A Novel Phospholipase C, PLCη2, Is a Neuron-specific Isozyme*

Twelve phospholipase C (PLC) isozymes have been cloned so far, and they are divided into six classes, β-, γ-, δ-, ϵ-, ζ-, and η-type, on the basis of structure and activation mechanisms. Here we report the identification of a novel PLC isozyme, PLCη2. PLCη2 is composed of conserved domains including pleckstrin homology, EF-hand, X and Y catalytic, and C2 domains and the isozyme-specific C-terminal region. PLCη2 consists of 1164 amino acids with a molecular mass of 125 kDa. The PLC activity of PLCη2 was more sensitive to calcium concentration than the PLC activity of the PLCδ-type enzyme, which is thought to be the most calcium-sensitive PLC. Immunofluorescence analysis showed that PLCη2 was localized predominantly to the plasma membrane at resting state via the pleckstrin homology domain. This observation was supported by Western blot analysis of cytosol and membrane fractions. In addition, expression of PLCη2 was detected after birth and showed a restricted distribution in the brain; it was particularly abundant in the hippocampus, cerebral cortex, and olfactory bulb. The pattern was similar to that of the neuronal marker microtubule-associated protein 2 by Western blot. Furthermore, in situ hybridization showed positive signals for PLCη2 in pyramidal cells of the hippocampus. Finally, we found that PLCη2 was expressed abundantly in neuron-containing primary culture but not in astrocyte-enriched culture. These results indicate that PLCη2 is a neuron-specific isozyme that may be important for the formation and/or maintenance of the neuronal network in the postnatal brain.

Recent data base information suggests the existence of an additional PLC-like protein in the mouse and human genomes. Therefore we attempted to isolate the cDNA for PLC-like protein, PLC2, from mouse brain by reverse transcription-PCR. We found PLC2 to be much enriched in the brain, particularly in the hippocampus, cerebral cortex, and olfactory bulb, beginning after birth. In addition, PLC2 was abundant in primary cultured neurons but not in astrocytes. These results suggest that PLC2 may be important for the formation and maintenance of the neuronal network in the postnatal brain.
Antibodies and Western Blot Analysis-A rabbit polyclonal antibody against a His 6 -tagged region between the X and Y domains (X-Y region) of PLC2 was developed. Affinity-purified anti-PLC2 antibody was prepared by passing the serum over a glutathione S-transferase (GST)tagged antigen-coupled Hi-Trap TM NHS-activated HP column (Amersham Biosciences). Anti-FLAG antibody was purchased from Sigma, and fluorescent secondary antibodies and Alexa 594-phalloidin were from Molecular Probes. Anti-microtubule-associated protein 2 (MAP2), anti-myelin basic protein (MBP), anti-glial fibrillary acidic protein (GFAP), and anti-actin antibodies were from Neomarkers, BD Bio-* This work was supported by a grant-in-aid for general scientific research and a High-Tech Research Center Project for Private Universities matching fund subsidy (2002)(2003)(2004)(2005)(2006) from the Japan Ministry of Education, Culture, Sports, Science and Technology. This study was also supported by the Naito Foundation and the Uehara Memorial Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AY966876.
Brain and other tissues were homogenized in lysis buffer containing 20 mM HEPES-KOH, pH 7.0, 120 mM KCl, complete protease inhibitor mixture (Roche Applied Science), and 0.1% sodium deoxycholate and centrifuged for 30 min at 100,000 ϫ g to remove insoluble debris. The resulting supernatants were subjected to SDS-PAGE and transferred electrophoretically onto polyvinylidene difluoride membranes (Millipore). Membranes were probed with antibodies against PLC2 or actin (Chemicon) and incubated with appropriate horseradish peroxidaseconjugated secondary antibodies (DAKO), and immunocomplexes were visualized by enhanced chemiluminescence (ECL, Amersham Biosciences).
