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. 2005 Jul 1;389(Pt 1):181-6.
doi: 10.1042/BJ20041677.

Molecular cloning and characterization of a novel phospholipase C, PLC-eta

Jong-Ik Hwang  1 , Yong-Seok Oh, Kum-Joo Shin, Hyun Kim, Sung Ho Ryu, Pann-Ghill Suh
Affiliations

Molecular cloning and characterization of a novel phospholipase C, PLC-eta

Jong-Ik Hwang et al. Biochem J. .

Abstract

PLC (phospholipase C) plays an important role in intracellular signal transduction by hydrolysing phosphatidylinositol 4,5-bisphosphate, a membrane phospholipid. To date, 12 members of the mammalian PLC isoforms have been identified and classified into five isotypes beta, gamma, delta, epsilon and zeta, which are regulated by distinct mechanisms. In the present study, we describe the identification of a novel PLC isoform in the brains of human and mouse, named PLC-eta, which contains the conserved pleckstrin homology domain, X and Y domains for catalytic activity and the C2 domain. The first identified gene encoded 1002 (human) or 1003 (mouse) amino acids with an estimated molecular mass of 115 kDa. The purified recombinant PLC-eta exhibited Ca2+-dependent catalytic activity on phosphatidylinositol 4,5-bisphosphate. Furthermore, molecular biological analysis revealed that the PLC-eta gene was transcribed to several splicing variants. Although some transcripts were detected in most of the tissues we examined, the transcript encoding 115 kDa was restricted to the brain and lung. In addition, the expression of the 115 kDa protein was defined in only nerve tissues such as the brain and spinal cord. In situ hybridization analysis with brain revealed that PLC-eta was abundantly expressed in various regions including cerebral cortex, hippocampus, zona incerta and cerebellar Purkinje cell layer, which are neuronal cell-enriched regions. These results suggest that PLC-eta may perform fundamental roles in the brain.

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Figures

Figure 1
Figure 1. Molecular cloning of PLC-η
(A) The predicted amino acid sequence of human PLC-η. (B) Schematic representation of the predicted domain features of human PLC-η. PH, X, Y and C2 represent their respective domains. (C) Alignment of amino acid sequences corresponding to the X and Y domains of PLCs. The amino acid sequence of PLC-η is compared with PLC-β1, PLC-γ1 and PLC-δ1. Identical amino acids are boxed. The amino acids indicated with arrows are known to be essential for PLC activity. (D) Dendrogram illustrating phylogeny of CLUSTAL W aligned mammalian PLC sequences.
Figure 2
Figure 2. PLC-η has PtdIns(4,5)P2-hydrolysing enzyme activity
(A) Expressions of FLAG-tagged PLC-η. Recombinant adenoviruses for Flag–PLC-η (wild), and its mutant form (mutant), in which a glutamine residue is substituted for His358, were infected into COS-7 cells. Cells were lysed, 2 days post-infection, and subjected to Western-blot analysis using anti-Flag antibody. (B) PLC-η has all amino acids necessary for enzymatic activity. Purified wild-type PLC-η (wild) and its mutant (H358Q) were assayed for the PtdIns(4,5)P2-hydrolysing activity. (C) The Ca2+-dependence of PLC-η enzyme activity. Purified PLC-η (0.5 pmol) was examined for PtdIns(4,5)P2-hydrolysing activity in the presence of various concentrations of free Ca2+.
Figure 3
Figure 3. Organization of the PLC-η gene
A linear presentation of the exon locus shows the relative positions and approximate sizes of 24 exons spanning 200 kb (human) or 160 kb (mouse) of chromosome 3. (A) The cDNA co-ordinates for the exons of human gene are as follows: 1, 1–43; 2, 44–190; 3, 191–435; 4, 436–564; 5, 565–735; 6, 736–829; 7, 830–1033; 8, 1034–1154; 9, 1155–1327; 10, 1328–1434; 11, 1435–1596; 12, 1597–1668; 13, 1669–1760; 14, 1761–1858; 15, 1859–2038; 16, 2039–2146; 17, 2147–2271; 18, 2272–2356; 19, 2357–2503; 20, 2504–2583; 21, 2584–2643; 22, 2644–2998; 23, 2999–3026; 24, 3027–6196; 25, 2999–3208. (B) Schematic presentation of splicing variants (see the Results and discussion section). (a) hPLC-ηa (GenBank® accession no. AY691170), (b) hPLC-ηb (GenBank® accession no. AY691171) and (c) hPLC-ηc (GenBank® accession no. XM_042635). (C) The cDNA co-ordinates for the exons of mouse gene are as follows: 1, 1–43; 2, 44–190; 3, 191–435; 4, 436–565; 5, 566–735; 6, 736–829; 7, 830–1033; 8, 1034–1154; 9, 1155–1326; 10, 1327–1434; 11, 1435–1596; 12, 1597–1668; 13, 1669–1763; 14, 1764–1861; 15, 1862–2040; 16, 2041–2149; 17, 2150–2274; 18, 2275–2359; 19, 2360–2506; 20, 2507–2586; 21, 2587–2646; 22, 2647–3001; 23, 3002–3024 or 3029; 24, 3025 or 3030–6273. (D) Schematic presentation of splicing variants (see the Results and discussion section). (a) mPLC-ηa (GenBank® accession no. AY691172), (b) mPLC-ηb (GenBank® accession no. AY691173) and (c) mPLC-ηc (GenBank® accession no. AY691174).
Figure 4
Figure 4. Tissue distributions of PLC-η
(A) Northern-blot analysis of human tissues detected by PLC-η cDNA. SK muscle, skeletal muscle. (B) PCR analysis of PLC-η in cDNA from mouse tissues. The primer pair for common nucleotides of three splicing forms (upper panel) and the specific primer for the 23rd exon-containing cDNA (lower panel) were used in each PCR reaction. (C) Immunoblot analysis of PLC-η with mouse tissue extracts. Mouse PLC-η protein expressed in HEK-293 cells was used as a control. Immunoblot of tubulin was used as a loading control.
Figure 5
Figure 5. Subcellular localization of PLC-η
(A) Expression pattern of GFP-tagged PLC isoenzymes. pEGFP-C1 plasmids carrying PLC genes were transfected into HEK-293 cells, using a liposome-mediated gene transfer method. After 24 h, cells were fixed with 4% (w/v) paraformaldehyde and observed under fluorescent microscope. (B) Subcellular fractionation of brain tissue. PN, postnuclear; C, cytosol; DS, detergent-soluble membrane; DI, detergent-insoluble membrane.
Figure 6
Figure 6. Expression pattern of PLC-η in mouse brain
In situ hybridization with cRNA probes detecting mouse PLC-η. (A) Parasagittal section (a, c, e and g). Sense probe as negative control; (b, d, f and h) antisense probe; (c, d) olfactory bulb; (e, f) hippocampus; and (g, h) cerebellum. (B) Coronal section (a) sense probe; (b–f) antisense probe; (c) hippocampus; (d) zona incerta; (e) habenular nuclei and (f) hypothalamus.
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