A Cytosolic, Galpha q- and beta gamma -insensitive Splice Variant of Phospholipase C-beta 4*

Myung Jong Kim, Do Sik MinDagger , Sung Ho Ryu, and Pann-Ghill Suh§

From the Department of Life Science, Pohang University of Science and Technology, Pohang, 790-784, South Korea

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Phospholipase C (PLC)-beta 4 has been considered to be a mammalian homolog of the NorpA PLC, which is responsible for visual signal transduction in Drosophila. We reported previously the cloning of a cDNA encoding rat phospholipase C-beta 4 (PLC-beta 4) (Kim, M. J., Bahk, Y. Y., Min, D. S., Lee, S. J., Ryu, S. H., and Suh, P.-G. (1993) Biochem. Biophys. Res. Commun. 194, 706-712). We report now the isolation and characterization of a splice variant (PLC-beta 4b). PLC-beta 4b is identical to the 130-kDa PLC-beta 4 (PLC-beta 4a) except that the carboxyl-terminal 162 amino acids of PLC-beta 4a are replaced by 10 distinct amino acids. The existence of PLC-beta 4b transcripts in the rat brain was demonstrated by reverse transcription-polymerase chain reaction analysis. Immunological analysis using polyclonal antibody specific for PLC-beta 4b revealed that this splice variant exists in rat brain cytosol. To investigate functional differences between the two forms of PLC-beta 4, transient expression studies in COS-7 cells were conducted. We found that PLC-beta 4a was localized mainly in the particulate fraction of the cell, and it could be activated by Galpha q, whereas PLC-beta 4b was localized exclusively in the soluble fraction, and it could not be activated by Galpha q. In addition, both PLC-beta 4a and PLC-beta 4b were not activated by G-protein beta gamma -subunits purified from rat brain. These results suggest that PLC-beta 4b may be regulated by a mechanism different from that of PLC-beta 4a, and therefore it may play a distinct role in PLC-mediated signal transduction.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Phosphoinositide-specific phospholipase C (PLC)1 plays a pivotal role in transmembrane signaling. In response to various extracellular stimuli such as numerous hormones, growth factors, and neurotransmitters, this enzyme catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) and thereby generates two second messengers, diacylglycerol and inositol 1,4,5-trisphosphate (IP3) (1, 2). Diacylglycerol is a direct activator of protein kinase C, whereas IP3 induces transient release of calcium from the endoplasmic reticulum into the cytoplasm (3).

Multiple PLC isozymes have been purified from a variety of mammalian tissues, and several PLC genes have been cloned (4, 5). As predicted from the cDNAs, the PLC isozymes vary in size, with molecular masses ranging from 85 to 150 kDa. Despite low overall homology among the predicted amino acid sequences, significant sequence similarity exists in two domains that are designated as the X- and the Y-domains. These domains appear to constitute regions important for catalytic activities such as the specific recognition of the substrate and the hydrolysis of its phosphodiester bond. On the basis of the relative locations of the X- and Y-domains in the primary structure, PLC isozymes are classified into three types: beta , gamma , and delta . All PLC-beta types have a carboxyl-terminal 400-amino acid domain that contains an unusually high number of charged residues. On the other hand, the gamma  type has a long stretch of sequence between the X- and Y-domains, and the delta  type contains neither of the two additional sequences (4-6).

As expected from their distinct structural features and their different cellular expression patterns, the PLC isozymes are distinct in their modes of activation in response to extracellular stimuli. The two gamma  type PLCs, PLC-gamma 1 and -gamma 2, but not the beta  and delta  type isozymes, are activated through tyrosyl phosphorylation by growth factor receptor tyrosine kinase or nonreceptor tyrosine kinases (6). On the other hand, the PLC-beta types (beta 1, beta 2, beta 3) have been shown in cotransfection assays and in in vitro reconstitution experiments to be activated by the alpha q-subunit of heterotrimeric G-protein (7-10) and also by the beta gamma -subunit (11-16, 39). Additionally, it is known that the carboxyl-terminal tail that follows the Y-domain is involved in the activation of PLC-beta type by Galpha q (17, 42-44).

Previously, Min et al. (18, 19) purified the 97-kDa and the 130-kDa PLC-beta 4 enzymes from bovine cerebellum. cDNA encoding a 130-kDa PLC-beta 4 has been isolated (20, 33, 38). Based on these studies, it has been suggested that PLC-beta 4 might be a mammalian homolog of the Drosophila NorpA PLC, which is responsible for photosignal transduction. Furthermore, recent results obtained from cotransfection assays and in vitro reconstitution experiments showed that PLC-beta 4 could be activated by Galpha q but not by beta gamma -subunits of G-proteins (21, 22).

Here we report the identification of a rat PLC-beta 4 variant with a different carboxyl-terminal region. We show by reverse transcription-PCR and immunoblot analysis that this new splice variant of PLC-beta 4 exists in vivo. Furthermore, we further demonstrate that this splice variant is neither associated with the particulate fraction of the cell, nor is it activated by Galpha q.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Isolation of cDNA-- In the course of isolating PLC-beta 4 cDNA from a rat brain lambda ZapII cDNA library (20), we identified cDNA clones (clone beta 4-53 and beta 4-52) which exhibited patterns of restriction enzyme digestion differing from the previously described 130-kDa PLC-beta 4 cDNA (Fig. 1A). Further sequence analysis of these cDNAs revealed that they were splice variants of the PLC-beta 4 gene.




