Subunit Oligomerization, and Topology of the Inositol 1,4,5-Trisphosphate Receptor
1999; Elsevier BV; Volume: 274; Issue: 41 Linguagem: Inglês
10.1074/jbc.274.41.29483
ISSN1083-351X
AutoresDaniel L. Galvan, Emma Borrego-Diaz, Pablo Jorge Pérez, Gregory A. Mignery,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoThe inositol 1,4,5-trisphosphate receptor (InsP3R) is a tetrameric assembly of highly conserved subunits that contain multiple membrane-spanning sequences in the C-terminal region of the protein. In studies aimed at investigating the oligomerization and transmembrane topology of the type-1 InsP3R, a series of membrane-spanning region truncation and deletion plasmids were constructed. These plasmids were transiently transfected in COS-1 cells, and the resulting expression products were analyzed for the ability to assemble into tetrameric structures. The topology of the membrane-spanning region truncations and the full-length receptor was determined by immunocytochemical analysis of transfected COS-1 cells using complete or selective permeabilization strategies. Our results are the first to experimentally define the presence of six membrane-spanning regions. These results are consistent with the current model for the organization of the InsP3R in the endoplasmic reticulum and show that the truncation mutants are properly targeted and oriented in the endoplasmic reticulum membrane, thus making them amenable reagents to study receptor subunit oligomerization. Fractionation of soluble and membrane protein components revealed that the first two membrane-spanning regions were necessary for membrane targeting of the receptor. Sedimentation and immunoprecipitation experiments show that assembly of the receptor subunits was an additive process as the number of membrane-spanning regions increased. Immunoprecipitations from cells co-expressing the full-length receptor and carboxyl-terminal truncations reveal that constructs expressing the first two or more membrane-spanning domains were capable of co-assembling with the full-length receptor. Inclusion of the fifth membrane-spanning segment significantly enhanced the degree of oligomerization. Furthermore, a deletion construct containing only membrane-spanning regions 5 and 6 oligomerized to a similar extent as that of the wild type protein. Membrane-spanning region deletion constructions that terminate with the receptor's 145 carboxyl-terminal amino acids were found to have enhanced assembly characteristics and implicate the carboxyl terminus as a determinant in oligomerization. Our results reveal a process of receptor assembly involving several distinct yet additive components and define the fifth and sixth membrane spanning regions as the key determinants in receptor oligomerization. The inositol 1,4,5-trisphosphate receptor (InsP3R) is a tetrameric assembly of highly conserved subunits that contain multiple membrane-spanning sequences in the C-terminal region of the protein. In studies aimed at investigating the oligomerization and transmembrane topology of the type-1 InsP3R, a series of membrane-spanning region truncation and deletion plasmids were constructed. These plasmids were transiently transfected in COS-1 cells, and the resulting expression products were analyzed for the ability to assemble into tetrameric structures. The topology of the membrane-spanning region truncations and the full-length receptor was determined by immunocytochemical analysis of transfected COS-1 cells using complete or selective permeabilization strategies. Our results are the first to experimentally define the presence of six membrane-spanning regions. These results are consistent with the current model for the organization of the InsP3R in the endoplasmic reticulum and