Characterizing protein phosphorylation downstream of the TrkA receptor
ABSTRACT: Phosphorylation twenty minutes after stimulation of the TrkA receptor was characterized in PC12 (rat) cells. The study was performed using chimeric receptors, where the endodomain of TrkA was fused to the extracellular domain of PDGFR. As PDGFR (and PDGF) are not expressed in these cells, addition of PDGF caused selective stimulation only of the chimeric receptor. Phosphorylation was compared between unstimulated cells, stimulated cells with the chimeric receptor, stimulated cells where Tyr490 in the chimeric receptor had been mutated to Phe, and stimulated cells where Tyr490 and Tyr785 were both mutated to Phe. This allowed characterization of protein phosphorylated by TrkA, and dissection of which proteins and sites are phosphorylated through the two key tyrosine residues in the TrkA receptor. This work led to two publications:
Biarc J, Chalkley RJ, Burlingame AL, Bradshaw RA. The induction of serine/threonine protein phosphorylations by a PDGFR/TrkA chimera in stably transfected PC12 cells. Mol Cell Proteomics. (2012) 11(5):15-30
Biarc J, Chalkley RJ, Burlingame AL, Bradshaw RA. Dissecting the roles of tyrosines 490 and 785 of TrkA in the induction of downstream protein phosphorylation using chimeric receptors. J Biol Chem. (2013) 288 (23):16606-16618
Project description:Despite a high degree of homology, insulin and IGF-1 receptors (IR/IGF1R) mediate distinct cellular and physiological functions. Here, using chimeric and site-mutated receptors, we demonstrate how domain differences between IR and IGF1R contribute the distinct functions of these receptors. Receptors with the intracellular domain of IGF1R show increased activation of Shc and Gab-1 and more potent regulation of genes involved in proliferation, corresponding to its higher mitogenic activity. Conversely, receptors with the intracellular domain of IR display higher IRS-1 phosphorylation, stronger regulation of genes in metabolic pathways and more dramatic glycolytic responses to hormonal stimulation. We generated mouse brown preadipocytes in which both insulin and IGF-1 receptors (IR and IGF1R) had been genetically inactivated using Cre-lox recombination. These IR and IGF1R DKO cells were then reconstituted with wild-type mouse IR, IGF1R, or one of two chimeric receptors: IR/IGF1R with the IR extracellular domain (ECD) fused to the IGF1R transmembrane and intracellular domains (ICD) and IGF1R/IR with the ECD of IGF1R fused to the ICD domains of IR. Three independent clones for each line were used for the study. For expression analysis, we serum-starved the preadipocytes clones overnight and stimulated cells with 100 nM insulin, IGF-1 or vehicle for 6 h, and subjected the cellular RNA to analysis using Affymetrix Mouse Gene 2.0 ST arrays.
Project description:Despite a high degree of homology, insulin receptor (IR) and IGF-1 receptor (IGF1R) mediate distinct cellular and physiological functions. Here, we demonstrate how domain differences between IR and IGF1R contribute to the distinct functions of these receptors using chimeric and site-mutated receptors. Receptors with the intracellular domain of IGF1R show increased activation of Shc and Gab-1 and more potent regulation of genes involved in proliferation, corresponding to their higher mitogenic activity. Conversely, receptors with the intracellular domain of IR display higher IRS-1 phosphorylation, stronger regulation of genes in metabolic pathways and more dramatic glycolytic responses to hormonal stimulation. Strikingly, replacement of leucine973 in the juxtamembrane region of IR to phenylalanine, which is present in IGF1R, mimics many of these signalling and gene expression responses. Overall, we show that the distinct activities of the closely related IR and IGF1R are mediated by their intracellular juxtamembrane region and substrate binding to this region.