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Fig. 1.   The cDNA clones and the deduced amino acid sequence of PLC-beta 4b. Panel A, comparative diagrams of the isolated cDNA clones of rat PLC-beta 4a and PLC-beta 4b. Overlapping clones, RACE-PCR product, and the cumulative cDNA structure are shown. The thick line and the box represent the untranslated region and the coding region, respectively. Shaded boxes marked X and Y denote the X- and Y-domains, respectively. P denotes the region that is the most lysine-rich in the entire PLC-beta 4 (PLC-beta 4a and PLC-beta 4b) molecules. Several restriction enzyme cleavage sites are indicated in the center. The asterisk marks the position where the PLC-beta 4b transcript differs from the PLC-beta 4a transcript. kb, kilobase pairs. Panel B, the deduced carboxyl-terminal amino acid sequences of PLC-beta 4b and PLC-beta 4a. The amino acid sequence of the amino-terminal region of PLC-beta 4b was deduced from that of the previously characterized PLC-beta 4a, and the corresponding carboxyl-terminal sequence of PLC-beta 4a (residues 1015-1176) is shown for comparison. The amino acid sequence of the 116-specific peptide used as immunogen is boxed.

DNA Sequencing-- Two clones, beta 4-53 and beta 4-52, were plaque purified and subcloned into Bluescript vectors by in vivo excision with R408 helper phage. The sequences were assembled from nested deletion clones generated by the Erase-a-base system (Promega, Madison, WI) and dideoxy chain termination sequencing (23).

Reverse Transcription-PCR Analysis-- Total RNA was prepared from adult Sprague-Dawley rat brain tissue using the guanidium thiocyanate phenol-based single-step method (24). cDNA was synthesized in a 50-µl reaction mixture containing 50 mM Tris-HCl, pH 8.3, 5 mM MgCl2, 75 mM KCl, 0.5 mM dNTPs, 10 mM dithiothreitol, 25 µg of oligo(dT)12-18/ml, 10 µg of total RNA, and 100 units of avian myeloblastosis virus reverse transcriptase. After a 2-h incubation at 42 °C, the reaction was terminated by heating at 94 °C for 5 min. One µl of the reaction mixture was used for PCR amplification. PCR was carried out in a 25-µl reaction mixture containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2 mM MgCl2, 0.2 mg of gelatin/ml, 1 mM dithiothreitol, 200 ng of each primer, 0.2 mM dNTPs, and 1.25 units of AmpliTaq DNA polymerase (Perkin-Elmer). The reaction proceeded for 30 cycles of 94 °C for 1 min, 58 °C for 1 min, and 72 °C for 1 min. The two primers used were: primer P1 (5'-AAGCAAAGAGATGCGAGC-3'), encompassing amino acids 1016-1021 of the PLC-beta 4b transcript, and primer P2 (5'-TGTGTTTGGGACACTGCATG-3'), which is the 3'-untranslated region specific for the PLC-beta 4b mRNA (Fig. 2A). The amplified PCR products were analyzed in a 2% agarose gel stained with ethidium bromide.


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Fig. 2.   Alternative splicing of PLC-beta 4. Panel A, schematic representation of the cDNA structures encoding the carboxyl-terminal regions of PLC-beta 4a and PLC-beta 4b. The solid box and thin line denote the coding region and 3'-untranslated region, respectively. The PLC-beta 4a cDNA has an additional 176 bp but lacks the 37 bp that are unique to PLC-beta 4b. Differently patterned boxes stand for the putative exons of the two transcripts. The lines that are linked denote common regions between the variant transcripts. The primers used in reverse transcription-PCR are indicated by arrows. nt, nucleotides. Panel B, cDNA sequence encoding the carboxyl-terminal region of PLC-beta 4b. Sequences shared with PLC-beta 4a are in lightface, and sequences unique to PLC-beta 4b are in boldface. The site where the 176-nucleotide deletion occurred is also indicated. The termination codon of the PLC-beta 4b transcript is boxed.

Isolation of Full-length cDNA Encoding PLC-beta 4b-- Whole rat brain poly(A) mRNA from CLONTECH was reverse transcribed with PLC-beta 4b-specific antisense primer (5'-CTTGTGTTTGGGACACTGCA-3'). The resulting single-stranded cDNA was used as template for PCR amplification using the sense primer (5'-ATCATGGCCAAACCTTACCGA-3') located at the initiation codon of PLC-beta 4a and another antisense primer (5'-CACTGCATGACAGGATTTCA-3'), which is also specific for PLC-beta 4b cDNA. The amplified PCR product was subcloned into T-vector (Novagen) and sequenced by the Sanger dideoxy method.