show that the truncation mutants are properly targeted and oriented in the endoplasmic reticulum membrane, thus making them amenable reagents to study receptor subunit oligomerization. Fractionation of soluble and membrane protein components revealed that the first two membrane-spanning regions were necessary for membrane targeting of the receptor. Sedimentation and immunoprecipitation experiments show that assembly of the receptor subunits was an additive process as the number of membrane-spanning regions increased. Immunoprecipitations from cells co-expressing the full-length receptor and carboxyl-terminal truncations reveal that constructs expressing the first two or more membrane-spanning domains were capable of co-assembling with the full-length receptor. Inclusion of the fifth membrane-spanning segment significantly enhanced the degree of oligomerization. Furthermore, a deletion construct containing only membrane-spanning regions 5 and 6 oligomerized to a similar extent as that of the wild type protein. Membrane-spanning region deletion constructions that terminate with the receptor's 145 carboxyl-terminal amino acids were found to have enhanced assembly characteristics and implicate the carboxyl terminus as a determinant in oligomerization. Our results reveal a process of receptor assembly involving several distinct yet additive components and define the fifth and sixth membrane spanning regions as the key determinants in receptor oligomerization. Inositol 1,4,5-trisphosphate (InsP3) 1The abbreviations used are:InsP3d-myoinositol 1,4,5-trisphosphateInsP3Rd-myoinositol 1,4,5-trisphosphate receptorCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidPMSF,phenylmethanesulfonyl fluorideSLO, streptolysin-OTMRtransmembrane regionPAGEpolyacrylamide gel electrophoresisPBSphosphate-buffered salineERendoplasmic reticulum is a well known second messenger that plays a pivotal role in the regulated release of intracellular calcium. Plasma membrane receptor-coupled activation of G-protein or tyrosine kinase-induced hydrolysis of phosphotidylinositol 4,5-bisphosphate results in the production of InsP3 and subsequent efflux of Ca2+ from endoplasmic reticulum stores. InsP3-mediated calcium release has been implicated in numerous, very diverse cellular processes including the initiation/propagation of Ca2+waves, cell growth, secretion, fertilization, and development (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6205) Google Scholar,2Berridge M.J. Nature. 1997; 386: 759-760Crossref PubMed Scopus (393) Google Scholar). d-myoinositol 1,4,5-trisphosphate d-myoinositol 1,4,5-trisphosphate receptor 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid SLO, streptolysin-O transmembrane region polyacrylamide gel electrophoresis phosphate-buffered saline endoplasmic reticulum The InsP3 receptor family consists of three or four highly homologous members with an overall sequence identity of ∼69% (3Südhof T.C. Newton C.L. Archer III B.T. Ushkaryov Y.A. Mignery G.A. EMBO J. 1991; 10: 3199-3206Crossref PubMed Scopus (322) Google Scholar). Most, if not all, cells express one or more of the InsP3R isoforms. The physiological significance of co-expression of multiple isoforms of the InsP3R within a given cell is not clear, but it has been generally thought to confer release channels that were functionally heterogeneous, differentially regulated, and differentially targeted or a combination of all three possibilities. Recent investigations, from this laboratory and others, have defined the intrinsic calcium channel properties for all three isoforms of the receptor (4Perez P.J. Ramos-Franco J. Fill M. Mignery G.A. J. Biol. Chem. 1997; 272:: 23961-23969Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 5Ramos-Franco J. Fill M. Mignery G.A. Biophys. J. 1998; 75: 834-839Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 6Ramos-Franco J. Caenepeel S. Fill M. Mignery G.A. Biophys. J. 1998; 75: 2783-2793Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 7Mak D-O.D. McBride S. Yue Y. Foskett J.K. Biophys. J. 1999; 76 (abstr.): 375Google Scholar, 8Hagar R.E. Burgstahler A.D. Nathanson M.H. Ehrlich B.E. Nature. 