cytochalasin-d and arginyl-glycyl-aspartyl-serine

Subcellular distribution of heterotrimeric GTP-binding proteins during thrombin receptor-mediated platelet activation was examined, revealing two phases of translocation to the cytoskeleton. A part of Gi2 alpha and Gs alpha shows first phase translocation to the low-speed pellet (15000 x g pellet) within 1 min after activation, suggesting involvement in platelet shape change or granule secretion. In the second phase, Gi2 alpha, Gs alpha, Gq alpha, and G beta translocate to the low-speed pellet, depending on platelet aggregation. These translocations correlated with the reorganization of the actin-cytoskeleton and were inhibited by cytochalasin D. Reconstitution experiments also revealed that G proteins are associated with the actin-cytoskeleton during platelet activation.

Topics: Actins; Amino Acid Sequence; Animals; Cattle; Cytochalasin D; Cytoskeleton; GTP-Binding Proteins; Humans; Molecular Sequence Data; Oligopeptides; Platelet Activation; Platelet Aggregation Inhibitors; Platelet Glycoprotein GPIIb-IIIa Complex; Receptors, Thrombin; Signal Transduction

To investigate a role of phospholipase C (PLC) isozymes in the integrin alphaIIbbeta3-mediated signaling, their location was examined in thrombin-activated human platelets, revealing different regulation of their translocation to the cytoskeleton (CSK). In resting platelets, the major PLCs such as PLCbeta2, PLCbeta3a (155 kDa), and PLCgamma2 and the minor PLCs (PLCbeta1 and PLCgamma1) were located in the Triton X-100-soluble (Tx.Sol) fraction and the membrane skeleton, whereas PLCbeta3b (140 kDa) was present only in Tx.Sol fraction when examined by Western immunoblotting. Thrombin stimulation caused a rapid and transient translocation of PLCbeta3a and PLCbeta3b and a slower accumulation of PLCbeta2 and PLCgamma2 in the reorganized CSK. The translocation to CSK of both PLCbeta3a and PLCbeta3b, but not PLCbeta2, was dependent on integrin alphaIIbbeta3-mediated aggregation. Furthermore, an actin polymerization inhibitor, cytochalasin D, or a protein tyrosine kinase inhibitor, genistein, abolished the CSK association of alphaIIbbeta3, PLCbeta3a, and PLCbeta3b. In the genistein-pretreated platelets, pp60(c-)src, Gq, and protein kinase Calpha were no longer able to associate with CSK. In contrast, these agents had no or marginal inhibitory effects on the CSK association of PLCbeta2 and Gi2. The late diacylglycerol generation induced by thrombin stimulation was significantly reduced by the genistein treatment. These results suggest that the integrin alphaIIbbeta3-mediated cytoskeletal association of PLCbeta3 is regulated by protein tyrosine kinase and also that the activation of the relocated PLC may play a role in the late platelet-to-platelet aggregation in thrombin-stimulated human platelets.

Topics: Actins; Antibodies, Monoclonal; Antigens, CD; Blood Platelets; Cell Fractionation; Cytochalasin D; Cytoskeleton; Enzyme Inhibitors; Genistein; Humans; In Vitro Techniques; Integrin beta3; Isoenzymes; Isoflavones; Kinetics; Octoxynol; Oligopeptides; Platelet Aggregation; Platelet Membrane Glycoproteins; Protein-Tyrosine Kinases; Thrombin; Type C Phospholipases

Attachment and neurite extension processes have been evaluated for an immortalized derivative cell of a rat dorsal root neuron after fusion with a mouse neuroblastoma cell (the clonal F11 hybrid cell line) and these processes compared with previous studies of neuroblastoma cells, since both cell types may be derived from the neural crest of the developing embryo. Biochemically defined substrata were provided by human plasma fibronectin (pFN), the heparan sulfate-binding protein platelet factor-4 (PF4), and the ganglioside GM1-binding protein cholera toxin B subunit (CTB). While some attachment of unsupplemented cells was noted on CTB substrata, GM1 supplementation permitted F11 cells to attach as well on CTB as on pFN or PF4. On PF4, very few neurite processes were observed while on pFN two morphologically distinct types of neurites could be identified: short, linear processes in a low percentage of cells resembling those of neuroblastoma cells and long, irregular and narrow processes in a higher percentage of cells resembling those of dorsal root neurons. On CTB, neurites of the latter class were even more prominent; however, cell bodies on CTB failed to spread by cytoplasmic extension as commonly observed in F11 cells on pFN and, to some extent, on PF4. The formation of both neurite classes on either pFN or CTB was completely inhibited by low concentrations of an RGDS (Arg-Gly-Asp-Ser) peptide in the medium of cultures, indicating the significance of pFN's binding to cell surface integrin or ganglioside GM1's possible interaction with integrin for mediating the differentiative process. In contrast, neurite formation of neuroblastoma cells is refractile to the soluble peptide as reported previously. Neurite extensions of F11 cells on either pFN or CTB were comparably sensitive to low concentrations of cytochalasin D, revealing the mediation of microfilament reorganization in these processes. Treatment of F11 cells with cycloheximide failed to inhibit neurite extension on pFN but did partially inhibit extension on CTB; this contrasts with the very high sensitivity of neurite formation by neuroblastoma cells on CTB substrata reported previously.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Animals; Cell Adhesion; Cell Movement; Cycloheximide; Cytochalasin D; Cytochalasins; Ganglia, Spinal; Hybrid Cells; Mice; Neuroblastoma; Neurons; Oligopeptides; Rats; Tumor Cells, Cultured