Membrane transport and in vitro metabolism of the Ras cascade messenger, glycerophosphoinositol 4-phosphate

The glycerophosphoinositols, phosphoinositide metabolites formed by Ras-dep endent activation of phospholipase A(2) 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 t ypes:after growth factor receptor stimulation of phosphatidylinositol 3-kin ase and the small G protein Rac, indicating the importance of defining furt her its cellular actions and metabolism. We show here that, in postnuclear membranes from Swiss 3T3 cells, there is no high-affinity 'receptor' bindin g of GroPIns4P. Instead, possibly through the interaction with a transporte r, GroPIns4P rapidly equilibrates between medium and cell,cytosol, and, at higher concentrations, can concentrate in the cell cytosol. GroPIns4P can b e dephosphorylated to GroPIns in vitro by an enzyme that is membrane-associ ated, Ca2+-dependent, GroPIns4P-selective and has a specific pH profile. Un der in vitro phosphorylating conditions, there is production of GroPIns(4,5 )P-2 and other inositol phosphates. As these in vitro enzyme activities do not fully correlate with the in vivo handling of GroPIns4P, the intracellul ar GroPIns4P levels may be controlled by its direct physical removal from t he cells.