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• W2068903566 abstract "A novel member of the G protein-coupled receptor (GPCR) family was cloned and characterized, which is unique, among the members, in its long extracellular domain comprising Ig-like repeats and in its high expression predominantly in the lung. The clone (Ig-Hepta) was first identified as a polymerase chain reaction product generated with primers designed to amplify secretin receptor family members including the parathyroid hormone-related peptide receptors. Analysis of the open reading frame of cDNAs isolated from a rat lung cDNA library indicated that Ig-Hepta is a protein of 1389 amino acid residues and has two Ig-like repeats in the N-terminal extracellular domain (exodomain) of 1053 amino acid residues and 7 transmembrane spans in the C-terminal region. Northern blot analysis revealed very high expression of its mRNA in the lung and low but detectable levels in the kidney and heart. The mRNA expression in the lung was found to be strongly induced postnatally. Biochemical analysis indicated that Ig-Hepta is a highly glycosylated protein and exists as a disulfide-linked dimer. Immunohistochemistry on rat lung and kidney sections revealed dense localization of Ig-Hepta in alveolar walls and intercalated cells in the collecting duct, respectively, suggesting a role in the regulation of acid-base balance. Ig-Hepta defines a new subfamily of GPCRs. A novel member of the G protein-coupled receptor (GPCR) family was cloned and characterized, which is unique, among the members, in its long extracellular domain comprising Ig-like repeats and in its high expression predominantly in the lung. The clone (Ig-Hepta) was first identified as a polymerase chain reaction product generated with primers designed to amplify secretin receptor family members including the parathyroid hormone-related peptide receptors. Analysis of the open reading frame of cDNAs isolated from a rat lung cDNA library indicated that Ig-Hepta is a protein of 1389 amino acid residues and has two Ig-like repeats in the N-terminal extracellular domain (exodomain) of 1053 amino acid residues and 7 transmembrane spans in the C-terminal region. Northern blot analysis revealed very high expression of its mRNA in the lung and low but detectable levels in the kidney and heart. The mRNA expression in the lung was found to be strongly induced postnatally. Biochemical analysis indicated that Ig-Hepta is a highly glycosylated protein and exists as a disulfide-linked dimer. Immunohistochemistry on rat lung and kidney sections revealed dense localization of Ig-Hepta in alveolar walls and intercalated cells in the collecting duct, respectively, suggesting a role in the regulation of acid-base balance. Ig-Hepta defines a new subfamily of GPCRs. G protein-coupled receptor brain-specific angiogenesis inhibitor cadherin EGF LAG seven-pass G-type receptor epidermal growth factor module-containing mucin-like receptor 1 epidermal growth factor polymerase chain reaction parathyroid hormone parathyroid hormone-related peptide polyacrylamide gel electrophoresis vasoactive intestinal polypeptide phosphate-buffered saline kilobase pair Since the cloning of rhodopsin (1Nathans J. Hogness D.S. Cell. 1983; 34: 807-814Abstract Full Text PDF PubMed Scopus (482) Google Scholar) and β-adrenergic receptor (2Dixon R.A. Kobilka B.K. Strader D.J. Benovic J.L. Dohlman H.G. Frielle T. Bolanowski M.A. Bennett C.D. Rands E. Diehl R.E. Munford R.A. Slater E.E. Sigal I.S. Caron M.G. Lefkowitz R.J. Strader C.D. Nature. 1986; 321: 75-79Crossref PubMed Scopus (866) Google Scholar), more than 1000 G protein-coupled receptors (GPCRs)1 have been cloned and characterized (3Ji T.H. Grossmann M. Ji I. J. Biol. Chem. 1998; 273: 17299-17302Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar, 4Gether U. Kobilka B.K. J. Biol. Chem. 1998; 273: 17979-17982Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar, 5Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar). They constitute one of the largest family of proteins, which have in common seven transmembrane domains, and are involved in broad spectrum of biological processes by mediating the signal of a wide variety of stimuli such as hormones, neurotransmitters, cytokines, light, and odorants. GPCRs can be grouped into various subfamilies based on their amino acid sequences. Recently a subfamily has emerged that shares the seven-transmembrane topology but has a low overall amino acid sequence similarity with other members of the GPCR superfamily. This subfamily, now referred to as the class II GPCR family (Figs. 2 and 3), comprises receptors for secretin, glucagon, VIP, calcitonin, PTH, PTHrP, glucagon-like peptide 1, gastric inhibitory polypeptide, growth hormone-releasing hormone, corticotropin-releasing factor, pituitary adenylate cyclase-activating peptide, and an insect diuretic hormone and is therefore also called the secretin receptor family or the glucagon/VIP/calcitonin receptor family (for review see Refs. 6Segre G.V. Goldring S.R. Trends Endocrinol. Metab. 