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• W1987157213 abstract "Ig-Hepta is a member of a new subfamily of the heptahelical receptors and has an unusually long N terminus extending toward the extracellular side of the plasma membrane. Pulse-chase experiments in 293T cells using antisera specifically recognizing its N- and C-terminal regions demonstrated that Ig-Hepta is core-glycosylated cotranslationally and proteolytically processed into a two-chain form in the endoplasmic reticulum, followed by maturation of oligosaccharide chains and dimerization. The cleavage occurs at two highly conserved sites: one in a “SEA” module (a module first identified in sperm protein, enterokinase, andagrin) near the N terminus and the other in the stalk region preceding the first transmembrane span, generating ∼20-, 130-, and 32-kDa fragments. The latter two remain tightly associated non-covalently even after cleavage as revealed by immunoprecipitation of native and myc-tagged Ig-Hepta constructs that were transiently expressed in 293T cells. The dimer consisting of four chains, (130 kDa + 32 kDa)2, is linked by disulfide bonds. A fusion protein of the extracellular domain of Ig-Hepta and the Fc domain of immunoglobulin was found to be a good substrate of the processing enzymes and used for determining the exact cleavage sites in the SEA module and juxtamembrane stalk region. Ig-Hepta is a member of a new subfamily of the heptahelical receptors and has an unusually long N terminus extending toward the extracellular side of the plasma membrane. Pulse-chase experiments in 293T cells using antisera specifically recognizing its N- and C-terminal regions demonstrated that Ig-Hepta is core-glycosylated cotranslationally and proteolytically processed into a two-chain form in the endoplasmic reticulum, followed by maturation of oligosaccharide chains and dimerization. The cleavage occurs at two highly conserved sites: one in a “SEA” module (a module first identified in sperm protein, enterokinase, andagrin) near the N terminus and the other in the stalk region preceding the first transmembrane span, generating ∼20-, 130-, and 32-kDa fragments. The latter two remain tightly associated non-covalently even after cleavage as revealed by immunoprecipitation of native and myc-tagged Ig-Hepta constructs that were transiently expressed in 293T cells. The dimer consisting of four chains, (130 kDa + 32 kDa)2, is linked by disulfide bonds. A fusion protein of the extracellular domain of Ig-Hepta and the Fc domain of immunoglobulin was found to be a good substrate of the processing enzymes and used for determining the exact cleavage sites in the SEA module and juxtamembrane stalk region. G protein-coupled receptor brain-specific angiogenesis inhibitor brefeldin A cadherin EGF LAG seven-pass G-type receptor calcium-independent receptor of α-latrotoxin epidermal growth factor module-containing mucin-like receptor 1 extracellular domain epidermal growth factor green fluorescent protein GPCR proteolytic site human epididymal gene product 6 long N-terminal type B peptidylN-glycanase F a module first identified insperm protein, enterokinase, andagrin transmembrane domain Dulbecco's modified Eagle's medium fetal bovine serum phosphate-buffered saline endoplasmic reticulum G protein-coupled receptors (GPCRs)1 comprise a large superfamily of proteins in the body and are involved in the recognition and transduction of a variety of extracellular signals. They share a common basic structure of seven transmembrane spans (TM7) with extracellular N and intracellular C termini. Mammalian GPCRs have been classified into three major groups on the basis of their sequence similarity to rhodopsin (class I or type A), secretin receptor (class II or