title: Phospholipase-C-independent inositol 1,4,5-trisphosphate formation in Dictyostelium cells. Activation of a plasma-membrane-bound phosphatase by receptor-stimulated Ca2+ influx.
abstract: Dictyostelium cells have enzyme activities that generate the inositol polyphosphate Ins(1,4,5)P3 from Ins(1,3,4,5,6)P5 via the intermediates Ins(1,3,4,5)P4 and Ins(1,4,5,6)P4. These enzyme activities could explain why cells with a deletion of the single phospholipase C gene (plc- cells) possess nearly normal Ins(1,4,5)P3 levels. In this study the regulation and the subcellular localization of the enzyme activities was investigated. The enzyme activities performing the different reaction steps from Ins(1,3,4,5,6)P5 to Ins(1,4,5)P3 are probably due to a single enzyme. Indications for this are the previously shown similar Ca2+ dependencies of the various reaction steps. Furthermore, the activities mediating the complete conversion of Ins(1,3,4,5,6)P5 to Ins(1,4,5)P3 co-purify after subcellular fractionation, solubilization, and chromatography of the proteins. Subcellular fractionation studies demonstrate that the enzyme is localized mainly at the inner face of the plasma membrane. The enzyme activity could not be stimulated in vitro by guanosine 5'-(3-thio)triphosphate, a procedure known to activate G-protein-coupled enzymes in Dictyostelium. Still, in plc- cells the level of Ins(1,4,5)P3 was increased significantly after stimulation with high concentrations of the extracellular ligand cAMP. This stimulation is most likely due to the influx of Ca2+ because no increase of Ins(1,4,5)P3 could be detected in the absence of extracellular Ca2+. The results demonstrate the existence of a new receptor-controlled route for the formation of Ins(1,4,5)P3 that is independent of phospholipase C.
authors.affiliation: Department of Biochemistry, University of Groningen, The Netherlands.
authors.forename: P
authors.initials: P
authors.lastname: Van Dijken
authors.forename: J C
authors.initials: JC
authors.lastname: Bergsma
authors.forename: P J
authors.initials: PJ
authors.lastname: Van Haastert
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D004023:Dictyostelium / Q000378:metabolism*; D004789:Enzyme Activation; D015544:Inositol 1,4,5-Trisphosphate / Q000096:biosynthesis* / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D011953:Receptors, Cyclic AMP / Q000502:physiology*; D012995:Solubility; D013347:Subcellular Fractions / Q000201:enzymology / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism*
chemical_list: D011953:Receptors, Cyclic AMP; D015544:Inositol 1,4,5-Trisphosphate; D010744:Phosphoric Monoester Hydrolases; C064119:multiple inositol-polyphosphate phosphatase; D010738:Type C Phospholipases; D002118:Calcium