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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

PROVIDER: MSV000078461 | MassIVE | Thu Jul 11 00:00:00 BST 2013

REPOSITORIES: MassIVE

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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.

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	PTR490785Snosilac.txt	Txt

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Characterizing protein phosphorylation downstream of the TrkA receptor

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