Membrane transport and in vitro metabolism of the Ras cascade messenger, glycerophosphoinositol 4-phosphate.
The glycerophosphoinositols, phosphoinositide metabolites formed by Ras-dependent activation of phospholipase A2 and a lysophospholipase, have been proposed to be markers of Ras-induced cell transformation. These compounds can have important cellular effects; GroPIns4P is an inhibitor of G protein-stimulated adenylate cyclase and is transiently produced in several cell types after growth factor receptor stimulation of phosphatidylinositol 3-kinase and the small G protein Rac, indicating the importance of defining further its cellular actions and metabolism. We show here that, in postnuclear membranes from Swiss 3T3 cells, there is no high-affinity 'receptor' binding of GroPIns4P. Instead, possibly through the interaction with a transporter, GroPIns4P rapidly equilibrates between medium and cell cytosol, and, at higher concentrations, can concentrate in the cell cytosol. GroPIns4P can be dephosphorylated to GroPIns in vitro by an enzyme that is membrane-associated, Ca2+-dependent, GroPIns4P-selective and has a specific pH profile. Under in vitro phosphorylating conditions, there is production of GroPIns(4,5)P2 and other inositol phosphates. As these in vitro enzyme activities do not fully correlate with the in vivo handling of GroPIns4P, the intracellular GroPIns4P levels may be controlled by its direct physical removal from the cells.
Berrie
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Department of Cell Biology and Oncology, Istituto de Richerche Farmacologiche, Mario Negri, Cheti, Italy. berrie@cmns.mnegri.it
Iurisci
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Corda
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D016475:3T3 Cells; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007295:Inositol Phosphates / Q000737:chemistry*; D051379:Mice; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D010766:Phosphorylation; D011485:Protein Binding; D015398:Signal Transduction; D013997:Time Factors; D018631:ras Proteins / Q000737:chemistry* / Q000378:metabolism
D007295:Inositol Phosphates; C062528:glycero-3-phosphoinositol 4-phosphate; D010741:Phospholipases A; D054467:Phospholipases A2; D018631:ras Proteins; D002118:Calcium