Immunoprecipitation and Measurement of PLC Activity in Vitro-Brain was homogenized as described above. Lysates were centrifuged at 100,000 ϫ g for 30 min at 4°C, and the supernatants were used for immunoprecipitation with nonimmune IgG (Santa Cruz Biotechnology), anti-PLC␦1 (a gift from Dr. Pann-Ghill Suh, POSTECH, Pohang, Korea), or PLC␦4 antibody (16) followed by incubation with protein A-Sepharose CL-4B (Amersham Biosciences). PLC activity of the precipitates was measured as described elsewhere (17) with some modifications. In brief, precipitates were incubated in a reaction mixture of 50 M phosphatidylethanolamine (Doosan Serdary Research Laboratories), 40 M PIP 2 (Sigma), 1 Ci/ml (100 nM) [ 3 H]PIP 2 (PerkinElmer Life Sciences), 50 mM HEPES-KOH, pH 7.0, 76 mM KCl, 100 M Ca 2ϩ , and 0.5 mg/ml bovine serum albumin at 37°C for 5 min, and the reaction was terminated by adding chloroform:methanol (2:1, v/v). Resulting [ 3 H]IP 3 was extracted by adding 1 N HCl and measured with a liquid scintillation counter. For mutation analysis and assay of calcium dependence and pH dependence, GST-tagged PLC2 and GST-tagged PLC␦1 were prepared with a baculovirus/Sf9 cell expression system (Invitrogen). For mutation analysis and pPH dependence, assays were performed in the presence of 10 M Ca 2ϩ , and then the amounts of [ 3 H]IP 3 production by these enzymes in vitro were measured.
Northern Blot Analysis-Mouse multiple Northern blot membrane was purchased from Clontech. The membrane was probed with radiolabeled probe comprising 426 bp of the following region of the C2 domain (nucleotides 2623-3048 of the open reading frame) as recommended by the manufacturer's protocol.
Cell Culture and Immunostaining-HeLa S3 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Invitrogen) at 37°C under 5% CO 2 . For immunostaining, HeLa S3 cells were transfected with various plasmids with the use of Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer's protocol. Twenty-four h after transfection, cells were fixed in 3.7% formaldehyde at room temperature for 5 min, permeabilized with 0.2% Triton X-100, and blocked with 5% skim milk. Then the cells were incubated with anti-PLC2 polyclonal antibody or anti-FLAG polyclonal antibody and treated with the appropriate Alexa 488-conjugated goat secondary antibody. To visualize the actin cytoskeleton, Alexa 594-conjugated phalloidin (Molecular Probes) was used.
Hippocampal primary culture and cortical astrocyte-enriched culture were carried out as described previously (18,19). In brief, the hippocampal cells were prepared from embryonic day 18 or 19 Wistar rats by enzymatic treatment and plated onto 12-mm polyethyleneimine-coated coverslips in DMEM containing 50 units/ml penicillin, 50 g/ml streptomycin, 5% horse serum, and 5% FBS. The day after plating, the medium was replaced with DMEM containing B27 supplement (Invitrogen), and cells were cultured for 14 -21 days. To obtain astrocytes, cortical cells were prepared from postnatal day 1 rat by enzymatic treatment and cultured for 12 days in basal Eagle's medium containing 10% FBS with a medium change every 3 days, and the resulting mixed glia culture was shaken at 260 rpm for 18 h at 37°C and then rinsed with medium to remove nonastrocytic cells. Adherent cells were subcultured in DMEM containing 25 mM HEPES-KOH, pH 7.4, and 10% FBS. After 48 h, if necessary the medium was changed to astrocyte-defined medium (ADM), and culture continued for an additional 48 -96 h. Cultures were fixed in 3.7% formaldehyde, permeabilized with 0.2% Triton X-100, and blocked with 5% skim milk. Cells were then incubated with anti-MAP2 monoclonal antibody and anti-GFAP polyclonal antibody and treated with the appropriate Alexa 488-or Alexa 594-conjugated goat secondary antibodies. Cells were observed with confocal laser scanning microscopy (Leica Microsystems).