1998; 396: 81-84Crossref PubMed Scopus (231) Google Scholar). These studies reveal that the three isoforms of InsP3R have channels with remarkably similar permeation properties. All channels have similar Ca2+ conductances (∼60 pS) and almost identical reversal potentials using Cs+ or Ca2+, indicating similar ionic selectivity. Despite the similarities observed at the single channel level, the individual receptor types appear to be differentially regulated by calcium and InsP3. These studies demonstrated that the type-1 isoform from cerebellum is modulated by calcium in a biphasic manner, whereas the type-2 and type 3 isoforms are not (5Ramos-Franco J. Fill M. Mignery G.A. Biophys. J. 1998; 75: 834-839Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 6Ramos-Franco J. Caenepeel S. Fill M. Mignery G.A. Biophys. J. 1998; 75: 2783-2793Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar,8Hagar R.E. Burgstahler A.D. Nathanson M.H. Ehrlich B.E. Nature. 1998; 396: 81-84Crossref PubMed Scopus (231) Google Scholar, 9Bezprozvanny I. Watras J. Ehrlich B.E. Nature. 1991; 351: 751-754Crossref PubMed Scopus (1454) Google Scholar). The type 2 receptor Ca2+ channel from cardiac myocytes activates at a lower calcium concentration and sustains this activity over a wide range of Ca2+ concentrations. Similar results were observed with the type 3 receptor from rat insulinoma cells (RIN-5F) (8Hagar R.E. Burgstahler A.D. Nathanson M.H. Ehrlich B.E. Nature. 1998; 396: 81-84Crossref PubMed Scopus (231) Google Scholar). In addition to the differences in calcium sensitivities observed between the type 1 and 2 receptors, the type 2 homologue exhibits approximately a 3-fold increased efficacy to a similar dose of InsP3. Together, these results imply that the intrinsic calcium channels between isoforms are very similar, yet they appear to be regulated quite differently and may help explain the heterogeneity observed in calcium release events in cells and tissue expressing multiple forms of the receptor. The InsP3R channel protein is a tetrameric structure resulting from the homo- or hetero-oligomerization of the receptor isoform subunits. Each subunit can be divided into three principle domains consisting of an amino-terminal ligand binding region, a carboxyl-terminal channel domain, and a central coupling or regulatory domain (3Südhof T.C. Newton C.L. Archer III B.T. Ushkaryov Y.A. Mignery G.A. EMBO J. 1991; 10: 3199-3206Crossref PubMed Scopus (322) Google Scholar, 10Mignery G.A. Südhof T.C. EMBO J. 1990; 9: 3893-3898Crossref PubMed Scopus (277) Google Scholar). Within the carboxyl-terminal region of the receptors there are numerous arrays of hydrophobic amino acids that form membrane-spanning helices that are thought to form the receptor's intrinsic calcium channel. The role of these multiple membrane-spanning domains in the oligomerization and membrane targeting of the receptor were demonstrated in mutagenesis studies. These studies revealed that the elimination of residues 2205–2225 encompassing the putative membrane-spanning regions resulted in the expression of soluble, monomeric protein that binds InsP3 with similar affinity and specificity as that of full-length native or recombinant receptor (10Mignery G.A. Südhof T.C. EMBO J. 1990; 9: 3893-3898Crossref PubMed Scopus (277) Google Scholar). Studies using a green fluorescent protein chimera