type B), and metabotropic receptor (class III or type C) (1Bockaert J. Pin J.P. EMBO J. 1999; 18: 1723-1729Crossref PubMed Scopus (1236) Google Scholar). During the past few years, a subgroup of the class II GPCRs has emerged whose members have unusually large N-terminal extracellular domains that contain a number of well-defined protein modules (2Stacey M. Lin H.H. Gordon S. McKnight A.J. Trends Biochem. Sci. 2000; 25: 284-289Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 36Hayflick J.S. J. Recept. Signal Transduct. Res. 2000; 20: 119-131Crossref PubMed Scopus (27) Google Scholar). Identification of cell types expressing these molecules and their potential roles in cell adhesion and signaling have become a focus of research in immunology, neuroscience, and developmental biology. This subgroup is referred to as LNB-TM7, where LNB means long N-terminal and type B (2Stacey M. Lin H.H. Gordon S. McKnight A.J. Trends Biochem. Sci. 2000; 25: 284-289Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). The members of the LNB-TM7 family include (i) EMR1 (3Baud V. Chissoe S.L. Viegas-Pequignot E. Diriong S. N′Guyen V.C. Roe B.A. Lipinski M. Genomics. 1995; 26: 334-344Crossref PubMed Scopus (99) Google Scholar), CD97 (4Hamann J. Eichler W. Hamann D. Kerstens H.M. Poddighe P.J. Hoovers J.M. Hartmann E. Strauss M. van Lier R.A. J. Immunol. 1995; 155: 1942-1950PubMed Google Scholar), F4/80 (5McKnight A.J. Macfarlane A.J. Dri P. Turley L. Willis A.C. Gordon S. J. Biol. Chem. 1996; 271: 486-489Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar), and ETL (6Nechiporuk T. Urness L.D. Keating M.T. J. Biol. Chem. 2001; 276: 4150-4157Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), which contain EGF modules; (ii) HE6 (7Osterhoff C. Ivell R. Kirchhoff C. DNA Cell Biol. 1997; 16: 379-389Crossref PubMed Scopus (74) Google Scholar) and GPR56 (8Liu M. Parker R.M. Darby K. Eyre H.J. Copeland N.G. Crawford J. Gilbert D.J. Sutherland G.R. Jenkins N.A. Herzog H. Genomics. 1999; 55: 296-305Crossref PubMed Scopus (66) Google Scholar) with mucin-like regions; (iii) α-latrotoxin receptors CL1, CL2, and CL3, which have a galactose-binding lectin homologous region and an olfactomedin homologous region (9Krasnoperov V.G. Bittner M.A. Beavis R. Kuang Y. Salnikow K.V. Chepurny O.G. Little A.R. Plotnikov A.N., Wu, D. Holz R.W. Petrenko A.G. Neuron. 1997; 18: 925-937Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar); (iv) BAI1, BAI2, and BAI3 with thrombospondin type-1 repeats (10Futamura M. Oda K. Nishimori H. Nakamura Y. Tokino T. Biochem. Biophys. Res. Commun. 1998; 247: 597-604Crossref PubMed Scopus (83) Google Scholar, 11Shiratsuchi T. Nishimori H. Ichise H. Nakamura Y. Tokino T. Cytogenet. Cell Genet. 1997; 79: 103-108Crossref PubMed Scopus (83) Google Scholar); (v) Celsr1–3 (12Hadjantonakis A.K. Sheward W.J. Harmar A.J. de Galan L. Hoovers J.M. Little P.F. Genomics. 1997; 45: 97-104Crossref PubMed Scopus (59) Google Scholar, 13Formstone C.J. Little P.F. Mech. Dev. 2001; 109: 91-94Crossref PubMed Scopus (72) Google Scholar) and Flamingo (14Usui T. Shima Y. Shimada Y. Hirano S. Burgess R.W. Schwarz T.L. Takeichi M. Uemura T. Cell. 1999; 98: 585-595Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar) with cadherin repeats; and (vi) Ig-Hepta with immunoglobulin repeats (15Abe J. Suzuki H. Notoya M. Yamamoto T. Hirose S. J. Biol. Chem. 1999; 274: 19957-19964Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Despite these variations in the membrane-distal region of the extracellular domain, their membrane-proximal regions or stalks are highly conserved. Namely, they contain a characteristic Cys-box motif close to the extracellular face of the membrane, suggesting a common role. For the α-latrotoxin receptors, it has been shown that proteolytic cleavage takes place immediately downstream to the conserved Cys-box (9Krasnoperov V.G. Bittner M.A. Beavis R. Kuang Y. Salnikow K.V. Chepurny O.G. Little A.R. Plotnikov A.N., Wu, D. Holz R.W. Petrenko A.G. Neuron. 