Construction of Mammalian Expression Vectors-- A pBluescript KS plasmid containing the whole open reading frame of PLC-beta 4a was constructed by splicing an ApaI-SalI fragment of clone beta 4-19 and a SalI-XhoI fragment of clone beta 4-54 (the XhoI site used was originated from 3'-end linker of lambda ZapII). The resulting cDNA was then subcloned into the EcoRV site of pBluescript KS (Stratagene, La Jolla, CA) and named pKS/beta 4a. The construction of a pBluescript KS vector containing the whole cDNA for PLC-beta 4b (pKS/beta 4b) was accomplished by replacing the SalI-ApaI fragment of pKS/beta 4a with the SalI-ApaI fragment of pKS/beta 4-53, which had been constructed by inserting an SalI-XhoI (XhoI in 3'-end linker of lambda ZapII) fragment of clone beta 4-53 between the SalI-XhoI sites of pBluescript KS. The mammalian expression vectors for PLC-beta 4a and PLC-beta 4b were constructed by inserting the blunt ended SmaI-ApaI fragments of pKS/beta 4a and pKS/beta 4b into the EcoRV site of pcDNAI. The resulting plasmids were named pcDNAI/PLC-beta 4a and pcDNAI/PLC-beta 4b, respectively. A mammalian expression vector for mouse Galpha q was made utilizing the PCR technology with the GeneAMP kit from Perkin-Elmer using mouse Galpha q cDNA as template together with the sense primer 5'-CGCGGATCCATGACTCTGGAGTCCATCAT-3' and the antisense primer 5'-GCCGGATCCTTAGACCAGATTGTACTCCT-3' (BamHI sites are underlined). PCR primers were designed to amplify region corresponding to the open reading frame of mouse Galpha q cDNA. So, amplified PCR product contain no untranslated regions of 5'-end and 3'-end. The amplified product was digested with BamHI and inserted into the BamHI site of pcDNAI, and the construct was named pcDNAI/Galpha q. All DNA sequences were verified by sequencing.

Transient Transfection of COS-7 Cells-- COS-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Transfection of the COS-7 cells was by the DEAE-dextran method (25). Cells were seeded at 1 × 106 cells/100-mm dish and transfected 24 h later by incubation with 2 ml of transfection mixture (4 µg of plasmid DNA and 500 µg of DEAE-dextran in PBS) for 1 h. Then, 7 ml of serum-free Dulbecco's modified Eagle's medium containing 100 µM chloroquine was added. After 2.5 h the medium was aspirated, and the cells were treated with 10% dimethyl sulfoxide in Dulbecco's modified Eagle's medium for 2.5 min, washed with PBS, and incubated in a CO2 incubator. The cells were harvested 48 h after transfection.

Antibodies-- Peptide beta 4-N (MAKPYEFNWQKE, corresponding to residues 1-12 of PLC-beta 4a or PLC-beta 4b) and peptide 116-specific (GKQRDASPSG, corresponding to residues 1013-1022 of PLC-beta 4b) were synthesized by Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry, conjugated to keyhole limpet hemocyanin with glutaraldehyde, and injected into rabbits as described before (26). Antisera were affinity purified on protein A-agarose (Pierce Chemical Co.). The anti-Galpha q rabbit polyclonal antibody used in immunoblotting was a generous gift from Dr. Y. S. Kim (Seoul National University, South Korea).

Identification of PLC-beta 4b in the Cytosolic Fraction of Rat Brain-- Twenty frozen rat brains were homogenized in a homogenizer (Brinkmann) with 200 ml of buffer A (20 mM Tris-HCl, pH 7.6, 1 mM EDTA, 1 mM EGTA, and 0.1 mM dithiothreitol) containing 1 mM phenylmethylsulfonyl fluoride. The homogenate was centrifuged at 23,000 × g for 1 h. The supernatant was adjusted to pH 7.4 with 1 M Tris solution and applied to a DE52-cellulose column (5 cm × 10 cm2) preequilibrated with buffer A. The proteins were eluted with a 1-liter linear gradient from 0 to 1 M NaCl in buffer A. All fractions collected were tested by immunoblots probed with the antibody generated against the 116-kDa PLC-beta 4b-specific sequence GKQRDASPSG. Fractions that contained protein recognized by the antibody were eluted with 70-120 mM NaCl. The peak fraction showing the strongest immunoreactivity eluted with 90 mM NaCl and was used for analysis.

Intracellular Localization of PLC-beta 4a and -beta 4b-- COS-7 cells, transfected with expression plasmids carrying the cDNA of PLC-beta 4a or -beta 4b, were lysed in homogenization buffer (20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, and 0.2 mM phenylmethylsulfonyl fluoride) by pestle strokes. The suspension was then centrifuged at 100,000 rpm for 20 min at 4 °C in a Beckman TL-100s ultracentrifuge. The cytosolic fraction (supernatant) was separated from the particulate fraction. Samples were resolved by 6% SDS-polyacrylamide gel electrophoresis and then electroblotted. The blots were incubated initially with rabbit antibody (1:5,000 dilution) raised against the NH2-terminal 12 amino acids of PLC-beta 4, and then the blot was incubated with a 1:10,000 dilution of horseradish peroxidase-conjugated anti-rabbit antibody and processed using the ECL (enhanced chemiluminescence) system (Amersham). The expression of recombinant PLC-beta 4b protein in HeLa cell by using recombinant vaccinia virus expression system was done as described previously (40).