In Situ Hybridization-In situ hybridization was performed with digoxigenin-labeled RNA probes (Roche Applied Science). Probes for a 401-base pair fragment were designed from position 1482 to 1882 of the open reading frame of PLC2. Hybridization was carried out as de-scribed previously (20), under contract by Genostaff. The probes were hybridized at 60°C with 60% formamide. Sections were treated with anti-digoxigenin antibody at room temperature and visualized with diaminobenzidine.

RESULTS
Isolation of Mouse PLC2 cDNA-The mouse genome-sequencing project has recently been completed. We searched the Celera, Inc. data base and found two genes on mouse chromosomes 3 (mCG6375) and 4 (mCG3920) that encode PLC-like proteins. The former gene was recently reported as PLC (21). The latter gene encoded five PLC-specific functional domains, including PH, EF-hand, PLCX, PLCY, and C2, and a long extension at the C terminus, but unfortunately this gene lacked the N-terminal sequence. We then carried out a BLAST search analysis of mCG3920 and found a clone that might contain the N-terminal region of this protein (RIKEN cDNA A930027K05). With this putative full-length sequence, a complete open reading frame region was isolated from mouse brain by reverse transcription-PCR, and correct nucleotide and amino acid sequences were confirmed (Fig. 1A). The five PLC-specific domains, PH, EF-hand, PLCX, PLCY, and C2, are shown boxed in Fig. 1A. Because there were two N-terminal methionine residues that could be used as the first methionine, we attempted to determine which was the correct one. First, we developed a rabbit polyclonal antibody against this PLC-like protein. The antigen was designed against the linker region between PLCX and PLCY (underlined in Fig. 1A). This antibody showed no cross-reaction with other PLC isozymes (data not shown). With this antibody, we immunoprecipitated the endogenous protein from mouse brain, and the molecular mass was compared with the molecular mass of ectopically expressed short-type (⌬N) or long-type (Full) novel PLC-like protein (Fig. 1B). The molecular mass of the endogenous protein coincided with that of long-type PLC-like protein. The estimated number of amino acids was 1164, and the predicted molecular mass was 125 kDa. The domain structures of PLC subtypes PLC␤, -␥, -␦, -⑀, -, and -1 and the novel PLC-like protein are illustrated in Fig. 1C. Although the novel protein appeared to be a member of the PLC␤ family with a long C terminus, amino acid comparison showed that it is similar to PLC␦ and has the highest similarity to PLC1 (Fig. 1D). Therefore, we named the protein PLC2 (GenBank TM accession number AY966876). We could not find any similarity against the PH domain and EF-hand motif of PLC␤ and ⑀. A dendrogram based on total amino acid similarity is shown in Fig. 1E, in which PLC2 locates closer to the PLC␦ than to the PLC␤ family.
Distribution of PLC2 in Various Tissues by Western and Northern Analysis-We examined the distribution of PLC2 in various mouse tissues. First, Western blot analysis with PLC2 antibody was carried out. As shown in Fig. 2A, a 125-kDa protein was specifically detected in mouse brain. We occasionally detected a degraded protein of ϳ95 kDa. No bands were detected in heart, skeletal muscle, kidney, liver, small intestine, lung, testis, or spleen. Actin was used as a loading control. Northern blot analysis also showed a specific signal of ϳ6.5 kb in brain (Fig. 2B), indicating that expression of PLC2 is limited to the brain.
Activity of PLC2 Is Very Sensitive to the Calcium Concentration-Two PLC-like proteins (PLC-L-type) that lack PLC activity (PIP 2 -hydrolyzing activity) have been reported (22,23). Thus, we examined whether PLC2 possesses PLC activity. Endogenous PLC2 immunoprecipitated from mouse brain with anti-PLC2 antibody showed relatively high activity (Fig. 3A). The activity of PLC2 was similar to that of PLC␦4 and lower than that of PLC␦1, which possesses high PLC activity. Immunoprecipitate with normal IgG (Fig. 3A, N) showed little activity.
