CHARACTERIZATION OF A CDNA-ENCODING THE 43-KDA MEMBRANE-ASSOCIATED INOSITOL-POLYPHOSPHATE 5-PHOSPHATASE

Agonist stimulation of cells results in phosphatidylinositol turnover and the generation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P-3), wh ich mobilizes intracellular calcium. The inositol-polyphosphate 5-phos phatase (5-phosphatase) enzymes hydrolyze Ins(1,4,5)P-3 in a signal-te rminating reaction. We have isolated a 2.7-kilobase (kb) composite cDN A, encoding the 43-kDa membrane-associated 5-phosphatase, by screening a human placental lambda gt11 library, using degenerate oligonucleoti des. The 2.7-kb cDNA contains a 1.1-kb open reading frame, comprising 363 amino acids, which encodes a protein of a predicted molecular mass of 42 kDa. Amino acid sequence analysis demonstrates a number of pote ntial sites for phosphorylation by protein kinase C and a CAAX motif i n the COOH terminus, which may mediate membrane localization. The reco mbinant enzyme was expressed in COS-7 cells, resulting in a 50-fold in crease in enzyme activity in the detergent-soluble membrane fraction o f the cell (nanomole of Ins(1,4,5)P-3 hydrolyzed per min/mg), but only a 2.5-fold increase in 5-phosphatase activity in the total cell homog enate. Sequence analysis demonstrated a 73-amino acid domain in the CO OH terminus of the 43-kDa membrane-associated 5-phosphatase, which had 30% sequence identity and 67% similarity to a region in the 75-kDa 5- phosphatase and 34% identity and 70% similarity to a sequence in the p rotein that is encoded by the gene, defective in Lowe's oculocerebrore nal syndrome. As shown by RNA analysis the 43-kDa membrane-associated 5-phosphatase appears to be predominantly expressed in heart, brain, a nd skeletal muscle.