with amino-terminally truncated receptor confirmed these results and proposed that the membrane spanning domains confer reticular targeting and oligomerization. (11Sayers L.G. Miyawaki A. Muto A. Takeshita H. Yamamoto A. Michikawa T. Furuichi T. Mikoshiba K. Biochem. J. 1997; 323: 273-280Crossref PubMed Scopus (47) Google Scholar). Joseph et al. (12Joseph S.K. Boehning D. Pierson S. Nicchitta C.V. J. Biol. Chem. 1997; 272: 1579-1588Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) used cell-free systems to examine the processing of truncated receptor isoforms (types 1 and 3) and concluded that there were two determinants encompassed by this region required for subunit assembly. The number and topological organization of the transmembrane-spanning regions of the InsP3R subunit has been difficult to determine. A six-transmembrane-helix model has emerged from numerous computer predictions and sequence homology comparisons, which are supported by immunological and N-linked glycosylation studies (13Takei K. Mignery G.A. Mugnaini E. Südhof T.C. De Camilli P. Neuron. 1994; 12: 327-342Abstract Full Text PDF PubMed Scopus (127) Google Scholar, 14Michikawa T. Hamanaka H. Otsu H. Yamamoto A. Miyawaki A. Furuichi T. Tashiro Y. Mikoshiba K. J. Biol. Chem. 1994; 269: 9184-9189Abstract Full Text PDF PubMed Google Scholar). The details regarding the receptor's intrinsic calcium channel structure and organization are limited. The calcium channel pore is thought to form as a result of subunit monomer assembly into tetramers. Thus, a detailed analysis of the components involved in tetramerization and topological organization of the membrane-spanning sequences should provide new insights into the formation of the receptors intrinsic calcium release channel. In this study, we use type 1 InsP3R expression constructions containing differing numbers and combinations of putative membrane-spanning sequences with or without the carboxyl terminus in a mammalian expression system to evaluate the elements involved in subunit oligomerization. In addition, these expression plasmids were used to determine the topological orientation of the membrane-spanning helices through the endoplasmic reticulum using detergent and streptolysin-O permeabilization/immunofluorescence analysis. These experiments confirm the six-membrane-spanning hypothesis of the receptors topological orientation. We show that the oligomerization of the receptor is dependent upon several unique, yet additive determinants. We find that InsP3R mutants expressing the first two transmembrane sequences are sufficient to form high molecular weight complexes and that when coupled to the carboxyl-terminal 145 amino acids, the assembly is enhanced. In addition, a construction lacking the first four transmembrane regions, possessing only the fifth and sixth membrane-spanning helices and the intervening luminal loop, is very efficient at forming tetramers. Dulbecco's modified Eagle's medium was obtained from Mediatech. Fetal bovine serum and penicillin-streptomycin were purchased from Life Technologies, Inc. Goat serum and streptolysin-O (SLO) were purchased from Sigma. Fluorescein isothiocyanate-conjugated goat anti-rabbit IgG was obtained from Organon Teknika. Restriction enzymes, DNA modifying enzymes and DNA polymerase were purchased from New England Biolabs, Roche Molecular Biochemicals, and U.S. Biochemical Corp. The Fluoromount-G was purchased from Southern Biotechnology Associates, Inc. Protein-A-Sepharose CL4B was obtained from Amersham Pharmacia Biotech. All other chemicals were of reagents grade and used without further purification. Expression plasmids harboring increasing numbers of membrane-spanning regions that terminate with a common immunological tag were prepared