1997; 18: 925-937Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar), and similar processing has been suggested for other members (2Stacey M. Lin H.H. Gordon S. McKnight A.J. Trends Biochem. Sci. 2000; 25: 284-289Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Ig-Hepta cDNA was cloned from a rat lung cDNA library in our laboratory. It was shown to contain two C2-type immunoglobulin-like domains and to be highly glycosylated (15Abe J. Suzuki H. Notoya M. Yamamoto T. Hirose S. J. Biol. Chem. 1999; 274: 19957-19964Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Northern blot analysis subsequently demonstrated that mRNA transcripts are expressed abundantly in the lung and significantly in the kidney (15Abe J. Suzuki H. Notoya M. Yamamoto T. Hirose S. J. Biol. Chem. 1999; 274: 19957-19964Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Immunohistochemistry demonstrated alveolar wall and intercalated cell localizations in the rat lung and kidney, respectively (15Abe J. Suzuki H. Notoya M. Yamamoto T. Hirose S. J. Biol. Chem. 1999; 274: 19957-19964Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). One striking and unusual feature of the Ig-Hepta molecule is that it exists as a disulfide-linked dimer; although there are a growing number of GPCRs that can be detected as homo- or heterodimers, many of them are non-covalently associated except the disulfide-linked dimers between calcium-sensing and metabotropic glutamate receptors (16Romano C. Yang W.L. O'Malley K.L. J. Biol. Chem. 1996; 271: 28612-28616Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar, 17Bai M. Trivedi S. Brown E.M. J. Biol. Chem. 1998; 273: 23605-23610Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar) and between κ and δ opioid receptors (18Jordan B.A. Devi L.A. Nature. 1999; 399: 697-700Crossref PubMed Scopus (983) Google Scholar) (for review, see Refs. 19Bouvier M. Nat. Rev. Neurosci. 2001; 2: 274-286Crossref PubMed Scopus (584) Google Scholar and20Gomes I. Jordan B.A. Gupta A. Rios C. Trapaidze N. Devi L.A. J. Mol. Med. 2001; 79: 226-242Crossref PubMed Scopus (153) Google Scholar). While performing Western blot analysis of mature Ig-Hepta to characterize the nature of the dimer, we noticed that N- and C-terminal-directed antisera stained distinct bands of ∼130 and ∼32 kDa, respectively, suggesting that Ig-Hepta undergoes proteolytic processing during the biosynthetic process. In the present study, therefore, we performed pulse-chase experiments to confirm this possibility. We further determined the site of cleavage and found it is located in the highly conserved stalk region mentioned above, implying that similar processing also occurs in other members of the LNB-TM7 family. N-terminal sequencing of Ig-Hepta also revealed another proteolytic processing occurring at a “SEA” module that is located close to the N terminus. The SEA module was first described as a motif present in an ectodomain of a number of mucin-like membrane proteins and so named after the first three proteins in which it was identified (sperm protein, enterokinase, andagrin). The number of SEA module-containing proteins now exceeds 73 (available at dylan.embl-heidelberg.de/). The function of the SEA module is not clear, but it serves as a site for proteolytic cleavage (21Parry S. Silverman H.S. McDermott K. Willis A. Hollingsworth M.A. Harris A. Biochem. Biophys. Res. Commun. 