Localization of PLC-beta 4s in the Purkinje Cells in the Rat Brain by Immunocytochemistry-- Male Sprague-Dawley rats weighing 200-250 g were anesthetized with pentobarbital (60 mg/kg, intraperitoneally) and perfused with fixative solution that contained 2% paraformaldehyde, 0.075 M lysine, 0.01 M sodium m-periodate in 0.05 M sodium phosphate buffer, pH 7.4, at room temperature. The brain was dissected, postfixed overnight in the perfusion fixative without sodium m-periodate at 4 °C, and then cut on a vibratome (TPI, vibratome series 1000) into frontal sections (30 µm). Sections of the cerebellum were incubated for 30 min with 2% normal goat serum in TBS to block the nonspecific binding sites of protein. The sections were incubated with preimmune sera and antibodies against PLC-beta 4a and -beta 4b (diluted 1:2,000 in TBS and 2% BSA) for 16 h at 4 °C. For avidine-biotin-peroxidase immunostaining, the sections were washed three times for 10 min each with TBS, incubated for 2 h with biotin-labeled goat anti-rabbit IgG or anti-rabbit IgG (Vector), diluted 1:400 in TBS and 2% BSA, washed three times for 10 min each with TBS, and then incubated for 1 h with peroxidase-labeled streptavidin (Vector), diluted 1:400 in TBS. After washing three times with same buffer, the preparations were reacted with 0.05% 3,3'-diaminobenzidine (Sigma) in 50 mM TBS, pH 7.6, containing 0.006% H2O2.

Responsiveness of PLC-beta 4a and PLC-beta 4b to Galpha q Activation-- COS-7 cells were cotransfected with pcDNAI/Galpha q and pcDNAI/beta 4a or pcDNAI/beta 4b. After 24 h, the cells were labeled overnight with 2 µCi/ml myo-[3H]inositol in inositol-free Dulbecco's modified Eagle's medium. The monolayered cells were washed twice with PBS and preincubated in serum free medium for 1 h at 37 °C. During the last 10 min of the preincubation period, 20 mM LiCl was added. The cells were then treated with 30 µM AlF4- (10 mM NaF and 30 µM AlCl3) at 37 °C for 30 min. The reaction was terminated by removal of the medium and washing the cell with ice-cold PBS. Total inositol formation was measured as described previously (27). Cells were incubated with 3 ml of ice-cold 20 mM formic acid for 30 min on ice. The cells were scraped off the dishes, and cell debris was removed by centrifugation. One ml of supernatant was neutralized with 0.5 ml of 50 mM ammonium hydroxide and loaded onto a 1-ml Bio-Rad Dowex AG 1-X8 anion exchange column (formate form, 200-400 mesh). Free inositol was washed three times with 3 ml of distilled water (free inositol fraction, Ins), and then the column was washed three times with 3 ml of 60 mM ammonium formate and 5 mM sodium tetraborate removing the glycerophosphoinositol fraction. Finally, total inositol phosphate was eluted with 6 ml of 1 M ammonium formate and 0.1 M formic acid (total inositol phosphate fraction, IPs). 0.5 ml of each of the Ins and IP fractions was mixed with 10 ml of scintillation mixture and counted (27). The data are presented as the quotient of IP divided by Ins plus IP. In addition, by measuring total radio activities of cell lysates extracted with formic acid, the incorporation of precursor in the transfected cell was normalized.

Activation by G-protein beta gamma -Subunits-- Mammalian expression vectors for PLC-beta 4a, PLC-beta 4b, or PLC-beta 2 were transiently transfected into COS-7 cells. After 48 h, transfected COS-7 cells were rinsed twice with PBS and extracted with extraction buffer (20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 10 mM EGTA, 37 mM sodium cholate, 43 mM 2-mercaptoethanol, and 0.1 mM phenylmethylsulfonyl fluoride) for 30 min at 4 °C. After centrifugation, the extracted proteins (~5 mg/ml) were quick-frozen in liquid nitrogen. For the in vitro PLC assay, detergent extracts of transfected COS-7 cells were diluted 200-fold with buffer containing 20 mM Tris-HCl, pH 7.5, 10 mM EGTA, 43 mM 2-mercaptoethanol, and 0.1 mM phenylmethylsulfonyl fluoride. PIP2 lipid substrates were made as follows. Phosphatidylethanolamine and [3H]phosphatidylinositol 4,5-bisphosphate were mixed in a molar ratio of 10:1. The lipids were evaporated to dryness under a stream of N2 and then sonicated in a bath type sonicator for 10 min in buffer containing 87.5 mM Tris-maleate, 17.5 mM LiCl, 17.5 mM EDTA, and 1.6 mM sodium deoxycholate. The final concentration of [3H]phosphatidylinositol 4,5-bisphosphate in the 70-µl assay mixture was 28 µM, with 38,000-40,000 cpm/single assay. The PLC activity was assayed at 25 °C in a mixture (70 µl) containing 40 µl of lipid substrate, 5 µl (125 ng) of the transfected COS-7 cells lysates, 5 µl (final 2 µM) of the beta gamma -subunits (provided by Dongeun Park (Kwang-Joo Institute of Science and Technology, South Korea), and 5-10 µl of 0.1 M CaCl2, adjusting the concentration of free Ca2+ to 0.1 µM. The reaction was started with the addition of the transfected cell lysate and stopped by adding 350 µl of chloroform/methanol/concentrated HCl (500:500:3, v/v/v) followed by vortex mixing. Samples were then supplemented with 100 µl of 1 M HCl containing 5 mM EGTA. After centrifugation in an Eppendorf microcentrifuge for 5 min at 4 °C, the amount of [3H]IP3 in the supernatant was assayed for radioactivity by liquid scintillation counting.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

