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In addition to these pathways, LPA induces the Rho-dependent formation of focal adhesions and actin stress fibers in quiescent Swiss 3T3 fibroblasts (4, 10).We report direct coupling of the LPA receptor to proteins of the G Swiss 3T3 cells, kindly provided by Dr.Proteins were transferred to nitrocellulose as described (16).To obtain quiescent and serum-starved Swiss 3T3 cells, cultures were rinsed three times in serum-free DMEM and incubated in DMEM supplemented with 25% Ham’s F-12, 0.2% Na HCO phenylmethylsulfonylfluoride.LPA-induced activation of the Ras/Raf/mitogen-activated protein kinase cascade also occurs in a PTX-sensitive fashion (3, 6), and Gβγ dimers of heterotrimeric G-proteins are thought to transduce this effect via intermediary protein-tyrosine kinases (7, 8).However, the G-proteins coupling the LPA receptor to Rho activation have not been specified.Cells were disrupted by forcing the suspension 10 times through a 26-gauge needle.The pelleted cells were resuspended in the buffer described above devoid of Na Cl but containing 20 μg/ml leupeptin.LPA-induced cytoskeletal reorganizations are PTX-insensitive and therefore presumably not transmitted via G into Swiss 3T3 cells triggers actin polymerization in a Rho-dependent manner (13).A tyrosine kinase has been implicated in the LPA-induced Rho stimulation, since the tyrosine kinase inhibitor tyrphostin A 25 blocks the effects of LPA but not of microinjected Rho on actin polymerization (11).Lysophosphatidic acid (LPA) utilizes a G-protein-coupled receptor to activate the small GTP-binding protein Rho and to induce rapid remodeling of the actin cytoskeleton.We studied the signal transduction from LPA receptors to Rho activation.The tyrosine kinase inhibitor tyrphostin A 25 and the epidermal growth factor (EGF) receptor-specific tyrphostin AG 1478 completely blocked actin stress fiber formation caused by LPA or activated Gα Upon stimulation, a number of heterotrimeric G-protein-coupled receptors initiate cellular responses involving small GTP-binding proteins.Alan Hall (London), and COS-7 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum.

In fibroblasts, LPA lowers c AMP levels in a pertussis toxin (PTX)-sensitive manner, suggesting the coupling of the LPA receptor to G-proteins of the G subfamily (5).Interestingly, the LPA-dependent cytoskeletal reorganization was suppressed by microinjected antibodies directed against Gα-specific antibodies showed no inhibition.In contrast, in a large variety of cells, the stimulation of phospholipase C induced by LPA was shown to be PTX-insensitive (9), suggesting a coupling of the LPA receptor to members of the G subfamily.Undisrupted cells were pelleted (750 × For immunological detection of G-proteins, SDS-PAGE using 10% (w/v) polyacrylamide separating gels was employed.For instance, the water-soluble phospholipid lysophosphatidic acid (LPA)1 binds to a G-protein-coupled heptahelical receptor (1, 2) and stimulates the activation of Ras and Rho proteins via different pathways (3, 4).

In the present study, we determined the G-protein coupling pattern of the LPA receptor in membrane preparations and characterized the pathway leading from LPA receptor activation to stress fiber formation in intact cells.