2001; 283: 715-720Crossref PubMed Scopus (116) Google Scholar, 22Wreschner D.H. McGuckin M.A. Williams S.J. Baruch A. Yoeli M. Ziv R. Okun L. Zaretsky J. Smorodinsky N. Keydar I. Neophytou P. Stacey M. Lin H.H. Gordon S. Protein Sci. 2002; 11: 698-706Crossref PubMed Scopus (96) Google Scholar). Based on this fact, Wreschner et al. (22Wreschner D.H. McGuckin M.A. Williams S.J. Baruch A. Yoeli M. Ziv R. Okun L. Zaretsky J. Smorodinsky N. Keydar I. Neophytou P. Stacey M. Lin H.H. Gordon S. Protein Sci. 2002; 11: 698-706Crossref PubMed Scopus (96) Google Scholar) have proposed a mechanism whereby a combination of ligand and receptor is generated from a single precursor molecule by its SEA module-mediated cleavage. The significance of the cleavage of Ig-Hepta at its SEA module is discussed in relation to this hypothesis. Restriction enzymes were from Takara, Kyoto, Japan; Pro-mix l-[35S] in vitrocell-labeling mix and protein G-Sepharose were obtained fromAmersham Biosciences, Uppsala, Sweden; Complete protease inhibitor mixture was from Roche Molecular Biochemicals, Mannheim, Germany; BL21(DE3)pLysS was from Stratagene, La Jolla, CA; pcDNA3, pSecTag, pRSET, pZErO-2, Zeocin, and LipofectAMINE Plus were from Invitrogen, San Diego, CA; anti-myc monoclonal antibody (9E10) was from Santa Cruz Biotechnology, Santa Cruz, CA; brefeldin A (BFA) was from Sigma Chemical Co., Munich, Germany; Immobilon polyvinylidene difluoride membrane was from Millipore, Tokyo, Japan; nickel-nitrilotriacetic acid resin was from Qiagen, Valencia, CA; BCA Protein Assay Reagent Kit was from Pierce, Rockford, IL. 293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin. All the cells were maintained in a humidified atmosphere at 37 °C under 5% CO2. Expression vectors encoding wild type Ig-Hepta and Ig-Hepta-ECD were described previously (15Abe J. Suzuki H. Notoya M. Yamamoto T. Hirose S. J. Biol. Chem. 1999; 274: 19957-19964Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). To generate the C-terminally myc-His6-tagged full-length Ig-Hepta constructs, the transmembrane sequence of Ig-Hepta without the stop codon was amplified by PCR using Pfu DNA polymerase with the primers IgH7TMF and IgH7TMR shown below, digested byApaI restriction enzyme, and ligated into theEcoRV and ApaI sites of pSecTag-Ig-Hepta-ECD, in-frame at the 3′-end of the extracellular domain (amino acid residues 25–1016) of Ig-Hepta. DNA sequencing was used to verify the sequence. The expression vector encoding an Ig-Hepta-GFP fusion protein (pEGFP-N3-Ig-Hepta) was generated by ligating a 4.0-kbSpeI/KpnI fragment of pcDNA3-Ig-Hepta-myc comprising the full-length Ig-Hepta into the NheI/KpnI site of pEGFP-N3. To generate expression vector for the soluble rat Ig-Hepta-human immunoglobulin Fc chimera (sIg-Hepta-Fc), the Fc region of human IgG1 were amplified from human spleen cDNA with the primers hIg-G1FcF (EcoRV) and hIg-G1FcR (ApaI) shown below, and cloned into pZErO-2 cloning vector and verified by sequencing. A cDNA cassette encoding the Fc region of human IgG1 was inserted between the EcoRV and ApaI sites of pSecTag-Ig-Hepta-ECD, in-frame at the 3′-end of the extracellular domain (residues 25–1016) of Ig-Hepta. The primers used for constructing the expression vectors were as follows: IgH7TMF (EcoRV), 5′-ATCATTTCTTACATCGGGTT-3′; IgH7TMR (ApaI), 5′-GCGCGGGCCCGTTGAGCAATGAGTAAGCAC-3′; hIg-G1FcF (EcoRV), 5′-ATCAAATCTTGTGACAAAACTCA-3′; and hIg-G1FcR (ApaI), 5′-GGGCCCTCATTTACCCGGAGACAGGG-3′. Transient transfections were performed with the LipofectAMINE Plus system according to the manufacturer's protocol. In brief, 293T cells at 80% confluence in 35-mm dishes were transfected with 1 μg of DNA, 6 μl of Plus reagent, and 4 μl of LipofectAMINE in Opti-MEM medium for 3 h. Cells were harvested 48 h after transfection. For pulse-chase analysis, transiently transfected 293T cells were starved in