PLC-beta 4b cDNA Cloning-- We isolated two clones (clone beta 4-52 and beta 4-53) that had restriction enzyme digestion patterns differing from the 130-kDa PLC-beta 4 reported previously. Subsequent sequence analysis revealed that these clones could be a splice variant of the 130-kDa PLC-beta 4 mRNA (Fig. 1A). Clone beta 4-53 was selected for further analysis because it contained the longer cDNA insert. The overall cDNA structure of clone beta 4-53 was almost identical to the 130-kDa PLC-beta 4 mRNA except that this clone had a 176-nucleotide deletion from the region encoding the carboxyl-terminal part of the 130-kDa PLC-beta 4 (Figs. 1A and 2A). This deletion caused a frameshift compared with the reading frame of the 130-kDa PLC-beta 4 cDNA. As a result the carboxyl-terminal 162 amino acids are replaced with 10 distinct amino acids in beta 4-53 (Fig. 1B). As seen in Fig. 2, the variant mRNA shares most of its sequence with the 130-kDa PLC-beta 4 mRNA, but it also has its own 37 nucleotides that are not present in the 130-kDa PLC-beta 4 mRNA and are inserted 247 nucleotides downstream from where the 176-nucleotide deletion occurred. Because the difference between the two mRNAs is restricted to the presence or absence of only those two specific regions, it is possible that the variant mRNA originated from an alternative mRNA processing event, although it cannot be ruled out that each mRNA is the product of a different gene.

The longest clone (clone beta 4-53) encoding the novel splice variant of PLC-beta 4 encompassed the whole region downstream of the X-domain (Fig. 1A). In an effort to isolate a clone encoding the whole open reading frame of this splice variant, we screened extensively two other rat brain cDNA libraries, but we failed to obtain any clone that was longer than clone beta 4-53. However, based on the results obtained from studies using sequence-specific antipeptide antibodies, we could predict that the protein encoded by the variant transcript shared the amino-terminal region with the 130-kDa PLC-beta 4 protein. Therefore, we used the long distance PCR cloning method to test whether PLC-beta 4b shared an identical amino-terminal primary structure with PLC-beta 4a. Whole rat brain mRNA was reverse transcribed with PLC-beta 4b-specific antisense primer. The resulting single-stranded cDNA was then used as template for long distance PCR amplification using the sequence located near the initiation codon of PLC-beta 4a as sense primer and an antisense primer corresponding to the 3'-untranslated region of PLC-beta 4b. PCR amplification generated approximately a 3.3-kilobase pair DNA fragment (RACE-PCR product) (Fig. 1A and Fig. 3, lane 2). The PstI digestion pattern of this RACE-PCR product was the same as the PCR product amplified with pKS/beta 4b as a template (Fig. 3, lanes 5 and 6). By sequencing this RACE-PCR product, we can conclude that PLC-beta 4b has an amino-terminal structure identical to the corresponding PLC-beta 4a structure.


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Fig. 3.   Isolation of full-length cDNA encoding rat PLC-beta 4b. Whole rat brain poly(A) mRNA from CLONTECH was reverse transcribed with PLC-beta 4b-specific antisense primer (5'-CTTGTGTTTGGGACACTGCA-3'). The resulting single-stranded cDNA was used as template for PCR amplification using the sense primer (5'-ATCATGGCCAAACCTTACCGA-3') located at the initiation codon of PLC-beta 4a and the antisense primer (5'-CACTGCATGACAGGATTTCA-3') specific for PLC-beta 4b cDNA. The resulting RACE-PCR product was electrophoresed on a 2% agarose gel and stained with ethidium bromide (lane 2). Lanes 1 and 4 are DNA size markers (lambda  DNA-HindIII digest from New England Biolabs); lane 3, PCR amplification using DNA prepared from pKS/beta 4b; lane 5, DNA fragments generated by PstI-digested RACE-PCR product; lane 6, DNA fragments generated by PstI-digested PCR product using DNA prepared from pKS/beta 4b

The open reading frame predicted by the cumulative sequences of the clones beta 4-53 and the long distance PCR product codes for a polypeptide of 1022 amino acids with a calculated molecular mass of 115,965 Da (GenBank accession number AF031370). The protein predicted by this sequence was designated PLC-beta 4b, whereas the previously reported PLC-beta 4 was now renamed PLC-beta 4a.

Detection of the Alternative Transcript by Reverse Transcription-PCR-- To verify whether a transcript of the spliced variant of PLC-beta 4 exists in vivo, we performed reverse transcription- PCR. We used total RNA from rat brain for reverse transcription with oligo(dT)12-18. The primers used in the PCR were designed to amplify specifically the region corresponding to the 3'-untranslated region of the PLC-beta 4b transcript (Fig. 2A). As shown in Fig. 4, the PCR product obtained from reverse-transcribed rat brain RNA, and clone beta 4-53 was the expected 279 bp. This suggests that a PLC-beta 4b transcript does exist in vivo and that our clone was not just an entity generated by recombination during lambda  phage amplification.


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Fig. 4.   Detection of the alternative transcript by reverse transcription-PCR. RNA from rat brain was reverse transcribed using oligo(dT)12-18. PCR was done with primer P1 (sense) and primer P2 (antisense), which were designed to amplify specifically a PLC-beta 4b sequence (see "Experimental Procedure" and Fig. 2). PCR products were electrophoresed on a 2% agarose gel and stained with ethidium bromide. Lane 1, DNA size markers as a 100-bp ladder (Research Genetics and Life Technologies, Inc.); lane 2, PCR amplification using DNA prepared from clone beta 4-54; lane 3, PCR positive control using DNA prepared from clone beta 4-53, showing that a 279-bp PCR product is obtained; lane 4, PCR amplification from reverse transcribed rat brain RNA showing the presence of a PLC-beta 4b transcript in the rat brain.

