Isoprenylated Human Brain Type I Inositol 1,4,5-Trisphosphate 5-Phosphatase Controls Ca²⁺ Oscillations Induced by ATP in Chinese Hamster Ovary Cells*

Abstract

d-myo-Inositol 1,4,5-trisphosphate (InsP₃) 5-phosphatase and 3-kinase are thought to be critical regulatory enzymes in the control of InsP₃ and Ca²⁺ signaling. In brain and many other cells, type I InsP₃ 5-phosphatase is the major phosphatase that dephosphorylates InsP₃ andd-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 Ca²⁺ ([Ca²⁺]_i) responses induced by ATP, thapsigargin, and ionomycin in Chinese hamster ovary (CHO-K1) cells transfected with the intact InsP₃5-phosphatase and with a mutant in which the C-terminal cysteine cannot be farnesylated. [Ca²⁺]_i was also measured in cells transfected with an InsP₃ 3-kinase construct encoding the A isoform. The Ca²⁺ oscillations detected in the presence of 1 μm ATP in control cells were totally lost in 87.5% of intact (farnesylated) InsP₃5-phosphatase-transfected cells, while such a loss occurred in only 1.1% of the mutant InsP₃ 5-phosphatase-transfected cells. All cells overexpressing the InsP₃ 3-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 thapsigargin 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 InsP₃-induced Ca²⁺release nor in the threshold for InsP₃ action were observed among the four clones and no differences in the expression levels of the various InsP₃ receptor isoforms could be shown between the clones. Our data support the contention that the ATP-induced increase in InsP₃ concentration in transfected CHO-K1 cells is essentially restricted to the site of its production near the plasma membrane, where it can be metabolized by the type I InsP₃5-phosphatase. This enzyme directly controls the [Ca²⁺]_i response and the Ca²⁺oscillations in intact cells.