Isoprenylated human brain type I inositol 1,4,5-trisphosphate 5- phosphatase controls Ca 2+ oscillations induced by ATP in Chinese hamster ovary cells

Abstract

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 Ca 2+ signaling. In brain and many other cells, type I InsP 3 5-phosphatase is the major phosphatase that dephosphorylates InsP 3 and D-myo-inositol 1,3,4,5-tetrakisphosphate. The type I 5-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 Ca 2+ ([Ca 2+](i)) responses induced by ATP, thapsigargin, and ionomycin in Chinese hamster ovary (CHO-K1) cells transfected with the intact InsP 3 5- phosphatase and with a mutant in which the C-terminal cysteine cannot be farnesylated. [Ca 2+](i) was also measured in cells transfected with an InsP 3 3-kinase construct encoding the A isoform. The Ca 2+ oscillations detected in the presence of 1 μM ATP in control cells were totally lost in 87.5% of intact (farnesylated) InsP 3 5-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 3-kinase also responded with an oscillatory pattern. However, in contrast to control cells, the [Ca 2+](i) returned to base-line levels in between a couple of oscillations. The [Ca 2+](i) responses to thapsigargin and ionomycin were identical for all cells. The four cell clones compared in this study also behaved similarly with respect to capacitative Ca 2+ entry. In permeabilized cells, no differences in extent of InsP 3-induced Ca 2+ 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-K1 cells is essentially restricted to the site of itsproduction near the plasma membrane, where it can be metabolized by the type I InsP 3 5-phosphatase. This enzyme directly controls the [Ca 2+](i) response and the Ca 2+ oscillations in intact cells.