A Cytosolic, Gαq- and βγ-insensitive Splice Variant of Phospholipase C-β4
1998; Elsevier BV; Volume: 273; Issue: 6 Linguagem: Inglês
10.1074/jbc.273.6.3618
ISSN1083-351X
AutoresMyung Jong Kim, Do Sik Min, Sung Ho Ryu, Pann‐Ghill Suh,
Tópico(s)Neurobiology and Insect Physiology Research
ResumoPhospholipase C (PLC)-β4 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-β4 (PLC-β4) (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-β4b). PLC-β4b is identical to the 130-kDa PLC-β4 (PLC-β4a) except that the carboxyl-terminal 162 amino acids of PLC-β4a are replaced by 10 distinct amino acids. The existence of PLC-β4b transcripts in the rat brain was demonstrated by reverse transcription-polymerase chain reaction analysis. Immunological analysis using polyclonal antibody specific for PLC-β4b revealed that this splice variant exists in rat brain cytosol. To investigate functional differences between the two forms of PLC-β4, transient expression studies in COS-7 cells were conducted. We found that PLC-β4a was localized mainly in the particulate fraction of the cell, and it could be activated by Gαq, whereas PLC-β4b was localized exclusively in the soluble fraction, and it could not be activated by Gαq. In addition, both PLC-β4a and PLC-β4b were not activated by G-protein βγ-subunits purified from rat brain. These results suggest that PLC-β4b may be regulated by a mechanism different from that of PLC-β4a, and therefore it may play a distinct role in PLC-mediated signal transduction. Phospholipase C (PLC)-β4 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-β4 (PLC-β4) (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-β4b). PLC-β4b is identical to the 130-kDa PLC-β4 (PLC-β4a) except that the carboxyl-terminal 162 amino acids of PLC-β4a are replaced by 10 distinct amino acids. The existence of PLC-β4b transcripts in the rat brain was demonstrated by reverse transcription-polymerase chain reaction analysis. Immunological analysis using polyclonal antibody specific for PLC-β4b revealed that this splice variant exists in rat brain cytosol. To investigate functional differences between the two forms of PLC-β4, transient expression studies in COS-7 cells were conducted. We found that PLC-β4a was localized mainly in the particulate fraction of the cell, and it could be activated by Gαq, whereas PLC-β4b was localized exclusively in the soluble fraction, and it could not be activated by Gαq. In addition, both PLC-β4a and PLC-β4b were not activated by G-protein βγ-subunits purified from rat brain. These results suggest that PLC-β4b may be regulated by a mechanism different from that of PLC-β4a, and therefore it may play a distinct role in PLC-mediated signal transduction. Phosphoinositide-specific phospholipase C (PLC) 1The 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). 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) (1Berridge M.J. Helslop J.P. Irvine R.F. Brown K.D. Biochem. J. 1984; 222: 195-201Crossref PubMed Scopus (317) Google Scholar, 2Berridge M.J. Irvine R.F. Nature. 1984; 312: 315-321Crossref PubMed Scopus (4254) Google Scholar). Diacylglycerol is a direct activator of protein kinase C, whereas IP3 induces transient release of calcium from the endoplasmic reticulum into the cytoplasm (3Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4232) Google Scholar). Multiple PLC isozymes have been purified from a variety of mammalian tissues, and several PLC genes have been cloned (4Rhee S.G. Suh P.-G. Ryu S.H. Lee S.Y. Science. 1989; 244: 546-550Crossref PubMed Scopus (699) Google Scholar, 5Rhee S.G. Choi K.D. Adv. Second Messenger Phosphoprotein Res. 1992; 26: 35-61PubMed Google Scholar). 