Phosphoinositide-3 kinase activity is implicated in diverse cellular responses triggered by mammalian cell surface receptors and in the regulation of protein sorting in yeast. Receptors with intrinsic and associated tyrosine kinase activity recruit heterodimeric phosphoinositide-3 kinases that consist of p110 catalytic subunits and p85 adaptor molecules containing Src homology 2 (SH2) domains. A phosphoinositide-3 kinase isotype, p110 gamma, was cloned and characterized. The p110 gamma enzyme was activated in vitro by both the alpha and beta gamma subunits of heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins) and did not interact with p85. A potential pleckstrin homology domain is located near its amino terminus. The p110 gamma isotype may link signaling through G protein-coupled receptors to the generation of phosphoinositide second messengers phosphorylated in the D-3 position.
Class I phosphoinositide 3-kinases (PI3Ks) regulate important cellular processes such as mitogenesis, apoptosis, and cytoskeletal functions. They include PI3K␣, -, and -␦ isoforms coupled to receptor tyrosine kinases and a PI3K␥ isoform activated by receptor-stimulated G proteins. This study examines the direct interaction of purified recombinant PI3K␥ catalytic subunit (p110␥) and G␥ complexes. When phosphatidylinositol was used as a substrate, G␥ stimulated p110␥ lipid kinase activity more than 60-fold (EC 50 , ϳ20 nM). Stimulation was inhibited by G␣ o -GDP or wortmannin in a concentration-dependent fashion. Stoichiometric binding of a monoclonal antibody to the putative pleckstrin homology domain of p110␥ did not affect G␥-mediated enzymatic stimulation, whereas incubation of G␥ with a synthetic peptide resembling a predicted G␥ effector domain of type 2 adenylyl cyclase selectively inhibited activation of p110␥. G␥ complexes bound to N-as well as C-terminal deletion mutants of p110␥. Correspondingly, these enzymatically inactive N-and C-terminal mutants inhibited G␥-mediated activation of wild type p110␥. Our data suggest that (i) p110␥ directly interacts with G␥, (ii) the pleckstrin homology domain is not the only region important for G␥-mediated activation of the lipid kinase, and (iii) G␥ binds to at least two contact sites of p110␥, one of which is close to or within the catalytic core of the enzyme.
Signalling via seven transmembrane helix receptors can lead to a massive increase in cellular PtdIns(3,4,5)P3, which is critical for the induction of various cell responses and is likely to be produced by a trimeric G-protein-sensitive phosphoinositide 3-kinase (PI3Kgamma). We show here that PI3Kgamma is a bifunctional lipid kinase and protein kinase, and that both activities are inhibited by wortmannin at concentrations equal to those affecting the p85/p110alpha heterodimeric PI3K (IC50 approx. 2 nM). The binding of wortmannin to PI3Kgamma, as detected by anti-wortmannin antisera, closely followed the inhibition of the kinase activities. Truncation of more than the 98 N-terminal amino acid residues from PI3Kgamma produced proteins that were inactive in wortmannin binding and kinase assays. This suggests that regions apart from the core catalytic domain are important in catalysis and inhibitor interaction. The covalent reaction of wortmannin with PI3Kgamma was prevented by preincubation with phosphoinositides, ATP and its analogues adenine and 5'-(4-fluorosulphonylbenzoyl)adenine. Proteolytic analysis of wortmannin-prelabelled PI3Kgamma revealed candidate wortmannin-binding peptides around Lys-799. Replacement of Lys-799 by Arg through site-directed mutagenesis aborted the covalent reaction with wortmannin and the lipid kinase and protein kinase activities completely. The above illustrates that Lys-799 is crucial to the phosphate transfer reaction and wortmannin reactivity. Parallel inhibition of the PI3Kgamma-associated protein kinase and lipid kinase by wortmannin and by the Lys-799-->Arg mutation reveals that both activities are inherent in the PI3Kgamma polypeptide.
Nucleotides signal through purinergic receptors such as the P2 receptors, which are subdivided into the ionotropic P2X receptors and the metabotropic P2Y receptors. The diversity of functions within the purinergic receptor family is required for the tissue-specificity of nucleotide signalling. In the present study, hetero-oligomerization between two metabotropic P2Y receptor subtypes is established. These receptors, P2Y1 and P2Y11, were found to associate together when co-expressed in HEK293 cells. This association was detected by co-pull-down, immunoprecipitation and FRET (fluorescence resonance energy transfer) experiments. We found a striking functional consequence of the interaction between the P2Y11 receptor and the P2Y1 receptor where this interaction promotes agonist-induced internalization of the P2Y11 receptor. This is remarkable because the P2Y11 receptor by itself is not able to undergo endocytosis. Co-internalization of these receptors was also seen in 1321N1 astrocytoma cells co-expressing both P2Y11 and P2Y1 receptors, upon stimulation with ATP or the P2Y1 receptor-specific agonist 2-MeS-ADP. 1321N1 astrocytoma cells do not express endogenous P2Y receptors. Moreover, in HEK293 cells, the P2Y11 receptor was found to functionally associate with endogenous P2Y1 receptors. Treatment of HEK293 cells with siRNA (small interfering RNA) directed against the P2Y1 receptor diminished the agonist-induced endocytosis of the heterologously expressed GFP-P2Y11 receptor. Pharmacological characteristics of the P2Y11 receptor expressed in HEK293 cells were determined by recording Ca2+ responses after nucleotide stimulation. This analysis revealed a ligand specificity which was different from the agonist profile established in cells expressing the P2Y11 receptor as the only metabotropic nucleotide receptor. Thus the hetero-oligomerization of the P2Y1 and P2Y11 receptors allows novel functions of the P2Y11 receptor in response to extracellular nucleotides.