by polymerase chain reaction. Briefly, a host plasmid was generated by digesting pIP3R-Stop1267 (10Mignery G.A. Südhof T.C. EMBO J. 1990; 9: 3893-3898Crossref PubMed Scopus (277) Google Scholar) with KpnI/BstBI followed by insertion of the KpnI–BstBI fragment of pCMVI-9 (15Mignery G.A. Newton C.L. Archer III B.T. Südhof T.C. J. Biol. Chem. 1990; 265: 12679-12685Abstract Full Text PDF PubMed Google Scholar). This plasmid was then digested with BstBI, and BstBI/ClaI cut polymerase chain reaction products containing defined membrane-spanning regions were inserted. The 5′ oligonucleotide primer, corresponding to nucleotides 6945–6968 (GAATCGAAACTTCGAATATATTAC), was constant, and the 3′ antisense primers were designed to terminate C-terminal to the query sequence. The specific 3′ oligonucleotide primer sequences were as follows: transmembrane region (TMR) 1 (CGAATCGATGGTCTTGTTGGCTCCTCCTCTCACTCCTTTA), TMR 2 (CGAATCGATGGTCTTGTTGGCCCGGATGCCATGGGGCTT), TMR 3 (CGCATCGATGGTCTTGTTGGCGGTCCCACAGTTGCC), TMR 4 (CGCATCGATGGTCTTGTTGGCATAGAAAAACTCATG), TMR 5 (CGCATCGATGGTCTTGTTGGCCAAGATAAAGTCATCCTT), and TMR 6 (CGCATCGATGGTCTTGTTGGCCAGGTCAGCAAAGGTGTC). In all cases, the full-length receptor pCMVI-9/pInsP3R-T1 (6Ramos-Franco J. Caenepeel S. Fill M. Mignery G.A. Biophys. J. 1998; 75: 2783-2793Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 15Mignery G.A. Newton C.L. Archer III B.T. Südhof T.C. J. Biol. Chem. 1990; 265: 12679-12685Abstract Full Text PDF PubMed Google Scholar) was used as the amplification template. The construct lacking all membrane-spanning domains (TMR0-C) was prepared by digesting the host plasmid withBstBI, repair with Klenow DNA polymerase, and religation, resulting in the restoration of the reading frame between the InsP3R and the proton pump immunological tag. Constructions that included the InsP3R carboxyl-terminal 145 amino acids were prepared using similar polymerase chain reaction strategies. In these cases, amplified product was digested withBstBI/BglII and inserted into a similarly digested intermediate plasmid spanning nucleotides 6659–9467. These plasmids were digested with BstBI/XbaI, and the resulting fragments were inserted into the full-length type 1 receptor (pCMVI-9/pInsP3R-T1) at theBstBI/XbaI sites. The 5′ oligonucleotide primer, corresponding to nucleotides 6945–6968 (GAATCGAAACTTCGAATATATTAC), was constant, and the 3′ antisense primers were as follows: TMR 1 (ACTAGTCGACGAGATCTTGGTCTTGTTTCCTCCTCTCACTCC); TMR 2 (ACTAGTCGACGAGATCTTGGTCTTGTTGCCATGGGGCTTGGGCAG); TMR 3 (ACTAGTCGACGAGATCTTGGTCTTGTTGGTCCCACAGTTGCC); TMR 4 (ACTAGTCGACGAGATCTTGGTCTTGTTATAGAAAAACTCATGTACGAAGAGGCC); TMR 5 (ACTAGTCGACGAGATCTTGGTCTTGTTCAAGATAAAGTCATCCTT); and TMR 6 (ACTAGTCGACGAGATCTTGGTCTTGTTCAGGTCAGCAAAGGTGTC). The construct lacking all membrane spanning sequences (TMR0+C) was described previously and is analogous to pIP3RΔ2225–2604 (10Mignery G.A. Südhof T.C. EMBO J. 1990; 9: 3893-3898Crossref PubMed Scopus (277) Google Scholar). Plasmid constructions containing membrane-spanning regions 3 and 4 and regions 5 and 6 were prepared by polymerase chain reaction using the primer pairs GCGCCATGGTTCGAATATTTTCAGTTGGATTACAGCCC/ACTAGTCGACGAGATCTTGGTCTTGTTATAGAAAAACTCATGTACGAAGAGGCC and GCGCCATGGTTCGAAGTGTCACCCGCAATGGACGGCCC/ACTAGTCGACGAGATCTTGGTCTTGTTCAGGTCAGCAAAGGTGTC, respectively. The amplification products were digested withBstBI/BglII and inserted into similarly digested intermediate plasmid-spanning nucleotides 6659–9467. The resulting plasmid was digested with BstBI/XbaI, and the TMR-containing fragments were ligated intoBstBI/XbaI-digested full-length receptor. COS-1 cells were transiently transfected with InsP3R plasmid DNA using the DEAE-dextran method. Sheared salmon sperm DNA was used to mock transfect