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: β, γ, and δ. All PLC-β types have a carboxyl-terminal 400-amino acid domain that contains an unusually high number of charged residues. On the other hand, the γ type has a long stretch of sequence between the X- and Y-domains, and the δ type contains neither of the two additional sequences (4Rhee S.G. Suh P.-G. Ryu S.H. Lee S.Y. Science. 1989; 244: 546-550Crossref PubMed Scopus (699) Google Scholar, 5Rhee S.G. Choi K.D. Adv. Second Messenger Phosphoprotein Res. 1992; 26: 35-61PubMed Google Scholar, 6Rhee S.G. Bae Y.S. J. Biol. Chem. 1997; 272: 15045-15048Abstract Full Text Full Text PDF PubMed Scopus (817) Google Scholar). 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 γ type PLCs, PLC-γ1 and -γ2, but not the β and δ type isozymes, are activated through tyrosyl phosphorylation by growth factor receptor tyrosine kinase or nonreceptor tyrosine kinases (6Rhee S.G. Bae Y.S. J. Biol. Chem. 1997; 272: 15045-15048Abstract Full Text Full Text PDF PubMed Scopus (817) Google Scholar). On the other hand, the PLC-β types (β1, β2, β3) have been shown in cotransfection assays and in in vitro reconstitution experiments to be activated by the αq-subunit of heterotrimeric G-protein (7Smrcka A.V. Hepler J.R. Brown K.O. Sternweis P.C. Science. 1991; 252: 804-807Crossref Scopus (706) Google Scholar, 8Taylor S.J. Chae H.Z. Rhee S.G. Exton J.H. Nature. 1991; 350: 516-518Crossref PubMed Scopus (616) Google Scholar, 9Wu D.Q. Lee C.H. Rhee S.G. Simon M.I. J. Biol. Chem. 1992; 267: 1811-1817Abstract Full Text PDF PubMed Google Scholar, 10Jhon D.-Y. Lee H.-H. Park D. Lee C.-W. Lee K.-H. Yoo O.J. Rhee S.G. J. Biol. Chem. 1993; 268: 6654-6661Abstract Full Text PDF PubMed Google Scholar) and also by the βγ-subunit (11Camps M. Carozzi A. Schnabel P. Scheer A. Parker P.J. Gierschik P. Nature. 1992; 360: 684-686Crossref PubMed Scopus (515) Google Scholar, 12Katz A. Wu D. Simon M.I. Nature. 1992; 360: 686-689Crossref PubMed Scopus (419) Google Scholar, 13Park D. Jhon D.-Y. Lee C.-W. Lee K.-H. Rhee S.G. J. Biol. Chem. 1993; 268: 4573-4576Abstract Full Text PDF PubMed Google Scholar, 14Smrcka A.V. Sternweis P.C. J. Biol. Chem. 1993; 268: 9667-9674Abstract Full Text PDF PubMed Google Scholar, 15Boyer J.L. Graber S.G. Waldo G.L. Harden T.K. Garrison J.C. J. Biol. Chem. 1994; 269: 2814-2819Abstract Full Text PDF PubMed Google Scholar, 16Ueda N. Iniguez-Lluhi J.A. Lee E. Smrcka A.V. Robishaw J.D. Gilman A.G. J. Biol. Chem. 1994; 269: 4388-4395Abstract Full Text PDF PubMed Google Scholar, 39Wu D. Katz A. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5297-5301Crossref PubMed Scopus (174) Google Scholar). Additionally, it is known that the carboxyl-terminal tail that follows the Y-domain is involved in the activation of PLC-β type by Gαq (17Wu D. Jiang H. Katz A. Simon M.I. J. Biol. Chem. 1993; 268: 3704-3709Abstract Full Text PDF PubMed Google Scholar,42Park D. Jhon D.Y. Lee C.W. Ryu S.H. Rhee S.G. J. Biol. Chem. 1993; 268: 3710-3714Abstract Full Text PDF PubMed Google Scholar, 43Lee S.B. Shin S.H. Hepler J.R. Gilman A.G. Rhee S.G. J. Biol. Chem. 1993; 268: 25952-25957Abstract Full Text PDF PubMed Google Scholar, 44Kim C.G. Park D. Rhee S.G. J. Biol. Chem. 1996; 271: 21187-21192Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Previously, Min et al. (18Min D.S. Kim D.M. Lee Y.H. Seo J. Suh P.G. Ryu S.H. J. Biol. Chem. 1993; 268: 12207-12212Abstract Full Text PDF PubMed Google Scholar, 19Min D.S. Kim Y. Lee Y.H. Suh P.G. Ryu S.H. FEBS Lett. 1993; 331: 38-42Crossref PubMed Scopus (13) Google Scholar) purified the 97-kDa and the 130-kDa PLC-β4 enzymes from bovine cerebellum. cDNA encoding a 130-kDa PLC-β4 has been isolated (20Kim M.J. Bahk Y.Y. Min D.S. Lee S.J. Ryu S.H. Suh P.G. Biochem. Biophys. Res. Commun. 1993; 194: 706-712Crossref PubMed Scopus (24) Google Scholar, 33Ferreira P.A. Shortridge R.D. Pak W.