanism whereby Ser145 within intracellular domain (IC)2 has been identified as the key phospho-target. In addition, TPβ also undergoes more profound and sustained agonist-induced desensitization involving G protein-coupled receptor kinase (GRK)2/3-phosphorylation of both Ser239 and Ser357 within its IC3 and carboxyl-terminal C-tail domains, respectively. Inhibition of phosphorylation of either Ser239 or Ser357, through site directed mutagenesis, impaired desensitization while mutation of both Ser239 and Ser357 almost completely abolished desensitization of signalling, GRK phosphorylation and β-arrestin association, thereby blocking TPβ internalization. These data suggest a model whereby agonist-induced PKC phosphorylation of Ser145 partially impairs. TPβ signalling while GRK2/3 phosphorylation at both Ser239 and Ser357 within its IC3 and C-tail domains, respectively, sterically inhibits G-protein coupling, profoundly desensitizing signalling, and promotes β-arrestin association and, in turn, facilitates TPβ internalization. Thromboxane (TX) A2 is a potent stimulator of platelet aggregation and smooth muscle contraction and contributes to a variety of vascular pathologies. Herein the mechanism whereby the cellular responses to TXA2 mediated through the TPβ isoform of the human TXA2 receptor (TP) are dynamically regulated was investigated by examining the mechanism of its agonist-induced desensitization of intracellular signalling and second messenger generation. TPβ is subject to profound agonist-induced homologous desensitization of signalling (intracellular calcium mobilization and inositol 1,3,5 trisphosphate generation) in response to stimulation with the TXA2 mimetic U46619 and this occurs through two key mechanisms: TPβ undergoes partial agonist-induced desensitization that occurs through a GF 109203X-sensitive, protein kinase (PK)C mechanism whereby Ser145 within intracellular domain (IC)2 was identified as the key phospho-target. In addition, TPβ also undergoes more profound and sustained agonist-induced desensitization involving G protein-coupled receptor kinase (GRK)2/3-phosphorylation of both Ser239 and Ser357 within its IC3 and carboxyl-terminal C-tail domains, respectively. Inhibition of phosphorylation of either Ser239 or Ser357, through site directed mutagenesis, impaired desensitization while mutation of both Ser239 and Ser357 almost completely abolished desensitization of signalling, GRK phosphorylation and β-arrestin association, thereby blocking TPβ internalization. These data suggest a model whereby agonist-induced PKC phosphorylation of Ser145 partially impairs TPβ signalling while GRK2/3 phosphorylation at both Ser239 and Ser357 within its IC3 and C-tail domains, respectively, sterically inhibits G-protein coupling, profoundly desensitizing signalling, and promotes β-arrestin association and, in turn, facilitates TPβ internalization.
Keywords: Thromboxane receptor; Beta; Desensitization; Phosphorylation; Phospholipase C; G protein coupled receptor; G protein coupled receptor kinase; Arrestin;