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Molecular cloning and characterization of PLC‐η2
Author(s) -
Zhou Yixing,
Wing Michele R,
Sondek John,
Harden T. Kendall
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a693-c
Subject(s) - biology , alternative splicing , exon , isozyme , microbiology and biotechnology , rna splicing , expressed sequence tag , phospholipase c , coding region , peptide sequence , gene , complementary dna , genetics , biochemistry , enzyme , signal transduction , rna
Phospholipase C isozymes catalyze the conversion of phosphatidylinositol‐(4,5)P 2 to the Ca 2+ ‐mobilizing second messenger, Ins(1,4,5)P 3 , and the protein kinase C‐activating second messenger, diacylglycerol. With the goal of identifying additional mammalian PLC isozymes, we screened the NCBI non‐redundant database for novel sequences with homology to the conserved PLC catalytic core. Two unique sequences corresponding to two unknown PLC isozymes were identified, and one of these, designated PLC‐η2, was cloned and characterized. Most of the coding sequence of PLC‐η2 was constructed from two ESTs, which included overlapping sequence confirmed by multiple ESTs and mRNAs. 5′‐RACE also identified an upstream exon not deduced from available sequences. Sequence analysis of PLC‐η2 revealed the canonical domains of a PLC isozyme with an additional long carboxyl terminus that contains a PDZ binding motif. Genomic analyses indicated that PLC‐η2 is encoded by 23 exons. RT‐PCR analyses illustrated expression of PLC‐η2 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 splice variants in both mouse and human. PLC‐η2‐specific antisera recognized one of these splice variants as a 155 kDa species when expressed in COS‐7 cells; PLC‐η2 natively expressed in 1321N1 human astrocytoma cells also migrated as a 155 kDa species. PLC activity was observed in vitro and in vivo for three constructs of PLC‐η2 each containing possible alternatively spliced first exon. Coexpression of PLC‐η2 with Gβ1γ 2 dimers of heterotrimeric G proteins resulted in marked stimulation of inositol lipid hydrolysis. Thus PLC‐η2 may in part function downstream of G‐protein‐coupled receptors.