Peroxisome proliferator-activated receptor (PPAR) transcription factors are pharmaceutical drug targets for treating diabetes, atherosclerosis, and inflammatory degenerative diseases. The possible mechanism of interaction between the three PPAR isotypes (␣, /␦, and ␥) is not yet clear. However, this is important both for understanding transcription factor regulation and for the development of new drugs. The present study was designed to compare the effects of combinations of synthetic agonists of PPAR␣ [2-[4-[2-[4-cyclohexylbutyl (cyclohexylcarbamoyl), and PPAR␥ (rosiglitazone, ciglitazone) on inflammatory gene regulation in rat primary astrocytes. We measured cyclooxygenase-2 (COX-2) expression and prostaglandin E 2 synthesis in lipopolysaccharide (LPS)-stimulated cells. PPAR␣, PPAR/␦, and PPAR␥ knockdown models served to delineate the contribution of each PPAR isotype. Thiazolidinediones enhanced the LPSinduced COX-2 expression via PPAR␥-dependent pathway, whereas L-165041 and GW7647 had no influence. However, the addition of L-165041 potentiated the effect of PPAR␥ activation through PPAR/␦-dependent mechanism. On the contrary, PPAR␣ activation (GW7647) suppressed the effect of the combined L-165041/rosiglitazone application. The mechanism of the interplay arising from combined applications of PPAR agonists involves changes in PPAR expression levels. A PPAR/␦ overexpression model confirmed that PPAR/␦ expression level is the point at which PPAR␥ and PPAR␣ pathways converge in control of COX-2 gene expression. Thus, we discovered that in primary astrocytes, PPAR␥ has a positive influence and PPAR␣ has a negative influence on PPAR/␦ expression and activity. A positive/negative-feedback loop is formed by PPAR/␦-dependent increase in PPAR␣ expression level. These findings elucidate a novel principle of regulation in the signaling by synthetic PPAR agonists that involves modulating the interaction between PPAR␣, -/␦, and -␥ isoforms on the level of their expression.
Nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) belongs to the family of ecto-nucleotidases, which control extracellular nucleotide, nucleoside, and (di)phosphate levels. To study the (patho)physiological roles of NPP1 potent and selective inhibitors with drug-like properties are required. Therefore, a compound library was screened for NPP1 inhibitors using a colorimetric assay with p-nitrophenyl 5'-thymidine monophosphate (p-Nph-5'-TMP) as an artificial substrate. This led to the discovery of 2-(3H-imidazo[4,5-b]pyridin-2-ylthio)-N-(3,4-dimethoxyphenyl)acetamide (5a) as a hit compound with a Ki value of 217 nM. Subsequent structure-activity relationship studies led to the development of purine and imidazo[4,5-b]pyridine analogues with high inhibitory potency (Ki values of 5.00 nM and 29.6 nM, respectively) when assayed with p-Nph-5'-TMP as a substrate. Surprisingly, the compounds were significantly less potent when tested versus ATP as a substrate, with Ki values in the low micromolar range. A prototypic inhibitor was investigated for its mechanism of inhibition and found to be competitive versus both substrates.
Extracellular nucleotides exert a large number of physiological effects through activation of P2Y receptors. We expressed rat P2Y2 (rP2Y2) receptor, tagged with green fluorescent protein (GFP) in HEK-293 cells and visualized receptor translocation in live cells by confocal microscopy. Functional receptor expression was confirmed by determining [Ca2+]i responses. Agonist stimulation caused a time-dependent translocation of the receptor from the plasma membrane to the cytoplasm. Rearrangement of the actin cytoskeleton was observed during agonist-mediated rP2Y2-GFP receptor internalization. Colocalization of the internalized receptor with early endosomes, clathrin and lysosomes was detected by confocal microscopy. The inhibition of receptor endocytosis by either high-density medium or chlorpromazine in the presence of UTP indicates that the receptor was internalized by the clathrin-mediated pathway. The caveolin-mediated pathway was not involved. Targeting of the receptor from endosomes to lysosomes seems to involve the proteasome pathway, because proteasomal inhibition increased receptor recycling back to the plasma membrane.
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