Molecular cloning of magnesium-independent type 2 phosphatidic acid phosphatases from airway smooth muscle

Members of the type 2 phosphatidic acid phosphatase (PAP2) family catalyse the dephosphorylation of phosphatidic acid (PA), lysophosphatidate and sphi ngosine 1-phosphate. Here, we demonstrate the presence of a Mg2+-independen t and N-ethymaleimide-insensitive PAP2 activity in cultured guinea-pig airw ay smooth muscle (ASM) cells. Two PAP2 cDNAs of 923 and 926 base pairs were identified and subsequently cloned from these cells. The ORF of the 923 ba se pair cDNA encoded a protein of 285 amino acids (Mr = 32.1 kDa), which ha d 94% homology with human PAP2a (hPAP2a) and which probably represents a gu inea-pig specific PAP2a (gpPAP2a1). The ORF of the 926 base pair cDNA encod ed a protein of 286 amino acids (Mr = 32.1 kDa) which had 84% and 91% homol ogy with hPAP2a and gpPAP2a1, respectively. This protein, termed gpPAP2a2, has two regions (aa 21-33 and 51-74) of marked divergence and altered hydro phobicity compared with hPAP2a and gpPAP2a1. This occurs in the predicted f irst and second transmembrane domains and at the extremes of the first oute r loop. Other significant differences between gpPAP2a1/2 and hPAP2a, hPAP2b and hPAP2c occur at the cytoplasmic C-terminal. Transient expression of gp PAP2a2 in Cos-7 cells resulted in an approx. 4-fold increase in Mg2+-indepe ndent PAP activity, thereby confirming that gpPAP2a2 is another catalytical ly active member of an extended PAP2 family. (C) 1999 Elsevier Science Inc.