Project description:Royal jelly (RJ), a hive product with versatile pharmacological activities, has been used as a traditional functional food to prevent or treat inflammatory diseases. However, little is known about the anti-inflammatory effect of RJ in microglial cells. The aim of this study is to assess the anti-inflammatory effects of RJ in lipopolysaccharide- (LPS-) induced murine immortalized BV-2 cells and to explore the underlying molecular mechanisms. Our results showed that in LPS-stimulated BV-2 cells, RJ significantly inhibited iNOS and COX-2 expression at mRNA and protein levels. The mRNA expression of IL-6, IL-1?, and TNF-? was also downregulated by RJ in a concentration-dependent manner. Additionally, RJ protected BV-2 cells against oxidative stress by upregulating heme oxygenase-1 (HO-1) expression and by reducing reactive oxygen species (ROS) and nitric oxide (NO) production. Mechanistically, we found that RJ could alleviate inflammatory response in microglia by suppressing the phosphorylation of I?B?, p38, and JNK and by inhibiting the nucleus translocation of NF-?B p65. These findings suggest that RJ might be a promising functional food to delay inflammatory progress by influencing the microglia function.
Project description:The epithelium of the small intestine plays an important role in detoxification processes due to the presence of various xenobiotic-metabolizing enzymes from phase I and II, as well as transport proteins of the ATP-binding cassette superfamily. Exposure to xenobiotics induces the expression of these proteins in the small intestine, with multiple signaling pathways stimulated by exogenous compounds converging at individual gene promoters by mechanisms which have not been fully understood yet. In this context the promoter region of the CYP1A1 gene, encoding the phase I monooxygenase cytochrome P450 1A1, was analyzed by chromatin immunoprecipitation with regard to binding of xeno-sensing receptors following stimulation of Caco-2 cells with agonists of the aryl hydrocarbon receptor (AHR) and retinoid receptors. Histone acetylation in the regulatory region of CYP1A1 was enhanced by treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or all-trans retinoic acid (at-RA). Binding of retinoid-X-receptor (RXR) ? to the promoter region was detected in response to at-RA, while AHR bound to the gene promoter following its activation by TCDD. Of note, enhanced RXR? binding was also detected after AHR stimulation, and increased AHR binding was observed after retinoid receptor activation by at-RA. Exposure of Caco-2 cells to mixtures of AHR and retinoid receptor agonists yielded synergistic induction of CYP1A1 mRNA. In conclusion, the present data improve our knowledge on retinoic acid-dependent effects on CYP1A1 expression and demonstrate unexpected mixture effects by cross-talk of the different receptors.
Project description:The role of Interleukin(IL)-6 in the pathogenesis of joint and systemic inflammation in rheumatoid arthritis (RA) and systemic juvenile idiopathic arthritis (s-JIA) has been clearly demonstrated. However, the mechanisms by which IL-6 contributes to the pathogenesis are not completely understood. This study investigates whether IL-6 affects, alone or upon toll like receptor (TLR) ligand stimulation, the production of inflammatory cytokines and chemokines in human peripheral blood mononuclear cells (PBMCs), synovial fluid mononuclear cells from JIA patients (SFMCs) and fibroblast-like synoviocytes from rheumatoid arthritis patients (RA synoviocytes) and signalling pathways involved. PBMCs were pre-treated with IL-6 and soluble IL-6 Receptor (sIL-6R). SFMCs and RA synoviocytes were pre-treated with IL-6/sIL-6R or sIL-6R, alone or in combination with Tocilizumab (TCZ). Cells were stimulated with LPS, S100A8-9, poly(I-C), CpG, Pam2CSK4, MDP, IL-1?. Treatment of PBMCs with IL-6 induced production of TNF-?, CXCL8, and CCL2, but not IL-1?. Addition of IL-6 to the same cells after stimulation with poly(I-C), CpG, Pam2CSK4, and MDP induced a significant increase in IL-1? and CXCL8, but not TNF-? production compared with TLR ligands alone. This enhanced production of IL-1? and CXCL8 paralleled increased p65 NF-?B activation. In contrast, addition of IL-6 to PBMCs stimulated with LPS or S100A8-9 (TLR-4 ligands) led to reduction of IL-1?, TNF-? and CXCL8 with reduced p65 NF-?B activation. IL-6/IL-1? co-stimulation increased CXCL8, CCL2 and IL-6 production. Addition of IL-6 to SFMCs stimulated with LPS or S100A8 increased CXCL8, CCL2 and IL-1? production. Treatment of RA synoviocytes with sIL-6R increased IL-6, CXCL8 and CCL2 production, with increased STAT3 and p65 NF-?B phosphorylation. Our results suggest that IL-6 amplifies TLR-induced inflammatory response. This effect may be relevant in the presence of high IL-6 and sIL-6R levels, such as in arthritic joints in the context of stimulation by endogenous TLR ligands.