The PLCX and PLCY domains, which confer PLC activity, are highly conserved among PLC isozymes. It is well known that a histidine residue in the PLCX domain is essential for PLC activity (Fig. 3B); substitution of histidine with alanine resulted in a dramatic reduction of enzyme activity (24). To determine whether histidine 341 in the PLCX domain of PLC2 is essential for PLC activity, we constructed a mutant in which this histidine is substituted with alanine (H341A). GST-fused recombinant PLC2 expressed in a baculovirus system showed specific activity of 54 nmol/min/mg protein, whereas the H341A mutant showed activity similar to that of the GST control (Fig. 3C), indicating that histidine 341 in the PLCX domain of PLC2 is essential for PLC activity.
PLC2 resembles PLC␦, which is very sensitive to [Ca 2ϩ ] (25). Therefore, we analyzed the dependence of PLC activity on [Ca 2ϩ ]. PLC activity was measured at [Ca 2ϩ ] between 10 Ϫ8 and 10 Ϫ4 M for PLC2 and PLC␦ 1 (Fig. 3D). Whereas PLC␦ 1 showed maximal PLC activity at 10 -100 M Ca 2ϩ , PLC2 showed maximal PLC activity at 1 M Ca 2ϩ and was sensitive to Ca 2ϩ at a concentration as low as 10 nM, indicating that the PLC activity of PLC2 is more sensitive to [Ca 2ϩ ] than is the PLC activity of PLC␦1. We also measured PLC activity under

FIG. 2. Distribution of PLC2 in various tissues by Western (A) and Northern (B) analysis.
A, expression of PLC2 in mouse tissues. Protein extracts from various tissues were subjected to SDS-PAGE, and Western blot was performed with anti-PLC2 antibody. B, brain; H, heart; Sk, skeletal muscle; K, kidney; Li, liver; Sm, small intestine; Lu, lung; T, testis; Sp, spleen. Actin was used as a loading control. B, Northern blot analysis of PLC2 in mouse tissues. A probe consisting of 426 nucleotides in the C-terminal region of PLC2 was radiolabeled and hybridized with 2 g of poly(A) RNA from various mouse tissues. Equal loading was confirmed by reprobing the membrane with ␤-actin. various pH conditions. Maximal PLC activity of PLC2 and PLC␦1 was obtained at pH 7.0 (Fig. 3E), similar to that observed for other PLC isozymes.
The N-terminal Region of PLC2 Contains a Localization Signal for the Plasma Membrane-Most PLC isozymes exist in the cytosol and translocate to the plasma membrane in response to cell stimulation through isozyme-specific anchoring mechanisms (26 -29). PLC␦1 expressed in HeLa S3 cells was localized predominantly in the cytosol (Fig. 4B, j). However, PLC2 was localized predominantly at the plasma membrane (Fig. 4B, a). Because the PH domain of PLC␦ provides a trigger for membrane targeting (26,29), we next examined whether membrane binding of PLC2 occurs via the PH domain. A deletion mutant of PLC2 PH domain (Fig. 4A, ⌬PH) localized predominantly to the cytosol (Fig. 4B, d), whereas the FLAGtagged PH region was restricted to the plasma membrane (Fig.  4B, g). Distribution of these proteins to the cytosol or plasma membrane in cell populations is shown in Fig. 4C. Approximately 85% of full-length PLC2 and 97% of FLAG-tagged PH protein localized to the membrane, whereas less than 6% of ⌬PH localized to the membrane. More than two-thirds of PLC␦1 was detected in the cytosol. These results strongly suggest that the N-terminal PH region of PLC2 contains a targeting signal for the plasma membrane. To confirm these results, membrane and cytosol fractions were separated, and Western blot analysis was performed (Fig. 4D). Bands of fulllength PLC2 and FLAG-tagged PH protein were detected in the membrane fraction, whereas bands of ⌬PH and PLC␦1 were detected mainly in the cytosol.