cysteine/methionine-free DMEM with dialyzed 5% FBS for 30 min, labeled for 15 min in the same culture medium supplemented with 0.2 mCi/ml [35S]cysteine/methionine. After the pulse, the radiolabeling medium was removed, the cell surface was washed three times with 5 ml of DMEM, and cells were chased with DMEM supplemented with 5 mm cysteine/methionine and 5% FBS. Cells were washed three times with ice-cold phosphate-buffered saline (PBS) and lysed in radioimmune precipitation buffer (0.1% SDS, 1% Nonidet P-40, 1% sodium deoxycholate, 150 mm NaCl, 10 mmsodium phosphate, pH 7.0) with Complete protease inhibitor mixture. The cell lysates were centrifuged at 15,000 rpm for 15 min to remove cellular debris and precleared with protein G-Sepharose beads. Proteins were immunoprecipitated by incubation with antibodies on ice for 2 h and then with protein G-Sepharose beads for another 2 h at 4 °C with rotation. Immunoprecipitates were washed five times with radioimmune precipitation buffer, boiled for 5 min at 95 °C in Laemmli sample buffer, and analyzed by SDS-PAGE and autoradiography. 293T cells transiently transfected with Ig-Hepta cDNA were pretreated with BFA (5 μg/ml) for 1 h and metabolically labeled for 15 min with [35S]methionine/cysteine and chased for 1 h with cold media in the continued presence of BFA. Cells were then washed with BFA-free medium three times and chased for additional 6 h. The cells were lysed at each time point and immunoprecipitated with a mixture of anti-NIg-Hepta and anti-CIg-Heptapolyclonal antibody. Immune complexes were analyzed by SDS-PAGE in the presence of 2-mercaptoethanol, and the radioactive bands were visualized. Membrane proteins from 293T cells transiently transfected with Ig-Hepta were solubilized with 0.5% SDS (w/v), and the extracts (20 μg of protein) were boiled for 3 min in 20 μl of 0.1 m 2-mercaptoethanol, and 50 mm sodium phosphate buffer, pH 7.2. After cooling to room temperature, Nonidet P-40 was added to give a final concentration of 1%. To the extracts, 2 milliunits of PNGase F (EC 3.5.1.52, Roche Molecular Biochemicals) were added, and the reaction mixture was incubated at 37 °C for 3 h. A control incubation was carried out in which 50 mm sodium phosphate buffer was added in place of the enzyme. The proteins were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and detected by immunoblotting as described above. Following immunoprecipitation, samples were washed with radioimmune precipitation buffer without proteinase inhibitors for three times and eluted from the beads by incubation in 30 μl of denaturing buffer (0.5% SDS, 1% 2-mercaptoethanol). Treatment with PNGase F was performed for 3 h at 37 °C as described above. Samples were electrophoresed on polyacrylamide. For antibody production, DNA fragments encoding the C-terminal cytosolic domain of Ig-Hepta (residues 1268–1349) and the N-terminal extracellular domain of Ig-Hepta (residues 571–1016) were amplified by PCR and cloned into the vector pRSET, and the constructs were transformed into Escherichia coli BL21(DE3)pLysS and used for fusion protein production. Fusion proteins were insoluble and purified under denaturing conditions on nickel-nitrilotriacetic acid resin columns. Rabbit polyclonal antibodies using the recombinant proteins were produced as previously described (15Abe J. Suzuki H. Notoya M. Yamamoto T. Hirose S. J. Biol. Chem. 1999; 274: 19957-19964Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). To establish cell lines that express sIg-Hepta-Fc, the expression vector encoding sIg-Hepta-Fc was linearized byScaI restriction enzyme and transfected into exponentially growing 293T cells using the LipofectAMINE Plus method. Selection of stable transfectants was carried out by adding 500 μg/ml