Molecular cloning and characterization of PLC-eta2.
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-eta2, was cloned and characterized. Most of the coding sequence of PLC-eta2 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-eta2 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-eta2 is encoded by 23 exons. RT-PCR (reverse transcriptase-PCR) analyses illustrated expression of PLC-eta2 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-eta2-specific antisera recognized one of these splice variants as an approx. 155 kDa species when expressed in COS-7 cells; PLC-eta2 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-eta2, each containing possible alternatively spliced first exons. Co-expression of PLC-eta2 with Gbeta1gamma2 dimers of heterotrimeric G-proteins resulted in marked stimulation of inositol lipid hydrolysis. Thus PLC-eta2 may in part function downstream of G-protein-coupled receptors.
Zhou
Yixing
Y
Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA.
Wing
Michele R
MR
Sondek
John
J
Harden
T Kendall
TK
D017398:Alternative Splicing / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004789:Enzyme Activation; D005091:Exons / Q000235:genetics; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry; D054801:Phosphoinositide Phospholipase C; D016415:Sequence Alignment; D010738:Type C Phospholipases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D007527:Isoenzymes; D009419:Nerve Tissue Proteins; D010738:Type C Phospholipases; C507516:PLCH2 protein, human; D054801:Phosphoinositide Phospholipase C; C503884:Plch2 protein, mouse; D020962:Heterotrimeric GTP-Binding Proteins