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6042-6046Crossref PubMed Scopus (51) Google Scholar, 38Lee C.W. Park D.J. Lee K.H. Kim C.G. Rhee S.G. J. Biol. Chem. 1993; 268: 21318-21327Abstract Full Text PDF PubMed Google Scholar). Based on these studies, it has been suggested that PLC-β4 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-β4 could be activated by Gαq but not by βγ-subunits of G-proteins (21Jiang H. Wu D. Simon M.I. J. Biol. Chem. 1994; 269: 7593-7596Abstract Full Text PDF PubMed Google Scholar,22Lee C.-W. Lee K.-H. Lee S.B. Rhee S.G. J. Biol. Chem. 1994; 269: 25335-25338Abstract Full Text PDF PubMed Google Scholar). Here we report the identification of a rat PLC-β4 variant with a different carboxyl-terminal region. We show by reverse transcription-PCR and immunoblot analysis that this new splice variant of PLC-β4 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 Gαq. In the course of isolating PLC-β4 cDNA from a rat brain λZapII cDNA library (20Kim M.J. Bahk Y.Y. Min D.S. Lee S.J. Ryu S.H. Suh P.G. Biochem. Biophys. Res. Commun. 1993; 194: 706-712Crossref PubMed Scopus (24) Google Scholar), we identified cDNA clones (clone β4–53 and β4–52) which exhibited patterns of restriction enzyme digestion differing from the previously described 130-kDa PLC-β4 cDNA (Fig. 1 A). Further sequence analysis of these cDNAs revealed that they were splice variants of the PLC-β4 gene. Two clones, β4–53 and β4–52, 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 (23Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52771) Google Scholar). Total RNA was prepared from adult Sprague-Dawley rat brain tissue using the guanidium thiocyanate phenol-based single-step method (24Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar). cDNA was synthesized in a 50-μl reaction mixture containing 50 mmTris-HCl, pH 8.3, 5 mm MgCl2, 75 mmKCl, 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-β4b transcript, and primer P2 (5′-TGTGTTTGGGACACTGCATG-3′), which is the 3′-untranslated region specific for the PLC-β4b mRNA (Fig. 2 A). The amplified PCR products were analyzed in a 2% agarose gel stained with ethidium bromide. Whole rat brain poly(A) mRNA from CLONTECH was reverse transcribed with PLC-β4b-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-β4a and another antisense primer (5′-CACTGCATGACAGGATTTCA-3′), which is also specific for PLC-β4b cDNA. The amplified PCR product was subcloned into T-vector (Novagen) and sequenced by the Sanger dideoxy method. A pBluescript KS plasmid containing the whole open reading frame of PLC-β4a was constructed by splicing an ApaI-SalI fragment of clone β4–19 and a SalI-XhoI fragment of clone β4–54 (the XhoI site used was originated from 3′-end linker of λZapII). The resulting cDNA was then subcloned into theEcoRV site of pBluescript KS (Stratagene, La Jolla, CA) and named pKS/β4a. The construction of a pBluescript KS vector containing the whole cDNA for PLC-β4b (pKS/β4b) was accomplished by replacing the SalI-ApaI fragment of pKS/β4a with the SalI-ApaI fragment of pKS/β4–53, which had been constructed by inserting anSalI-XhoI (XhoI in 3′-end linker of λZapII) fragment of clone β4–53 between theSalI-XhoI sites of pBluescript KS. The mammalian expression vectors for PLC-β4a and PLC-β4b were constructed by inserting the blunt ended SmaI-ApaI fragments of pKS/β4a and pKS/β4b into the EcoRV site of pcDNAI. The resulting plasmids were named pcDNAI/PLC-β4a and pcDNAI/PLC-β4b, respectively. A mammalian expression vector for mouse Gαq was made utilizing the PCR technology with the GeneAMP kit from Perkin-Elmer using mouse Gαq 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 Gαq 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/Gαq. All DNA sequences were verified by sequencing. 