Immunodetection of PLC-beta 4b in the Rat Brain Cytosol-- Although we successfully isolated a full-length cDNA encoding PLC-beta 4b by long distance PCR, we were not successful in isolating a lambda  phage clone containing the whole coding region of the splice variant, and thus we could not exclude the possibility that the PLC-beta 4b transcript was aberrant or a nonproductive mRNA. Therefore, we chose a different strategy to confirm the conclusion that PLC-beta 4b exists in vivo and shares the amino-terminal region with PLC-beta 4a. If we could detect the 116-kDa PLC in rat brain and if this protein would immunoreact with an antipeptide antibody generated against the 116-kDa PLC-beta 4b-specific sequence, and an antipeptide antibody generated against the amino-terminal sequence of PLC-beta 4a, then this would prove that PLC-beta 4b is an authentic entity in vivo. Thus, we made two antipeptide antibodies, one to the carboxyl-terminal region of PLC-beta 4b (anti-116-specific antibody) and the other to the amino-terminal region of PLC-beta 4a (anti-beta 4-N antibody). First, we used these antibodies in an experiment where we expressed PLC-beta 4b in HeLa cells after infection with vaccinia virus containing the corresponding cDNA to test whether the molecular mass of recombinant PLC-beta 4b would be the expected 116 kDa when expressed in a eukaryotic cell. As shown in Fig. 5, the molecular mass of the recombinant PLC-beta 4b expressed in HeLa cells was the predicted 116 kDa, and it was recognized by both the anti-beta 4-N antibody and the anti-116-specific antibody (Fig. 5, center lane in both panels).


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Fig. 5.   Immunodetection of PLC-beta 4b in rat brain cytosol. The rat brain cytosolic fraction was prepared as described under "Experimental Procedures" and analyzed with anti-beta 4-N antibody (1:5,000 dilution) or anti-116-specific peptide antibody (1:5,000 dilution) by immunoblotting. The 130-kDa PLC-beta 4 purified from bovine cerebellum and the 116-kDa PLC-beta 4b purified from extracts of HeLa cells that had been infected with vaccinia virus containing the corresponding cDNA were loaded to show specificities of anti-beta 4-N antibody and anti-116-specific peptide antibody.

Second, to identify PLC-beta 4b in vivo, we fractionated rat brain cytosol on a DE52-cellulose column. All column fractions were immunoblotted and probed with anti-116-specific antibody. The fractions containing protein recognized by the anti-116-specific antibody eluted with 70-120 mM NaCl. The peak fraction exhibiting the strongest immunoreactivity eluted with 90 mM NaCl (data not shown) and was used for the immunoblot analysis. This fraction was also recognized specifically by the anti-beta 4-N antibody (Fig. 5, right lane in both panels). Taken together with result from sequencing data of RACE-PCR product, we can, therefore, confirm that PLC-beta 4b shares a common amino-terminal region with PLC-beta 4a.

Intracellular Localization of PLC-beta 4a and PLC-beta 4b-- To assess functional differences of the two forms of PLC-beta 4, we compared their intracellular localization, since previous studies had suggested that the carboxyl-terminal region of the PLC-beta type is required for their association with the particulate fraction of the cell (17, 47-49). A mammalian expression vector carrying PLC-beta 4a or PLC-beta 4b was transiently transfected into COS-7 cells. The transfected cells were then fractionated into a soluble fraction and a particulate fraction, each of which was then immunoanalyzed with anti-beta 4-N antibody. As seen in Fig. 6, the majority of PLC-beta 4a is localized in the particulate fraction, whereas PLC-beta 4b is found exclusively in the soluble fraction. In addition, the distribution of PLC-beta 4b in the Purkinje cell of rat cerebellum was examined with immunocytochemistry. PLC-beta 4b immunoreactivity was distributed homogeneously in the cytoplasm of Purkinje cell (data not shown). Moreover, we have performed immunoblot analysis of the particulate fraction of the rat brain homogenate by using 116-kDa PLC-beta 4b-specific antibody. But 116 kDa PLC-beta 4b was not detected in the particulate fraction of the rat brain homogenate (data not shown). These results are consistent with our observation that 116-kDa PLC-beta 4b is found in the cytosolic fraction of the rat brain homogenate.


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Fig. 6.   Intracellular localizations of PLC-beta 4a and PLC-beta 4b. COS-7 cells transfected with pcDNAI/PLC-beta 4a or pcDNAI/PLC-beta 4b were fractionated as described under "Experimental Procedures." Supernatants (S) and pellets (P) derived from the same amount of homogenates (T) were subjected to 6% SDS-polyacrylamide gel electrophoresis and electroblotted. The blots were incubated with anti-peptide antibody (anti-beta 4-N antibody, 1:5,000 dilution) directed against the NH2-terminal portion of PLC-beta 4. Data are the representative of five independent experiments.