Increased Expression of PLC2 in Postnatal Brain-Although expression of PLC2 was abundant in adult brain, we did not detect it in embryos (data not shown). To understand the physiological function of PLC2, we examined the change in expression of PLC2 during brain development ( Fig. 5A). At 1 week after birth, PLC2 was barely detected, and expression was first observed at 2 weeks after birth. Expression of PLC2 gradually increased during brain development and was similar to that of MBP, a marker of mature oligodendrocytes.
PLC2 Is Abundant in the Hippocampus, Cerebral Cortex, and Olfactory Bulb-To further evaluate the distribution of PLC2, we examined the specific localization of PLC2 in the brain. PLC2 was detected at a high level in the hippocampus, cerebral cortex, and olfactory bulb and weakly in the telencephalon, diencephalon, mesencephalon, and cerebellum. No expression was observed in the pons, medulla oblongata, or spinal cord (Fig. 5B). Comparison of the expression pattern of PLC2 with that of MAP2 (a marker of neurons), GFAP (a marker of astrocytes), and MBP showed that the expression pattern of PLC2 was very similar to that of MAP2, implying that PLC2 expression is correlated with neural function. Olfactory organs function in odor discrimination, pheromone perception, and odor and pheromone memory. To determine whether PLC2 is coupled to the odor or pheromone receptor, we separated olfactory organs into regions and determined the distribution of PLC2 (Fig. 5C). We could not detect PLC2 in the olfactory epithelium or vomeronasal organs, where odor and pheromone receptors exist. However, PLC2 was abundant in the main olfactory bulb and subolfactory bulb, indicating that PLC2 is not coupled to odor or pheromone receptors but is involved in odor and pheromone signal transduction.
In situ hybridization supported the expression pattern of PLC2 detected by Western blot analysis (Fig. 6). Antisense signals for PLC2 were observed in the olfactory bulb (Fig. 6,  A-C), cerebral cortex (G-I), and hippocampus (M-O). The enlarged image of the hippocampus (Fig. 6, N and O) showed that pyramidal cells strongly express PLC2. The diencephalon, mesencephalon, pons, and medulla oblongata were not stained (data not shown). No positive signal was obtained with sense probes (Fig. 6, D-F, J-L, and P-R).
PLC2 Is a Neuron-specific Enzyme-The distinct expression of PLC2 in the brain and during development implies that PLC2 plays a role in nerve tissues. We attempted to confirm this in primary cultured neurons and astrocytes. Neuron-containing primary cultures in defined medium (B27) and astrocyte-enriched cultures in conventional serum-containing medium (FBS) or ADM were used. Cell population was verified by the expression of MAP2, GFAP, or MBP by immunohistochemistry (Fig. 7A) and Western blot analysis (Fig. 7B). Although astrocytes were observed in all of these cultures, neurons were detected only in primary cultures (B27). As shown in Fig. 7B, PLC2 was detected only in primary cultures (B27) but not in astrocyte-enriched cultures (FBS and ADM). Oligodendrocytes were not detected in these cultures. These results indicate that PLC2 is expressed specifically in neurons and may have important functions postnatally. DISCUSSION Herein we report the isolation of a novel PLC isozyme, PLC2, from mouse brain. The primary sequence of PLC2 suggests that it should be classified as a PLC␤-type enzyme because it includes the basic domains of PLC and an additional 290 amino acids at the C terminus (Fig. 1C). However, sequence comparison showed a similarity to PLC␦ (Fig. 1, D and  E). Recently, a novel class of PLC isozyme, PLC, was reported (21). Because PLC2 has the highest homology to PLC, we named this enzyme PLC2. One of the specific characteristics of PLC2 is the high sensitivity of PLC activity to [Ca 2ϩ ], which is similar to the sensitivity of PLC. PLC2 is activated at [Ca 2ϩ ] as low as 10 nM, indicating a sensitivity 10 times

FIG. 5. Expression of PLC2 by Western blot analysis.