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 (25Cullen B.R. Lomedico P.T. Ju G. Nature. 1984; 307: 241-245Crossref PubMed Scopus (214) Google Scholar). 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 μmchloroquine 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. Peptide β4-N (MAKPYEFNWQKE, corresponding to residues 1–12 of PLC-β4a or PLC-β4b) and peptide 116-specific (GKQRDASPSG, corresponding to residues 1013–1022 of PLC-β4b) were synthesized by Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry, conjugated to keyhole limpet hemocyanin with glutaraldehyde, and injected into rabbits as described before (26Bahk Y.Y. Lee Y.H. Lee T.G. Seo J. Ryu S.H. Suh P.-G. J. Biol. Chem. 1994; 269: 8240-8245Abstract Full Text PDF PubMed Google Scholar). Antisera were affinity purified on protein A-agarose (Pierce Chemical Co.). The anti-Gαq rabbit polyclonal antibody used in immunoblotting was a generous gift from Dr. Y. S. Kim (Seoul National University, South Korea). 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 mmphenylmethylsulfonyl 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-β4b-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. COS-7 cells, transfected with expression plasmids carrying the cDNA of PLC-β4a or -β4b, 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-β4, 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-β4b protein in HeLa cell by using recombinant vaccinia virus expression system was done as described previously (40Mackett M. Smith G.L. Moss B. J. Virol. 1984; 49: 857-864Crossref PubMed Google Scholar). 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-β4a and -β4b (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. COS-7 cells were cotransfected with pcDNAI/Gαq and pcDNAI/β4a or pcDNAI/β4b. After 24 h, the cells were labeled overnight with 2 μCi/mlmyo-[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 mmLiCl was added. The cells were then treated with 30 μmAlF4− (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 (27Yeo E.-J. Kazlauskas A. Exton J.H. J. Biol. Chem. 1994; 269: 27823-27826Abstract Full Text PDF PubMed Google Scholar). 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 mmammonium 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 (27Yeo E.-J. Kazlauskas A. Exton J.H. J. Biol. Chem. 1994; 269: 27823-27826Abstract Full Text PDF PubMed Google Scholar). 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. Mammalian expression vectors for PLC-β4a, PLC-β4b, or PLC-β2 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 mmphenylmethylsulfonyl 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 mmphenylmethylsulfonyl 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 mmsodium 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 βγ-subunits (provided by Dongeun Park (Kwang-Joo Institute of Science and Technology, South Korea), and 5–10 μl of 0.1m 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. We isolated two clones (clone β4–52 and β4–53) that had restriction enzyme digestion patterns differing from the 130-kDa PLC-β4 reported previously. Subsequent sequence analysis revealed that these clones could be a splice variant of the 130-kDa PLC-β4 mRNA (Fig. 1 A). Clone β4–53 was selected for further analysis because it contained the longer cDNA insert. The overall cDNA structure of clone β4–53 was almost identical to the 130-kDa PLC-β4 mRNA except that this clone had a 176-nucleotide deletion from the region encoding the carboxyl-terminal part of the 130-kDa PLC-β4 (Figs. 1 A and2 A). This deletion caused a frameshift compared with the reading frame of the 130-kDa PLC-β4 cDNA. As a result the carboxyl-terminal 162 amino acids are replaced with 10 distinct amino acids in β4–53 (Fig. 1 B). As seen in Fig. 2, the variant mRNA shares most of its sequence with the 130-kDa PLC-β4 mRNA, but it also has its own 37 nucleotides that are not present in the 130-kDa PLC-β4 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 β4–53) encoding the novel splice variant of PLC-β4 encompassed the whole region downstream of the X-domain (Fig. 1 A). 