PLC-beta 4b Is Not Activated by Galpha q-- Because PLC-beta 4b is localized exclusively in the cytosolic fraction and because the carboxyl-terminal region of PLC-beta 1 is essential for both its association with the particulate fraction and its activation by Galpha q (17), we tested PLC-beta 4a and PLC-beta 4b for their ability to be activated by Galpha q. COS-7 cells were cotransfected with the mammalian expression vector for Galpha q and a cDNA construct expressing either PLC-beta 4a or PLC-beta 4b. Aluminum fluoride (AlF4-) was added to activate the G-protein alpha -subunit (28). As seen in Fig. 7A, transfection with vector alone or with vector carrying PLC-beta 4a or PLC-beta 4b leads to a low level accumulation of total inositol phosphates. When Galpha q was transfected, there was some increase in total IP formation compared with vector alone. When the cells were cotransfected with the mammalian expression vectors for PLC-beta 4a and Galpha q, there was a significant increase in total inositol formation. However, cells cotransfected with the expression vectors for PLC-beta 4b and Galpha q showed no increase in inositol formation compared with that of cells transfected with the Galpha q expression vector alone. To exclude the possibility that the observed difference in the activating effect of Galpha q on PLC-beta 4a versus PLC-beta 4b was the result of a differential expression of intrinsic PLC-beta 4 s or Galpha q in COS-7 cells, we performed an immunoblot analysis using anti-beta 4-N antibody and anti-Galpha q antibody. It was confirmed that the amount of PLC-beta 4a was comparable to that of PLC-beta 4b and that the expression levels of intrinsic Galpha q and PLC-beta 4s in the COS-7 cells were not affected by the cotransfection (Fig. 7B). Furthermore, in a PI-PLC assay employing lipid vesicles the intrinsic PI-hydrolyzing activity of PLC-beta 4b purified from extracts of HeLa cells infected with vaccinia virus containing the corresponding cDNA was 2 µmol/min/mg of protein. This specific activity was comparable to that of the 130-kDa PLC-beta 4a purified from bovine cerebellum, suggesting that the 116-kDa PLC-beta 4b variant was as active as the 130-kDa PLC-beta 4a species (data not shown). It thus appears that the carboxyl-terminal region of PLC-beta 4a is important for activation by Galpha q, but it is not important for the intrinsic PLC activity of the PLC-beta 4 variants.


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Fig. 7.   PLC-beta 4b is not activated by Galpha q. Panel A, activation of PLC-beta 4a and PLC-beta 4b by Galpha q. COS-7 cells were transfected with the indicated mammalian expression vectors. The level of total IP was determined 48 h after transfection (see "Experimental Procedures"). Data are presented as the quotient of IP divided by Ins plus IP. The data shown are the means ± S.E. of three independent experiments. Panel B, expressions of Galpha q and the two forms of PLC-beta 4 in COS-7 cells. Cells were transfected with the indicated cDNA as described under "Experimental Procedures." The cells were solubilized in SDS sample buffer 48 h after transfection. Galpha q and the two forms of PLC-beta 4 were analyzed with polyclonal Galpha q antibody (1:5,000 dilution) and anti-beta 4-N antibody (1:5,000 dilution), respectively. The different lanes represent a comparable number of cells.

Both PLC-beta 4a and PLC-beta 4b Are Not Activated by G-Protein beta gamma -Subunits Purified from Rat Brain-- Previously, Banno et al. (36, 37) reported that the activation of a carboxyl-terminal truncated form of PLC-beta 3 generated by calpain cleavage can be enhanced by brain G-protein beta gamma -subunits over that of the intact PLC-beta 3. We examined whether PLC-beta 4b, having a shorter carboxyl-terminal tail, can be activated by G-protein beta gamma -subunits. For this purpose, mammalian expression vectors for PLC-beta 2, PLC-beta 4a, or PLC-beta 4b were transiently transfected into COS-7 cells. The transfected cell lysates were then reconstituted with G-protein beta gamma -subunits purified from rat brain, and in vitro PLC activity was measured. Fig. 8 shows that in accordance with previous reports of PLC-beta 2 being activated by G-protein beta gamma -subunits (21, 22), the PIP2-hydrolyzing activity of PLC-beta 2 is potentiated by the G-protein beta gamma -subunits, whereas PLC-beta 4a and PLC-beta 4b are not activated by the G-protein beta gamma -subunits. These results, therefore, suggest that PLC-beta 4a and PLC-beta 4b are not targets for regulation by G-protein beta gamma -subunits.


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Fig. 8.   Both PLC-beta 4a and PLC-beta 4b are not activated by G-protein beta gamma -subunits. COS-7 cells were transfected with cDNAs corresponding to PLC-beta 2, PLC-beta 4a, and PLC-beta 4b. Cell lysates were prepared as described under "Experimental Procedures" and assayed in the absence (dotted bars) or presence (hatched bars) of 2 µM G-protein beta gamma -subunits purified from rat brain with phospholipid vesicles containing [3H]PtInsP2. Counts/min (cpm) values were obtained from in vitro PLC assay using 125 ng of protein in transfected cell lysates. This result presents the mean ± S.E. of four experiments.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

PLC-beta 4 has been considered to be a mammalian homolog of the NorpA PLC, which is responsible for visual signal transduction in Drosophila (29, 30, 32). Recent results obtained from in situ hybridizations of rat brain tissue sections and from a mouse that lacks PLC-beta 4 suggest that PLC-beta 4 may play a significant role in mammalian visual processing (31, 41). In our study, we isolated a novel splice variant of rat PLC-beta 4 and named it PLC-beta 4b. This splice variant has a shorter carboxyl-terminal tail compared with the previously reported 130-kDa PLC-beta 4 (20, 33, 38). Based on the comparison of the cDNA sequences of the two forms of PLC-beta 4, we predict that the mechanism by which PLC-beta 4b is generated might be alternative processing of the mRNA.