Project description:Parathyroid hormone (PTH) and PTH-related protein (PTHrP) act via PTH receptors in bone to stimulate bone resorption. Bone resorption is also stimulated by certain cytokines, which are produced in bone and bone marrow. The effects of such cytokines on the PTH-receptor system were studied in the osteoblast-like osteosarcoma cell line UMR 106-06. 125I-labelled PTHrP-(1-84)-peptide bound specifically to the cells, and PTHrP-(1-34) and -(1-84) competed with equimolar affinity for binding to UMR 106-06 cells. The specific binding of 125I-PTHrP-(1-84) could be completely blocked by PTH. Therefore 125I-PTHrP-(1-84) bound to a classical receptor in UMR 106-06 cells. Preincubation for 3 days with either tumour necrosis factor alpha (TNF alpha) or retinoic acid (RA) both decreased the specific binding of 125I-PTHrP-(1-84) to about 40% of control levels. These effects were specific for PTH binding, since there was little effect on 125I-salmon-calcitonin binding. Both TNF alpha and RA required 24 h exposure to cells to produce a measurable effect. The decrease in 125I-PTHrP-(1-84) binding was due to a reduced number of binding sites, with little apparent change in affinity. Half-maximal effects were seen with 1 ng of TNF alpha/ml, whereas 1 microM-RA was needed to observe the loss of PTH receptors. Combinations of RA and TNF alpha produced a greater effect than that of either agonist alone. The loss of PTH receptors was accompanied by a specific loss of PTH-stimulated cyclic AMP production. Preincubation with TNF alpha increased the basal plasminogen activator (PA) activity in the cells and decreased the amplitude of the response of PA activity to PTH compared with control cells. Furthermore TNF alpha decreased sensitivity to PTH (50% stimulation of PA activity with 0.1 nM-PTH in control cells versus 50% stimulation with 0.3 nM-PTH in TNF alpha-treated cells). In contrast, TNF alpha pretreatment increased the amplitude of the response of PA activity to calcitonin, whereas sensitivity to calcitonin was not altered. These data are consistent with a specific down-regulation of PTH receptors in osteoblast-like UMR 106-06 cells after exposure to TNF alpha or RA. The loss of PTH receptors is accompanied by a decreased responsiveness to PTH, as measured with the PA system in these cells. A loss of PTH receptors could modulate PTH responses in osteoblasts, either in the local control of bone formation and resorption, or in pathological conditions such as humoral hypercalcaemia of malignancy.
Project description:Cadherin 11 is a homophilic cell-to-cell adhesion molecule expressed on joint synovial fibroblasts. Absence of cadherin 11 in a mouse rheumatoid arthritis (RA) model led to striking reductions in cartilage erosion. Matrix metalloproteinases (MMPs) are enzymes expressed by synovial fibroblasts important for cartilage erosion. The objective of this study was to determine if synovial fibroblast MMP production is regulated by cadherin 11.To mimic cadherin 11 engagement, human RA synovial fibroblasts were stimulated with a chimeric construct consisting of the cadherin 11 extracellular domain linked to the human IgG1 Fc domain (Cad-11-Fc). Effects on MMP production were measured by enzyme-linked immunosorbent assay, quantitative reverse transcription-polymerase chain reaction analysis, and immunoblotting.Human Cad-11-Fc up-regulated MMP-1 and MMP-3 protein production by RA synovial fibroblasts, both alone and in synergy with tumor necrosis factor ?. This up-regulation required cell cadherin 11 engagement, since a mutant Cad-11-Fc with reduced binding affinity stimulated significantly less MMP production. Also, short hairpin RNA (shRNA) cadherin 11 silencing almost completely inhibited Cad-11-Fc-induced MMP expression. Cad-11-Fc stimulation increased RA synovial fibroblast MMP messenger RNA levels. It also increased the phosphorylation of the MAPKs JNK, ERK, and p38 kinase, the phosphorylation of NF-?B p65, and the nuclear translocation of activator protein 1 transcription factor. MAPK and NF-?B inhibitors partially blocked RA synovial fibroblast MMP expression.Cadherin 11 engagement stimulates increased synthesis of several MMPs by RA synovial fibroblasts in a MAPK- and NF-?B-dependent manner. These results underscore the existence of a pathway by which cadherin 11 regulates MMP production and has important implications for joint destruction in RA.