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 β4–53. 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-β4 protein. Therefore, we used the long distance PCR cloning method to test whether PLC-β4b shared an identical amino-terminal primary structure with PLC-β4a. Whole rat brain mRNA was reverse transcribed with PLC-β4b-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-β4a as sense primer and an antisense primer corresponding to the 3′-untranslated region of PLC-β4b. PCR amplification generated approximately a 3.3-kilobase pair DNA fragment (RACE-PCR product) (Fig. 1 A and Fig. 3, lane 2). The PstI digestion pattern of this RACE-PCR product was the same as the PCR product amplified with pKS/β4b as a template (Fig. 3, lanes 5 and 6). By sequencing this RACE-PCR product, we can conclude that PLC-β4b has an amino-terminal structure identical to the corresponding PLC-β4a structure. The open reading frame predicted by the cumulative sequences of the clones β4–53 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-β4b, whereas the previously reported PLC-β4 was now renamed PLC-β4a. To verify whether a transcript of the spliced variant of PLC-β4 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-β4b transcript (Fig. 2 A). As shown in Fig. 4, the PCR product obtained from reverse-transcribed rat brain RNA, and clone β4–53 was the expected 279 bp. This suggests that a PLC-β4b transcript does exist in vivo and that our clone was not just an entity generated by recombination during λ phage amplification. Although we successfully isolated a full-length cDNA encoding PLC-β4b by long distance PCR, we were not successful in isolating a λ phage clone containing the whole coding region of the splice variant, and thus we could not exclude the possibility that the PLC-β4b transcript was aberrant or a nonproductive mRNA. Therefore, we chose a different strategy to confirm the conclusion that PLC-β4b exists in vivo and shares the amino-terminal region with PLC-β4a. 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-β4b-specific sequence, and an antipeptide antibody generated against the amino-terminal sequence of PLC-β4a, then this would prove that PLC-β4b is an authentic entityin vivo. Thus, we made two antipeptide antibodies, one to the carboxyl-terminal region of PLC-β4b (anti-116-specific antibody) and the other to the amino-terminal region of PLC-β4a (anti-β4-N antibody). First, we used these antibodies in an experiment where we expressed PLC-β4b in HeLa cells after infection with vaccinia virus containing the corresponding cDNA to test whether the molecular mass of recombinant PLC-β4b would be the expected 116 kDa when expressed in a eukaryotic cell. As shown in Fig. 5, the molecular mass of the recombinant PLC-β4b expressed in HeLa cells was the predicted 116 kDa, and it was recognized by both the anti-β4-N antibody and the anti-116-specific antibody (Fig. 5, center lane in bothpanels). Second, to identify PLC-β4b 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-β4-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-β4b shares a common amino-terminal region with PLC-β4a. To assess functional differences of the two forms of PLC-β4, we compared their intracellular localization, since previous studies had suggested that the carboxyl-terminal region of the PLC-β type is required for their association with the particulate fraction of the cell (17Wu D. Jiang H. Katz A. Simon M.I. J. Biol. Chem. 1993; 268: 3704-3709Abstract Full
Referência(s)