Isoprenylated human brain type I inositol 1,4,5-trisphosphate 5-phosphatase controls Ca2+ oscillations induced by ATP in Chinese hamster ovary cells

D-myo-Inositol 1,4,5-trisphosphate (InsP(3)) 5-phosphatase and 3-kinase are thought to be critical regulatory enzymes in the control of InsP(3) and Ca2+ signaling, In brain and many other cells, type I InsP(3) B-phosphatase is the major phosphatase that dephosphorylates InsP(3) and D-myo-inositol 1,3,4,5-tetrakisphosphate. The type I B-phosphatase appears to be associated with the particulate fraction of cell homogenates, Molecular cloning of the human brain enzyme identifies a C-terminal farnesylation site CVVQ. Post-translational modification of this enzyme promotes membrane interactions and changes in specific activity, We have now compared the cytosolic Ca2+ ([Ca2+](i)) responses induced by ATP, thapsigargin, and ionomycin in Chinese hamster ovary (CHO-K1) cells transfected with the intact InsP(3) B-phosphatase and with a mutant in which the C-terminal cysteine cannot be farnesylated, [Ca2+](i) was also measured in cells transfected with an InsP(3) 3-kinase construct encoding the A isoform, The Ca2+ oscillations detected in the presence of 1 mu M ATP in control cells were totally lost in 87.5% of intact (farnesylated) InsP(3) B-phosphatase-transfected cells, while such a loss occurred in only 1.1% of the mutant InsP(3) 5-phosphatase-transfected cells, All cells overexpressing the InsP(3) S-kinase also responded with an oscillatory pattern, However, in contrast to control cells, the [Ca2+](i) returned to base-line levels in between a couple of oscillations, The [Ca2+](i) responses to thapsigarin and ionomycin were identical for all cells, The four cell clones compared in this study also behaved similarly with respect to capacitative Ca2+ entry, In permeabilized cells, no differences in extent of InsP(3)-induced Ca2+ release nor in the threshold for InsP(3) action were observed among the four clones and no differences in the expression levels of the various InsP(3) receptor isoforms could be shown between the clones, Our data support the contention that the ATP-induced increase in InsP(3) concentration in transfected CHO-KI cells is essentially restricted to the site of its production near the plasma membrane, where it can be metabolized by the type I InsP(3) B-phosphatase. This enzyme directly controls the [Ca2+](i) response and the Ca2+ oscillations in intact cells.