COS-1 cells and served as a negative control. Cells were incubated at 37 °C, 5% CO2 for 48–72 h prior to harvesting for biochemical and immunocytochemical analysis. Transfected COS-1 cells were harvested 48–72 h post-transfection, and microsomes were prepared as described previously (15Mignery G.A. Newton C.L. Archer III B.T. Südhof T.C. J. Biol. Chem. 1990; 265: 12679-12685Abstract Full Text PDF PubMed Google Scholar). COS cells were washed with PBS; harvested by scrapping into 50 mm Tris-HCl, pH 8.3, 1 mm EDTA, 1 mm β-mercaptoethanol, 1 mm PMSF; and lysed by 40 passages through a 27-gauge needle. Membranes were pelleted by a 20-min centrifugation (289,000 × g av), resuspended in buffer, and either used immediately or frozen at −80 °C. Microsomal fractions were solubilized in 50 mmTris-HCl pH 8.3, 1 mm EDTA, 1 mmβ-mercaptoethanol, 1 mm PMSF, 1% CHAPS on ice for 1 h. Insoluble fractions were eliminated by a 10-min centrifugation at 289,000 × g av, and the supernatant containing solubilized receptor was fractionated through 5–20% sucrose (w/v) gradients. Sucrose gradients (2 ml) were centrifuged for 5 h, 4 °C at 166,320 ×g av using a Beckman TLS-55 swinging bucket rotor. Following centrifugation, the gradients were fractionated into 25 80-μl aliquots, and InsP3R protein(s) were detected by immunoblotting. Gradient performance and resolution were evaluated by applying standard proteins of known molecular weight and/or sedimentation coefficients. The standard proteins were bovine serum albumin (65 kDa, 4.36 S), alcohol dehydrogenase (150 kDa, 7.4 S), β-amylase (200 kDa), catalase (250, 11.3 S), and β-galactosidase (540 kDa, 16 S). In addition, CHAPS-solubilized cerebellar microsomes were applied to control gradients as a source of native InsP3R and RyR (30 S) (16Lai F.A. Erickson H.P. Rousseau E. Liu Q-Y. Meissner G. Nature. 1988; 331: 315-319Crossref PubMed Scopus (70) Google Scholar,17Marx S.O. Ondrias K. Marks A.R. Science. 1998; 281: 818-821Crossref PubMed Scopus (354) Google Scholar) sedimentation controls. Standard proteins were detected using Coomassie staining and immunostaining. COS cell microsomes and sucrose gradient fractions were analyzed by 5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as described previously (15Mignery G.A. Newton C.L. Archer III B.T. Südhof T.C. J. Biol. Chem. 1990; 265: 12679-12685Abstract Full Text PDF PubMed Google Scholar), followed by immunoblotting using chemiluminescence reagents (Amersham Pharmacia Biotech). Transiently transfected COS cells were harvested by brief trypsinization followed by plating onto poly-d-lysine-coated glass coverslips. Following an attachment interval, the cells were fixed with 2% paraformaldehyde in phosphate-buffered saline (PBS) and permeabilized with 0.3% Triton X-100 and/or streptolysin-O as described below. Transfected COS-1 cells were washed with PBS and fixed in 2% paraformaldehyde-PBS for 15 min at 25 °C. The coverslips were washed with PBS and incubated for 30 min with solution A (20 mm phosphate buffer, pH 7.2, 0.45m NaCl, 0.3% Triton X-100, and 1.0% goat serum). The Triton X-100 was removed by washing with PBS, and the cells were blocked in solution B (20 mm phosphate buffer pH 7.2, 0.45m NaCl) containing 10% goat serum and stored at 4 °C until use. Selective permeabilization using SLO was performed by the methods described by Otto and Smith (18Otto J.C. Smith W.L. J. Biol. Chem. 1994; 269: 19868-19875Abstract Full Text PDF PubMed Google Scholar). Transfected COS-1 cells were washed with PBS and fixed in 2% paraformaldehyde-PBS for 15 min. The coverslips were washed with PBS and incubated for 15 min at 4 °C with streptolysin-O (200 units/ml) that had been preactivated by a 5-min, 0 °C incubation with 10 mm dithiothreitol in