Zeocin into the medium. After 3 weeks, Zeocin-resistant cells were cloned by limiting dilution and expanded to obtain stable cell lines. Expression of sIg-Hepta-Fc was determined by Western blotting of cell supernatants. Stably transfected cells were grown for 7 days in serum-free Opti-MEM medium, and the supernatants were harvested, centrifuged (10,000 × g, 1 h), filtered through a 0.22-μm filter membrane to remove cell debris and membranes, and loaded as 40-ml aliquots onto 1-ml columns of protein G-Sepharose equilibrated in PBS. The columns were washed with 10 ml of PBS and eluted with 2.5 ml of 50 mm citric acid. The eluates were neutralized with 1 m Tris base and concentrated using Centriprep-10 concentrators. Protein concentration was determined by the bicinchoninic acid (BCA) method using bovine serum albumin as the standard, and the purity of the samples was assessed by SDS-PAGE and Coomassie Blue staining. For N-terminal sequencing, 5 μg of purified sIg-Hepta-Fc was electrophoresed on 10% SDS-polyacrylamide gel and electrotransferred onto polyvinylidene difluoride membranes. The protein band of interest was excised from the membrane, and N-terminal sequence was determined by automated Edman degradation using a PPSQ-21 sequencer (Shimadzu, Kyoto, Japan). A single Thr (ACG) to Ala (GCG) mutation at proteolytic cleavage site (P1′, residue 994) was introduced into pEGFP-N3-Ig-Hepta by PCR-based mutagenesis using mutated synthetic oligonucleotides. Briefly, a DNA fragment was amplified using the forward primer T994A-Xho-F shown below and the antisense mutant primer T994A-Nru-R, and an overlapping fragment was amplified using the sense mutant primer T994A-Nru-F and the downstream reverse primer T994A-Apa-R. Both fragments were gel-purified, cut withNruI, ligated, and re-amplified with the Pfu DNA polymerase with the T994A-Xho-F and T994A-Apa-R primers. The product was digested using Aor51HI-Bpu1102I restriction enzymes, gel-purified, and subcloned into pEGFP-N3-Ig-Hepta to replace the target segment. DNA sequencing was used to verify the sequence. The following primers were used for constructing the above mentioned point mutations (the underlined sequences indicate the mutated bases): T994A-Xho-F 5′-gcgcctcgcgagcttctccattctcatgtcc-3′; T994A-Nru-R 5′-gcgcgctcgcgaggtggttacacttgcagaa-3′; T994A-Nru-F 5′-gcgcctcgcgagcttctccattctcatgtcc-3′; T994A-Apa-R 5′-GCGCGGGCCCGTGCTGTCTCCCAGCTCGAG-3′. Cell extract was equilibrated with Laemmli sample buffer and kept at room temperature for 5 min. Ten μg were subjected to SDS-PAGE analysis at 4 °C, and fluorescent bands were detected by exposing the gel to Bioimage Analyzer FLA2000 (Fujifilm, Tokyo, Japan) at 473-nm excitation and 510-nm emission. While characterizing antisera, anti-NIg-Hepta and anti-CIg-Hepta, raised against the N- and C-terminal domains of rat Ig-Hepta, respectively, we noticed that they recognize distinct bands on Western blot analysis of Ig-Hepta (Fig.1 A). When extracts of 293T cells transiently expressing Ig-Hepta were analyzed, anti- NIg-Hepta antiserum reacted with broad bands of 30–160 kDa and their dimers (lanes 1 and 2), whereas anti-CIg-Hepta antiserum detected a band of 32-kDa and its dimer (lanes 3 and 4). In both cases, the dimer bands disappeared upon reduction (data not shown). The broad bands of 130–160 kDa can be explained by glycosylation; treatment with glycosidase PNGase F, which cleaves both high mannose and complexN-linked oligosaccharides, reduced their sizes to ∼95 kDa (1B, lane 3). These results suggest that Ig-Hepta is proteolytically processed into a mature two-chain form composed of the fully glycosylated 150-kDa N-terminal fragment and the 32-kDa C-terminal fragment. To see whether this proteolytic processing is a general