To date, identified splice variants of the PLC-beta type include rat PLC-beta 1 (26), bovine PLC-beta 4 (33), Drosophila PLC-p21 (34), and Drosophila NorpA PLC (35). The splicing in all of these PLC isozymes occurs outside the X- and Y-domains. Although alternative splicing in the carboxyl-terminal regions of the rat PLC-beta 1 and the Drosophila PLC-p21 has been reported (26, 34), the possibility that those splice variants may be regulated differentially based on differences in the primary structure is purely speculative.

Previously, Wu et al. (17) have identified regions in PLC-beta 1 which are involved in the activation process by Galpha q. By making a series of deletion mutants, they found that the region between residues Thr-903 and Gln-1030 (P-Box) is necessary for both association of PLC-beta 1 with the particulate fraction and activation by Galpha q. They also found that the region between residues Lys-1031 and Leu-1142 (G-box) is required for interaction with the G-protein alpha q-subunit but is not necessary for association of the PLC-beta 1 with the particulate fraction. The P-box in PLC-beta 1 is the most lysine-rich region in the entire molecule. In their study, the authors suggested that association with the particulate fraction might occur through phospholipid or some intermediate negatively charged membrane-bound proteins. In addition, the importance of the COOH-terminal basic residues of PLC-beta 1 and PLC-beta 2 for particulate association has been reported (42-44). We found that the most lysine-rich region in PLC-beta 4, the P-box, is located upstream of the carboxyl terminus in PLC-beta 4b (Fig. 1A). But only PLC-beta 4a, containing an additional 162 amino acids in the carboxyl-terminal region, was localized in the particulate fraction, and it could be activated by Galpha q. Therefore, it seems that the P-box in PLC-beta 4 is insufficient for the association of the protein with the particulate fraction, and the carboxyl-terminal 162 amino acids in PLC-beta 4a are necessary for the association with the particulate fraction and activation by Galpha q. Because the carboxyl-terminal 162 amino acids of PLC-beta 4a also contain a large number of charged residues (basic and acidic amino acids), a major determinant for the localization may be the appropriate spatial arrangement of the charged residues rather than the absolute strength of positive charge.

Banno et al. (36, 37) reported that a carboxyl-terminal truncated form of PLC-beta 3 generated by calpain cleavage can be activated to a greater extent by brain G-protein beta gamma -subunits than the intact PLC-beta 3. The authors suggested that the carboxyl-terminal region of PLC-beta 3 may inhibit its activation by G-protein beta gamma -subunits. Recently, Kuang et al. (45) suggested that the Glu-435 to Val-641 region of the PLC-beta 2 molecule is involved in the interaction with the G-protein beta gamma -subunits. In their report, they further narrowed the region down to 62 amino acids (residues Leu-580 to Val-641) after in vitro binding assays using glutathione S-transferase-fused PLC-beta 2 and pure G-protein beta gamma -subunits. These residues (Leu-580 to Val-641) of PLC-beta 2 are located inside the Y-domain, one of the most conserved regions among PLC isozymes. Although it has been known that PLC-beta 4a cannot be activated by G-protein beta gamma -subunits in vivo and in vitro, previously published observations raised the question of whether G-protein regulation of the 116-kDa PLC-beta 4b might occur by beta gamma -subunits. However, we found that PLC-beta 4b, having a short carboxyl-terminal tail, is nevertheless unresponsive to regulation by G-protein beta gamma -subunits. Therefore, our results suggest that PLC-beta 4 (PLC-beta 4a and PLC-beta 4b) does not have the sequence motif necessary to be activated by G-protein beta gamma -subunits.

In conclusion, we have isolated a splice variant of the previously reported rat brain PLC-beta 4 enzyme. The two subtypes of PLC-beta 4 (-beta 4a and -beta 4b) are most likely generated by alternative processing of mRNA. They differ in their intracellular localization and their susceptibility to activation by the alpha q-subunit of G-protein. We therefore propose that the two forms of PLC-beta 4 may have distinct roles in PLC-mediated signal transduction.

    FOOTNOTES

* This work was supported in part by Basic Research Institute Program BSRI-954434 from the Ministry of Education and by the Biotech 2000 Program from the Ministry of Science and Technology of Korea.The costs of publication of this article were defrayed in part by the payment of page charges. The 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 GenBankTM/EMBL Data Bank with accession number(s) AF031370.

Dagger Present address: Dept. of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232-0295.

§ To whom correspondence should be addressed. Tel.: 82-562-279-2293; Fax: 82-562-279-2199; E-mail: pgs{at}pop.postech.ac.kr.

1 The abbreviations used are: PLC, phospholipase C; IP, inositol phosphate; IP3, inositol 1,4,5-trisphosphate; PI, phosphatidylinositol; PIP2, phosphatidylinositol 4,5-bisphosphate; G-protein, heterotrimeric guanine nucleotide binding protein; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; Ins, free inositol fraction; RACE, rapid amplification of cDNA ends; bp, base pair(s).

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
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