Ligand stimulation of the platelet-derived growth factor (PDGF) P-receptor leads to activation of its intrinsic tyrosine kinase and autophosphorylation of the intracellular part of the receptor. The autophosphorylated tyrosine residues mediate interactions with downstream signal transduction molecules and thereby initiate different signalling pathways. A pathway leading to activation of the GTP-binding protein Ras involves the adaptor molecule GRB2. Here we show that Tyr-716, a novel autophosphorylation site in the PDGF ,8-receptor kinase insert, mediates direct binding of GRB2 in vitro and in vivo. In a panel of mutant PDGF ,-receptors, in which Tyr-716 and the previously known autophosphorylation sites were individually mutated, only PDGFRjY716F failed to bind GRB2. Furthermore, a synthetic phosphorylated peptide containing Tyr-716 bound GRB2, and this peptide specifically interrupted the interaction between GRB2 and the wild-type receptor. In addition, the Y716(P) peptide significantly decreased the amount of GTP bound to Ras in response to PDGF in permeabilized fibroblasts as well as in porcine aortic endothelial cells expressing transfected PDGF ,8-receptors. The mutant PDGFRjIY716F still mediated activation of mitogen-activated protein kinases and an increased DNA synthesis in response to PDGF, indicating that multiple signal transduction pathways transduce mitogenic signals from the activated PDGF j-receptor.Platelet-derived growth factor (PDGF) is a connective tissue cell mitogen, consisting of dimers of disulfide-bonded A and B polypeptide chains, which combine to form the three isoforms, PDGF-AA, -AB, and -BB (for a review, see reference 21). Two types of PDGF tyrosine kinase receptors have been identified and cloned. The ox-receptor binds both A and B chains with high affinity, whereas the p-receptor binds only the B chain.Signal transduction through tyrosine kinase receptors follows a general scheme whereby ligand binding induces dimerization or oligomerization of receptor molecules, activation of the receptor tyrosine kinase, and autophosphorylation in trans on multiple tyrosine residues in the intracellular region (reviewed in reference 44). Thereby, binding sites are created for intracellular signal transduction molecules, containing one or two copies of Src homology 2 (SH2) domains. There are two principal groups of signal transduction molecules: those which are equipped with a catalytic domain, and those which lack such domains but which serve as adaptors and associate with catalytically active molecules (50). For the PDGF P-receptor, considerable information has accumulated regarding the positions of autophosphorylation sites as well as which signal transduction molecules interact with individual sites. Thus, members of the Src family, i.e., phospholipase C--y, the Ras GTPase-activating protein (GAP), phosphatidylinositol 3'-kinase (P13-kinase), the phosphotyrosine phosphatase PTP1D/ Syp, and the adaptor molecules Shc in the PDGF P-receptor (reviewed in reference 9). The specificity of these in...

Activation of the b-receptor for platelet-derived growth factor (PDGF) by its ligand leads to autophosphorylation on a number of tyrosine residues. Here we show that Tyr763 in the kinase insert region is a novel autophosphorylation site, which after phosphorylation binds the protein tyrosine phosphatase SHP-2. SHP-2 has also previously been shown to bind to phosphorylated Tyr1009 in the PDGF b-receptor. Porcine aortic endothelial (PAE) cells transfected with a PDGF breceptor in which Tyr763 and Tyr1009 were mutated to phenylalanine residues failed to associate with SHP-2 after ligand stimulation. Morover, PDGF-BB-induced Ras GTP-loading and Erk2 activation were severely compromised in the receptor mutant. Whereas the mitogenic response to PDGF-BB remained at the same level as in cells expressing wild-type PDGF b-receptor, chemotaxis induced by PDGF-BB was signi®cantly decreased in the case of the Y763F/Y1009F mutant cells, suggesting an important role for SHP-2 in chemotactic signaling.

A selection of popular herbal medicinal products and food supplements were analysed for their potential to modulate the expression of the cytochrome P450 enzymes CYP1A2 and CYP3A4 and the transporter protein MDR1. A total of 31 products were analysed. Nine of the products have been approved by the Medical Products Agency (MPA) in Sweden and are marketed as herbal medicinal products. Twenty-two of the products have not been assessed by the MPA and are marketed as food supplements. LS180 cells were exposed to extracts from the different herbal products and real-time quantitative polymerase chain reaction, RT-QPCR, was subsequently used to analyse the relative mRNA levels of CYP1A2, CYP3A4 or MDR1 in treated and non-treated cells. Our results show that 17 of 31 products tested induced a two-fold expression or more for at least one of the genes analysed. Four products, of which a ginger-supplement was the most potent, induced all three genes.

Our results suggest that BTC in the GSE products is responsible for the increase in the INR value in a patient on warfarin treatment.

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Platelet-derived growth factor-stimulated actin rearrangement and edge ruffle formation have previously been shown to be dependent on activation of phosphatidylinositol 3′-kinase, the activity of which also is important for directed migration of cells. This lipid kinase binds to phosphorylated tyrosine residues Y740 and Y751 in the kinase insert of the human platelet-derived growth factor ss-receptor. We examined the role of two other tyrosine residues in the kinase insert of this receptor, Y775 and Y778, for ligand-induced actin rearrangement. Both were shown to be phosphorylation sites; Y775 was only marginally phosphorylated in cells expressing the wild-type ss-receptor, whereas Y778 was phosphorylated at higher stoichiometry. Mutant receptors Y775F, Y778F and Y775/778F were active kinases and mediated proliferative responses when expressed in porcine aortic endothelial cells. Fluorescence staining of actin in platelet-derived growth factor-stimulated PAE cells revealed that Y778 is involved in regulation of the actin cytoskeleton since the cells contained, apart from edge ruffles and circular ruffles, a novel type of giant ruffle on the dorsal side of the cell, which consisted of irregular multilayered actin structures. Mutation at Y778 had no effect on activation of phosphatidylinositol 3′-kinase, nor on the GTPase activating protein of Ras and phospholipase C(gamma), and the extent of directed migration towards platelet-derived growth factor of these cells was not changed. We conclude that actin rearrangement is regulated in part by Y778 in the platelet-derived growth factor ss-receptor, potentially through binding of a novel signaling molecule to this site.