CLONING, DIFFERENTIAL REGULATION AND TISSUE DISTRIBUTION OF ALTERNATIVELY SPLICED ISOFORMS OF ADP-RIBOSYLATION-FACTOR-DEPENDENT PHOSPHOLIPASE-D FROM RAT-LIVER

An alternatively spliced isoform of ADP-ribosylation-factor-dependent phospholipase D (PLD1) was previously shown to occur in rat C6 cells [ Yoshimura, Nakashima, Ohguchi, Sakai, Shinoda, Sakai and Nozawa (1996) Biochem. Biophys. Res. Commun. 225, 494-499] and human HeLa cells [Ha mmond, Jenco, Nakashima, Cadwallader, Gu, Cook, Nozawa, Prestwich, Fro hman and Morris (1997) J. Biol. Chem. 272, 3860-3868]. However, its co mplete sequence and the enzymological difference between the two PLD1 isoforms were unclear. Here we report the cloning, complete sequence, enzymological properties and tissue distribution of each of the two al ternatively spliced PLD1 isoforms, a and b, from rat liver. The major difference between the two isoforms was the deletion of 38 amino acids in the b isoform, but otherwise the two cDNA sequences were 99.9% ide ntical. The a-isoform sequence was 91% identical with the a form of hu man PLD1, and the 38-amino-acid deletion in the b form occurred at the same site as in the b form of human PLD1. Both of the rat PLD1 isofor ms expressed in the fission yeast Schizosaccharomyces pombe were depen dent on ADP-ribosylation factor 1 and phosphatidylinositol 4,5-bisphos phate. The a isoform was activated by RhoA in a synergistic manner wit h ADP-ribosylation factor 1, whereas the b isoform was less responsive to RhoA. Reverse transcription PCR showed that the b form was the pre dominant PLD1 isoform expressed in rat tissues. The b-form transcript occurred in various rat tissues, including lung, brain, liver, kidney, small intestine and colon, whereas the a-form transcript was only det ectable in lung, heart and spleen. Both transcripts were hardly detect able in thymus, stomach, testis and muscle. Thus the two PLD1 isoforms were differently regulated and expressed in rat tissues.