property of Ig-Hepta, we next performed Western blot analysis using whole detergent extracts of the rat lung. Although anti-NIg-Hepta detected 130- to 160-kDa bands corresponding to the N-terminal fragment, anti-CIg-Hepta yielded only a faint band of 32 kDa that was difficult to distinguish from nonspecific staining (data not shown). We therefore enriched the C-terminal fragment by immunoprecipitation and subjected it to Western blot analysis (Fig. 1 C), which demonstrated the presence of the processed C-terminal fragment of 32 kDa in the lung (lane 5) as well as in 293T cells (lane 4). To determine the time course of the post-translational modification of Ig-Hepta, we performed pulse-chase experiments. 293T cells expressing rat Ig-Hepta were pulse-labeled with [35S]methionine/cysteine for 15 min and chased for 0–120 min (Fig. 2). Immunoprecipitation of labeled products with either N- or C-terminal domain-directed antiserum revealed that rat Ig-Hepta is synthesized as a 170-kDa precursor and is rapidly cleaved into two chains: a 130-kDa N-terminal fragment (Fig.2 A, upper panel, arrowhead) and a 32-kDa C-terminal fragment (Fig. 2 A, lower panel). The size of the C-terminal fragment remained unchanged during the 2-h chase time. However, the size of the N-terminal fragment increased from 130 to 150 kDa after 1 h of chase (Fig.2 A, arrow) because of modification of oligosaccharide chains as demonstrated by deglycosylation of the pulse-chased products (Fig. 2 B). Deglycosylation experiments also showed that the initial glycosylation (attachment of a commonN-linked oligosaccharides or core-glycosylation) occurs almost cotranslationally as seen by a marked reduction (∼20 kDa) in size of the 170-kDa band as well as the band of 130 kDa (Fig. 2,A and B). The time courses of processing indicate that the cleavage occurs in the endoplasmic reticulum (ER) before the late steps in the processing of oligosaccharide chains is completed in the Golgi complex. To confirm this speculation, we performed the following experiment using BFA, an inhibitor that prevents the ER-to-Golgi vesicular trafficking. The identity of an ∼150-kDa band maximally seen at 20 min of chase (Fig. 2 A, double arrowhead on the left) will be addressed later. 293T cells were transiently transfected with an Ig-Hepta expression vector, preincubated for 1 h with BFA to achieve complete inhibition, pulse labeled for 15 min in the presence of BFA, and chased for 1 h in a medium containing BFA. BFA was then removed, the chase was further continued for 6 h, and cell lysates were assayed for the molecular species of Ig-Hepta accumulated during the chase by immunoprecipitation, SDS-PAGE, and autoradiography. As shown in Fig.3, Ig-Hepta that accumulated in ER in the presence of BFA was the proteolytically processed form consisting of the 130-kDa N-terminal fragment (Fig. 3, lanes 2–4, upper panel) and 32-kDa C-terminal fragment (Fig. 3, lower panel). However, the possibility cannot be excluded that the cleavage could be occurring due to the presence of enzymes normally found in the cis-Golgi, because some of the enzymes normally found in the early part of the Golgi are known to be present in ER through retrograde transport. On removal of BFA, the 130-kDa N-terminal extracellular domain was gradually converted to the fully glycosylated higher molecular weight species (lanes 5 and 6), suggesting that the complete maturation of the sugar chains occurs at a later stage in the Golgi. From the sizes of the cleaved fragments, the site of cleavage is predicted to be located in a juxtamembrane region of the N-terminal extracellular domain. The degree of cleavage was monitored by constructing a fluorescent derivative of Ig-Hepta termed Ig-Hepta-GFP that has a GFP tag at