PBS. Unbound streptolysin-O was removed by washing, and the coverslips were incubated at 37 °C for 20 min in PBS containing 10 mm dithiothreitol. The coverslips were washed in PBS, blocked in solution B (20 mmphosphate buffer, pH 7.2, 0.45 m NaCl) containing 10% goat serum and stored at 4 °C until use. Coverslips were incubated (45 min at room temperature) with primary antibody diluted in solution A (20 mm phosphate buffer, pH 7.2, 0.45 m NaCl, 0.3% Triton X-100, and 1.0% goat serum) for Triton X-100-permeabilized cells and solution C (1.0% goat serum in PBS) for SLO-treated cells. The cells were washed with solution B (20 mm phosphate buffer, pH 7.2, 0.45 m NaCl) for 15 min at room temperature. Coverslips were then incubated with goat anti-rabbit fluorescein isothiocyanate secondary antibody (12.5 μg/ml) diluted into solution A (for Triton X-100-permeabilized cells) and solution C (for SLO permeabilization) for 30 min at room temperature. The cells were washed with solution B, and the coverslips were fixed to microscope slides using Fluoromount-G. Coverslips were analyzed with a Nikon Diaphot 300 inverted microscope and photographed using T-Max 400 ASA film (Eastman Kodak Co.). The InsP3R-specific amino-terminal (T1NH) and carboxyl-terminal (T1C) polyclonal antibodies were generated against residues 308–326 of the type-1 SIb alternatively spliced isoform and the 19 C-terminal amino acids, respectively (6Ramos-Franco J. Caenepeel S. Fill M. Mignery G.A. Biophys. J. 1998; 75: 2783-2793Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The luminal loop antipeptide antibody (V753) is directed against residues 2463–2476 of the type-1 receptor (13Takei K. Mignery G.A. Mugnaini E. Südhof T.C. De Camilli P. Neuron. 1994; 12: 327-342Abstract Full Text PDF PubMed Scopus (127) Google Scholar). The proton pump tag antibody is directed against the 11 carboxyl-terminal residues of the 116-kDa subunit of the proton pump (15Mignery G.A. Newton C.L. Archer III B.T. Südhof T.C. J. Biol. Chem. 1990; 265: 12679-12685Abstract Full Text PDF PubMed Google Scholar, 19Perin M.S. Fried V.A. Stone D.K. Xie X.S. Südhof T.C. J. Biol. Chem. 1991; 266: 3877-3881Abstract Full Text PDF PubMed Google Scholar). All InsP3R peptide antibodies were affinity-purified using immunogenic peptide. The secondary antibody used in immunofluoresence staining was a fluorescein isothiocyanate-conjugated goat anti-rabbit antibody (Organon Teknika). Cotransfected COS-1 cells were harvested, and microsomes were prepared as described above. A small amount of the microsomes representing total expression products was removed and added to SDS-PAGE buffer. The remaining microsomes were solubilized by stirring on ice for 2 h in Buffer A (1% Triton X-100, 150 mm sodium chloride, 10 mm Tris-HCl, pH 7.4, 1 mm EDTA, 1 mm EGTA, 1 mmPMSF, and 2% bovine serum albumin). Samples were clarified by centrifugation at 106,120 × g av for 5 min at 4 °C. The supernatants were removed and mixed with 2 μl (∼2 μg), 1:250 dilution, of the immunoprecipitating antibody (T1C) directed against the carboxyl terminus of the InsP3R. The samples were incubated on ice for 4 h. Samples were clarified by centrifugation in a microcentrifuge (16,000 × g av) at 4 °C for 5 min, and the supernatants were retained. To each supernatant fraction, 20 μl of a 10% protein A-Sepharose CL4B (Amersham Pharmacia Biotech) slurry was added and incubated at 4 °C for 2 h with gentle agitation. The antigen-IgG-protein A-Sepharose conjugates were pelleted by 10-s centrifugation at 16,000 ×g av. The samples were washed three times for 5 min each in 0.5 ml of Buffer B (1% Triton X-100, 150 mm sodium chloride, 10 mm Tris-HCl, pH 7.4, 1 mm EDTA, 1 mm EGTA, and 1 mm PMSF), two times in Buffer C (1% Triton X-100, 300 mm sodium