Project description:Retinoic acid (RA), an active metabolite converted from vitamin A, plays an active role in immune function, such as defending against infections and immune regulation. Although RA affects various types of immune cells, including antigen-presenting cells, B lymphocytes, and T lymphocytes, whether it affects natural killer T (NKT) cells remain unknown. In this study, we found that RA decreased interferon (IFN)-? production by activated NKT cells through T-cell receptor (TCR) and CD28. We also found that RA reduced extracellular signal-regulated kinase (ERK) phosphorylation, but increased phosphatase 2A (PP2A) activity in TCR/CD28-stimulated NKT cells. The increased PP2A activity, at least partly, contributed to the reduction of ERK phosphorylation. Since inhibition of ERK activation decreases IFN-? production by TCR/CD28-stimulated NKT cells, RA may downregulate IFN-? production by TCR/CD28-stimulated NKT cells through the PP2A-ERK pathway. Our results demonstrated a novel function of RA in modulating the IFN-? expression by activated NKT cells.
Project description:We have identified two chemical series of compounds acting as selective positive allosteric modulators (enhancers) of native and recombinant metabotropic glutamate 1 (mGlu1) receptors. These compounds did not directly activate mGlu1 receptors but markedly potentiated agonist-stimulated responses, increasing potency and maximum efficacy. Binding of these compounds increased the affinity of a radiolabeled glutamate-site agonist at its extracellular N-terminal binding site. Chimeric and mutated receptors were used to localize amino acids in the receptor transmembrane region critical for these enhancing properties. Finally, the compounds potentiated synaptically evoked mGlu1 receptor responses in rat brain slices. The discovery of selective positive allosteric modulators of mGlu1 receptors opens up the possibility to develop a similar class of compounds for other family 3 G protein-coupled receptors.
Project description:A model of insulin-receptor down-regulation and desensitization has been developed and described. In this model, both insulin-receptor down-regulation and functional desensitization are induced in the human HepG2 cell line by a 16 h exposure of the cells to 0.1 microM-insulin. Insulin-receptor affinity is unchanged, but receptor number is decreased by 50%, as determined both by 125I-insulin binding and by protein immunoblotting with an antibody to the beta-subunit of the receptor. This down-regulation is accompanied by a disproportionate loss of insulin-stimulated glycogen synthesis, yielding a population of cell-surface insulin receptors which bind insulin normally but which are unable to mediate insulin-stimulated glycogen synthesis within the cell. Upon binding of insulin, the desensitized receptors are internalized rapidly, with characteristics indistinguishable from those of control cells. In contrast, this desensitization is accompanied by a loss of the insulin-sensitive tyrosine kinase activity of insulin receptors isolated from these cells. Receptors isolated from control cells show a 5-25-fold enhancement of autophosphorylation of the beta-subunit by insulin; this insulin-responsive autophosphorylation is severely attenuated after desensitization to a maximum of 0-2-fold stimulation by insulin. Likewise, the receptor-mediated phosphorylation of exogenous angiotensin II, which is stimulated 2-10-fold by insulin in receptors from control cells, is completely unresponsive to insulin in desensitized cells. These data provide evidence that the insulin-receptor tyrosine kinase activity correlates with insulin stimulation of an intracellular metabolic event. The data suggest that receptor endocytosis is not sufficient to mediate insulin's effects, and thereby argue for a role of the receptor tyrosine kinase activity in the mediation of insulin action.