A catalytically active fragment of the Rap-speci®c guanine-nucleotide exchange factor C3G was expressed in E coli. It was puri®ed and its interaction with GTPbinding proteins was investigated using¯uorescence spectroscopy. C3G stimulates GDP dissociation from Rap1, but not from Rap2, neither from Bud1, which is believed to be the yeast homologue of Rap1 nor from all other proteins of the human Ras-subfamily. Like the corresponding fragment from CDC25 Mm , the increase in the GDP dissociation rate is linear with increasing concentration of Rap1A . GDP up to 100 mM, indicating an apparent K M higher than 100 mM. Unlike the Ras-CDC25 Mm system, the Rap1A(S17N) mutant does not inhibit the C3G-activated guanine nucleotide dissociation from wild-type Rap1A in vitro. These data suggest that Rap1A(S17N) is unlikely to titrate away C3G in vivo, the proposed mechanism by which S17N-mutants exert their dominant negative e ects.
The role of fimbriae in the adherence of Haemophilus influenzae to oropharyngeal epithelial cells and the hemagglutination (HA) of human Anton-positive erythrocytes was examined. HA of bacteria was lost after shearing. Fimbriae purified from the extracellular fluid caused HA and bound to oropharyngeal epithelial cells, as analyzed with immunoperoxidase staining, in a way which was similar to the adherence of bacteria to these cells: binding was over the entire surface of the cells and showed cell-to-cell variation. The specific role of fimbriae in HA .nd adherence was further examined by inhibition experiments with inonoclonal antibodies elicited against the isolated fimbriae. These monoclonal antibodies bound along the entire length of the fimbriae, as seen by immunogold electron microscopy. The monoclonal antibodies and their Fab fragments inhibited HA (reduction in titer from 1:512 to 1:128 and 1:64, respectively) and inhibited the adherence of the homologous H. influenzae strain and of three of eight heterologous H. influenzae strains to oropharyngeal epithelial cells. These results indicate that fimbriae are involved in adherence and HA and that the binding site for the monoclonal antibodies on the fimbriae is not common on all strains.
Human Intestine 407 cells respond to hyposmotic stimulation by activating the conductive efflux of both Cl- and K+ (regulatory volume decrease) through pathways involving protein tyrosine phosphorylation (Tilly, B. C., N. van den Berghe, L. G. J. Tertoolen, M. J. Edixhoven, and H. R. de Jonge. J. Biol. Chem. 268: 19919-19922, 1993). Stimulation of the cells with hormones linked to the phospholipase C signaling cascade (e.g., bradykinin, histamine, or thrombin) or with the phosphotyrosine phosphatase inhibitor vanadate, potentiated the osmosensitive anion efflux by two- to threefold but did not affect anion efflux under isotonic conditions. No substantial increase in intracellular Ca2+ concentration ([Ca2+]i) was observed on mild hypotonicity-induced cell swelling. In addition, loading the cells with the intracellular Ca2+ chelator 1,2-bis(2-amino-phenoxy)ethane- N,N,N',N',-tetraacetic acid acetoxymethyl ester (BAPTA-AM) caused a partial reduction of the osmoshock-induced 125I- efflux but did not affect its potentiation by vanadate. In contrast, bradykinin transiently elevated [Ca2+]i, and its potentiation of the osmosensitive anion efflux was completely inhibited after BAPTA-AM loading. Both the Ca(2+)-mobilizing hormones as well as osmotic cell swelling rapidly triggered the phosphorylation of several proteins on tyrosine residues. However, the effects of the hormones, but not the effect of hypotonicity, on protein tyrosine phosphorylation was largely abolished in BAPTA-loaded cells. Taken together the results indicate a novel role for Ca(2+)-mobilizing hormones, although elevation of [Ca2+]i, in potentiating volume-sensitive ionic efflux even in cell types lacking the expression of Ca(2+)-activated Cl- channels in their plasma membrane.
The signal transduction pathway by which insulin stimulates glucose transport is largely unknown, but a role for tyrosine and serine/threonine kinases has been proposed. Since mitogen-activated protein (MAP) kinase is activated by insulin through phosphorylation on both tyrosine and threonine residues, we investigated whether MAP kinase and its upstream regulator, p21rs, are involved in insulin-mediated glucose transport. We did this by examining the time-and dose-dependent stimulation of glucose uptake in relation to the activation of Ras-GTP formation and MAP kinase by thrombin, epidermal growth factor (EGF), and insulin in 3T3-L1 adipocytes. Ras-GTP formation was stimulated transiently by all three agonists, with a peak at 5 to 10 min. Thrombin induced a second peak at '30 min. The activation of p2lr' was paralleled by both the phosphorylation and the activation of MAP kinase: transient for insulin and EGF and biphasic for thrombin. However, despite the strong activation of Ras-GTP formation and MAP kinase by EGF and thrombin, glucose uptake was not stimulated by these agonists, in contrast to the eightfold stimulation of 2-deoxy-D-['4C]glucose uptake by insulin. In addition, insulin-mediated glucose transport was not potentiated by thrombin or EGF.Although these results cannot exclude the possibility that p2lr' and/or MAP kinase is needed in conjunction with other signaling molecules that are activated by insulin and not by thrombin or EGF, they show that the Ras/MAP kinase signaling pathway alone is not sufficient to induce insulin-mediated glucose transport.Type 2 or non-insulin-dependent diabetes mellitus (NIDDM) is a disease in which insulin-mediated glucose uptake by adipocytes and cardiac and skeletal muscle cells is impaired (33). In these insulin-sensitive tissues, glucose uptake is stimulated by insulin-induced translocation and activation of a specific glucose transporter, GLUT 4 (2,12,24). GLUT 4 belongs to a family of glucose transporters composed of at least five isoforms (GLUT 1 to 5), which were identified by cDNA cloning (2,12,16). In the insulin-sensitive tissues, two types of glucose transporters are expressed: GLUT 4, which is restricted to these tissues, and GLUT 1, the more uniformly distributed isoform (15,16).Although the existence of genetic components in the etiology of NIDDM is well established, for the majority of NIDDM patients the gene defects have not been identified yet. A subgroup of NIDDM may be the result of mutations in genes encoding proteins involved in the intracellular signaling of insulin to glucose transport. Studies of their role in the pathogenesis of NIDDM are hampered by the poor characterization of these signaling intermediates. Some of the early effects of insulin are the phosphorylation of insulin receptor substrate 1 (IRS-1) (40) and the activation of the Ras/mitogenactivated protein (MAP) kinase signaling pathway (8). Treatment of adipocytes with the phosphatase inhibitor okadaic acid partly mimics the effect of insulin on 2-deoxy-D-glucose (2DOG) uptake (21 ...
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