chloride, 10 mm Tris-HCl, pH 7.4, 1 mm EDTA, 1 mm EGTA, and 1 mm PMSF), and once briefly in Buffer D (10 mm Tris-HCl, pH 7.4, 1 mm EDTA, 1 mm EGTA, and 1 mm PMSF). Bound antigen was released from the protein A beads by the addition of SDS-PAGE buffer and boiling for 3 min. It is well established that the multiple membrane-spanning sequences of the InsP3R that reside near the carboxyl terminus are involved in the targeting and assembly of receptor subunits into functional tetrameric calcium release channels (10Mignery G.A. Südhof T.C. EMBO J. 1990; 9: 3893-3898Crossref PubMed Scopus (277) Google Scholar, 11Sayers L.G. Miyawaki A. Muto A. Takeshita H. Yamamoto A. Michikawa T. Furuichi T. Mikoshiba K. Biochem. J. 1997; 323: 273-280Crossref PubMed Scopus (47) Google Scholar). In experiments designed to study the role of these transmembrane sequences as well as other components in the oligomerization and topology of the type-1 InsP3R, a series of expression plasmid mutations were constructed (Fig.1). In the initial mutation series, plasmids encoding carboxyl-terminally truncated InsP3R proteins containing increasing numbers of putative membrane-spanning sequences were prepared in a cytomegalovirus promoter-based mammalian expression plasmid. These truncations were transiently expressed in COS cells followed by analysis of oligomerization by sedimentation on 5–20% sucrose gradients. The density gradients were fractionated, and the migrations of receptor proteins through the gradient were detected by immunoblotting. The chimera in which no membrane-spanning sequences were present (TMR0-C) was soluble (Fig. 2) and sedimented to a position on the gradients consistent with that of a monomeric protein (Figs. 3 and4, upper panel) (10Mignery G.A. Südhof T.C. EMBO J. 1990; 9: 3893-3898Crossref PubMed Scopus (277) Google Scholar). A construct containing only the first membrane-spanning sequence was not efficiently targeted to the endoplasmic reticulum (ER), and a significant amount (∼50%) of the expressed protein was soluble (Fig.2). The reasons for the lack of efficient targeting to the ER is not clear, since an amino-terminally truncated protein containing similar transmembrane sequences, when expressed in an in vitroassay, was reported to be targeted to the pancreatic microsomes (12Joseph S.K. Boehning D. Pierson S. Nicchitta C.V. J. Biol. Chem. 1997; 272: 1579-1588Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). All of the additional expression products containing two or more putative transmembrane sequences encoded proteins that were efficiently targeted to the ER (Fig. 2).Figure 3Sedimentation of monomeric and tetrameric InsP3R proteins on sucrose gradients. Microsomes from transiently transfected COS-1 cells expressing TMR0±C and TMR1–6±C were solubilized with 1% CHAPS (in the case of TMR0±C, cytosolic proteins were used) and applied to 5–20% linear sucrose gradients (2 ml) to evaluate gradient performance. Samples were sedimented at 166, 320 × g av for 5 h, fractionated (n = 25), resolved on 5% SDS-PAGE, and immunoblotted with the T1C or proton pump antibodies to reveal the recombinant protein sedimentation profile. Gradient performance and resolution were evaluated by applying standard proteins of known molecular weight and/or sedimentation coefficients. The gradient position of standard proteins are indicated (lettered arrows) and correspond to bovine serum albumin (65 kDa, 4.36 S) (A), alcohol dehydrogenase (150 kDa, 7.4 S) (B), β-amylase (200 kDa) (C), catalase (250, 11.3 S) (D), β-galactosidase (540 kDa, 16 S) (E), and ryanodine receptor (2256 kDa, 30 S) (F). In addition, CHAPS-solubilized cerebellar microsomes were applied to control gradients as
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