The Journal of biological chemistry
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Comparative Study
Disrupted signaling in a mutant J2E cell line that shows enhanced viability, but does not proliferate or differentiate, with erythropoietin.
The immature erythroid J2E cell line proliferates and terminally differentiates following erythropoietin stimulation. In contrast, the mutant J2E-NR clone does not respond to erythropoietin by either proliferating or differentiating. Here we show that erythropoietin can act as a viability factor for both the J2E and J2E-NR lines, indicating that erythropoietin-initiated maturation is separable from the prevention of cell death. ⋯ However, protein interactions with the erythropoietin receptor and Grb2 were restricted in the mutant cells. Subsequent investigation of several other signaling molecules exposed numerous alterations in J2E-NR cells; phosphorylation changes to phosphatidylinositol 3-kinase, phospholipase Cgamma, p120 GAP, and mitogen-activated protein kinases (p42 and p44) observed in erythropoietin-stimulated J2E cells were not seen in the J2E-NR line. These data indicate that some pathways activated during erythropoietin-induced differentiation may not be essential for the prevention of apoptosis.
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Comparative Study
Chimeric vesicular monoamine transporters identify structural domains that influence substrate affinity and sensitivity to tetrabenazine.
The vesicular monoamine transporters (VMATs) 1 and 2 show close sequence similarity but substantial differences in apparent substrate affinity and drug sensitivity. To identify structural domains that determine these functional characteristics, chimeric transporters were constructed and their properties were analyzed in a heterologous expression system. The results implicate multiple regions in the recognition of serotonin and histamine and the sensitivity to tetrabenazine. ⋯ In addition, the extreme N terminus of VMAT2 alone suffices to confer a partial increase in substrate affinity and tetrabenazine sensitivity. Despite these similarities among the interactions with serotonin, histamine, and tetrabenazine, the region of VMAT2 from TMD3 through TMD4 increases serotonin affinity but not histamine affinity or tetrabenazine sensitivity, and whereas the region from TMD5 to TMD8 of VMAT2 increases serotonin affinity in the context of more C-terminal VMAT2 sequences, the region encompassing TMD5 through TMD7 reduces serotonin but not histamine affinity or tetrabenazine sensitivity in the context of more N-terminal VMAT2 sequences. Thus, the chimeric analysis also reveals differences between serotonin recognition and the recognition of both histamine and tetrabenazine that may account for the observed differences in their interaction with the transport protein.
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Distinctions between chemotaxis and haptotaxis of cells to extracellular matrix proteins have not been defined in terms of mechanisms or signaling pathways. Migration of A2058 human melanoma cells to soluble (chemotaxis) and substratum-bound (haptotaxis) vitronectin, mediated by alphav beta3, provided a system with which to address these questions. Both chemotaxis and haptotaxis were completely inhibited by treatment with RGD-containing peptides. ⋯ In contrast, soluble vitronectin (50-100 microg/ml) did not induce tyrosine phosphorylation of paxillin. The data suggest that soluble vitronectin stimulates chemotaxis predominantly through a G protein-mediated pathway that is functionally linked to alphavbeta3. Haptotaxis is analogous to directional cell spreading and requires alphavbeta3-mediated tyrosine phosphorylation of paxillin.