A, increased expression of PLC2 with brain development. Lysates from mouse brain at 1 day (1d), 3 days (3d), 1 week (1w), 2 weeks (2w), 3 weeks (3w), and 4 weeks (4w) after birth were subjected to SDS-PAGE and Western blot for PLC2, MAP2, GFAP, and MBP. Actin was used as a loading control. B, detailed distribution of PLC2 in mouse brain. Western blot analysis was performed with anti-PLC2, anti-MAP2, anti-GFAP, and anti-MBP antibodies on protein extracts from the indicated regions of mouse brain and spinal cord. Actin was used as a loading control. obl., oblongata. C, localization of PLC2 in olfactory organs. Protein extracts from the vomeronasal organ, subolfactory bulb, olfactory epithelium, and main olfactory bulb were subjected to SDS-PAGE and Western blot with anti-PLC2. Actin was used as a loading control.
greater than that of PLC␦ (Fig. 3D). PLC␦ is known as the most calcium-sensitive PLC and is activated only by calcium. Therefore, it is possible that the increased calcium dependence of PLC2 allows it to act as a calcium sensor and to be activated by small increases in intracellular calcium concentration under physiological conditions. It is also noteworthy that PLC2 is localized predominantly to the plasma membrane (Fig. 4B, a) in contrast to most other PLC isozymes, which commonly exist in the cytosol and translocate to the plasma membrane in response to receptor activation. We showed by analysis of cell staining and subcellular fractionation that the PH domain of PLC2 is essential for membrane anchoring. Constitutive PH domain-mediated localization of PLC2 to the plasma membrane suggests a novel role of PLC2 as an early response molecule.
We further found that the expression of PLC2 is restricted to the brain (Fig. 2) and occurs after birth (Fig. 5A). The expression continues throughout the life of the adult (Fig. 5B). Robust synapse formation occurs during a short period of postnatal development. Thus, it is likely that PLC2 functions in the formation and maintenance of the neuronal network in the brain. Detailed examination by Western blot and in situ hybridization revealed that PLC2 is particularly abundant in pyramidal cells of the hippocampus, cerebral cortex, and olfactory bulb (Figs. 5B and 6). These organs are known to contribute to memory formation, suggesting that PLC2 may be involved in this function. PLC2 is also expressed in olfactory organs (Fig. 5C). Although PLC2 is not apparently coupled to odor or pheromone receptors, we detected expression of PLC2 in the main olfactory bulb and subolfactory bulb, in which sensory neurons synapse with projection neurons, indicating that PLC2 may be involved in odor and pheromone signal transduction. Finally, we detected PLC2 abundantly in neuron-containing primary cultures but not in astrocytes (Fig. 7B).
These observations indicate that PLC2 may have important functions in neurons, such as neuronal network formation, a  Staining was visualized with appropriate Alexa 568 (MAP2, red) or Alexa 488 (GFAP, green)-conjugated secondary antibody. B, PLC2 distribution by Western blot analysis. Neuron-containing culture (B27) and astrocyte-enriched cultures (FBS and ADM) were subjected to SDS-PAGE and Western blot for PLC2, MAP2, GFAP, and MBP. Mouse brain lysate was used as a positive control. Actin was used as a loading control. process that extends continuously from late embryogenesis to adulthood.
The importance of calcium in the brain has been widely reported, including involvement of axon growth and retraction, growth cone guidance, synapse formation, and responses of various neurotransmitters. Because PLC is a key enzyme in cellular calcium mobilization, PLC2 may play a specific role in calcium detection and mobilization in neurons.