Molecular Cloning and Characterization of Atp6n1b

Abstract

PLC (phospholipase C) isoenzymes catalyse the conversion of PtdIns(4,5)P2 into the Ca2+-mobilizing second messenger, Ins(1,4,5)P3, and the protein kinase C-activating second messenger, diacylglycerol. With the goal of identifying additional mammalian PLC isoenzymes, we screened the NCBI non-redundant database using a BLAST algorithm for novel sequences with homology with the conserved PLC catalytic core. Two unique sequences corresponding to two unknown PLC isoenzymes were identified, and one of these, designated PLC-2, was cloned and characterized. Most of the coding sequence of PLC-2 was constructed from two ESTs (expressed sequence tags), which included an overlapping sequence that was confirmed by multiple ESTs and mRNAs. 5'-RACE (rapid amplification of cDNA ends) also identified an upstream exon not deduced from available EST or mRNA sequences. Sequence analysis of PLC-2 revealed the canonical domains of a PLC isoenzyme with an additional long C-terminus that contains a class II PDZ-binding motif. Genomic analyses indicated that PLC-2 is encoded by 23 exons. RT-PCR (reverse transcriptase-PCR) analyses illustrated expression of PLC-2 in human retina and kidney, as well as in mouse brain, eye and lung. RT-PCR with exon-specific primers also revealed tissue-specific expression of four splice variants in mouse that represent alternative use of sequences in exons 21, 22 and 23. PLC-2-specific antisera recognized one of these splice variants as an approx. 155 kDa species when expressed in COS-7 cells; PLC-72 natively expressed in 1321N1 human astrocytoma cells also migrated as an approx. 155 kDa species. PLC activity was observed in vitro and in vivo for three different constructs of PLC-2, each containing possible alternatively spliced first exons. Co-expression of PLC-2 with Gbeta12 dimers of heterotrimeric G-proteins resulted in marked stimulation of inositol lipid hydrolysis. Thus PLC-2 may in part function downstream of G-protein-coupled receptors. 2005 Biochemical Society.