its C terminus. Because GFP is stable, if not heated, in the Laemmli sample buffer for SDS-PAGE, this construct allowed us to detect the GFP-tagged C-terminal fragment by using a fluorescence gel scanner (Fig.4). A single band of ∼50 kDa was seen when extracts of 293T cells were transiently transfected with the Ig-Hepta-GFP construct (lanes 2 and 5,WT-GFP). The absence of higher molecular weight unprocessed species indicates that the processing occurs highly efficiently. Despite the efficient cleavage of the N-terminal extracellular domain of Ig-Hepta during its post-translational modification, the cleaved extracellular domain was not released into the culture medium (Fig. 5 A,lane 1) and was rather recovered from membrane fractions (lane 3). As expected, when a truncated form covering only the extracellular domain of Ig-Hepta (Ig-Hepta-ECD) was expressed in the same expression system, it was secreted and recovered from the culture medium (Fig. 5 A, lane 2). These pieces of experimental evidence suggest that the N-terminal fragment is tightly associated with certain membrane component(s), most likely with its C-terminal fragment. To explore this possibility, we prepared Ig-Hepta that has an myc tag at its C terminus (Ig-Hepta-myc) and performed immunoprecipitation analysis using a commercially available anti-myc monoclonal antibody. The anti-myc antibody precipitated the myc-tagged C-terminal domain together with the N-terminal fragment (Fig.5 B). These results clearly demonstrate non-covalent association of the cleaved N- and C-terminal fragments. In an attempt to determine the proteolytic cleavage site of Ig-Hepta, we first tried immunoaffinity purification of the 32-kDa C-terminal fragment. Such studies, however, have been hampered because of its membrane-bound nature, and hence, difficulties result in obtaining sufficient amounts for sequencing. As an alternative approach, we constructed a chimeric protein that contains a candidate cleavage site sequence and is easy to purify. The chimera was composed of the N-terminal extracellular domain of rat Ig-Hepta and the Fc domain of human IgG1 and named sIg-Hepta-Fc (s for soluble). When the chimeric construct was expressed in 293T cells, similar proteolytic processing occurred, yielding a 38-kDa fragment corresponding to the C-terminal Fc portion (Fig.6 A). The C-terminal fragment was purified from the culture medium by affinity chromatography on protein G-Sepharose and found to have the following N-terminal sequence by amino acid sequencing: TSFSILMSPD (Fig.6 B, upper sequence). This result strongly indicates that the cleavage site is Leu993-Thr994, which is located in a juxtamembrane region of the N-terminal extracellular domain (23 amino acid residues N-terminal to the first transmembrane span; Figs.6 B and 7).Figure 7Protein sequence alignment of the juxtamembrane region of LNB-TM7 family members. Amino acid sequences obtained from the NCBI data base for LNB-TM7 members were aligned with the ClustalW program followed by manual adjustment. Thelower two lines of sequences represent similar juxtamembrane sequences found in other family members of transmembrane proteins. The conserved juxtamembrane cysteine residues (Cys-box) areboxed, and the proteolytic cleavage site is indicated by anarrow. TM1, the" @default.
• W1987157213 created "2016-06-24" @default.
• W1987157213 creator A5014981700 @default.
• W1987157213 creator A5047378769 @default.
• W1987157213 creator A5069739406 @default.
• W1987157213 date "2002-06-01" @default.
• W1987157213 modified "2023-09-30" @default.
• W1987157213 title "Cleavage of Ig-Hepta at a “SEA” Module and at a Conserved G Protein-coupled Receptor Proteolytic Site" @default.
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