1993; 4: 309-314Abstract Full Text PDF PubMed Scopus (303) Google Scholar, 7Laburthe M. Couvineau A. Gaudin P. Maoret J.J. Rouyer-Fessard C. Nicole P. Ann. N. Y. Acad. Sci. 1996; 805: 94-109Crossref PubMed Scopus (123) Google Scholar, 8Ulrich C.D. Holtmann M. Miller L.J. Gastroenterology. 1998; 114: 382-397Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). The class II receptors are characterized not only by the lack of the structural signature sequences present in the class I rhodopsin/β-adrenergic receptor family but also by the presence of a large N-terminal extracellular domain (exodomain).Figure 3Phylogenetic relationship of Ig-Hepta and other members of the class II GPCR family. Based on alignment of 14 members of the secretin receptor family shown in Fig. 2, a phylogenetic tree was constructed using the neighbor-joining method. The abbreviations are as defined in Fig. 2 legend. The most recent addition to this family of receptors, methuselah (52Lin Y.J. Seroude L. Benzer S. Science. 1998; 282: 943-946Crossref PubMed Scopus (680) Google Scholar), is also included.View Large Image Figure ViewerDownload (PPT) Recently Baud et al. (9Baud 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), Hamann et al. (10Hamann 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), and McKnight et al. (11McKnight 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 (188) Google Scholar) have identified a novel subtype of the class II receptors through the structural analyses of a cDNA clone (EMR1) of neuroectodermal origin, of a leukocyte activation antigen (CD97), and of a macrophage-restricted cell-surface glycoprotein (F4/80), respectively, that has an extraordinarily long N-terminal domain containing EGF-like repeats (for review see Refs. 12McKnight A.J. Gordon S. Immunol. Today. 1996; 17: 283-287Abstract Full Text PDF PubMed Scopus (73) Google Scholar and 13McKnight A.J. Gordon S. J. Leukocyte Biol. 1998; 63: 271-280Crossref PubMed Scopus (122) Google Scholar). In addition, Hadjantonakis et al. (14Hadjantonakis 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, 15Hadjantonakis A.K. Formstone C.J. Little P.F.R. Mech. Dev. 1998; 78: 91-95Crossref PubMed Scopus (52) Google Scholar) have reported the presence of a developmentally regulated gene, Celsr1, that encodes an orphan receptor of the class II GPCR family with an extended exodomain containing a block of cadherin repeats. Although the exact physiological functions are unknown, these receptors are gaining a great deal of attention because of their unusual structural properties; they are composed of two well characterized protein motifs usually seen in distinct protein superfamilies as follows: one is the motifs (EGF and cadherin repeats) found in many cell-surface molecules with a single membrane-spanning domain, and the other is the transmembrane bundle that is composed of seven helices. Here we report another unusual member of the class II GPCR family whose hepta-helical transmembrane region is similar to those of the secretin receptor family, but its large exodomain (1053 amino acid residues) is unique in having immunoglobulin-like repeats, a motif characteristic of the members of the immunoglobulin superfamily of cell-surface proteins (16Williams A.F. Barclay A.N. Annu. Rev. Immunol. 1988; 6: 381-405Crossref PubMed Scopus (1791) Google Scholar,17Smith D.K. Xue H. J. Mol. Biol. 1997; 274: 530-545Crossref PubMed Scopus (88) Google Scholar). Based on this chimeric structural feature, we term the protein “Ig-Hepta.” Its localization and regulation of expression are also very unique; Ig-Hepta is predominantly expressed in the lung, and its expression was found to be markedly induced postnatally. Immunostaining of the lung and kidney, which exhibited, by Northern analysis, strongly and weakly positive signals, respectively, revealed specific staining in the alveolar walls of the lung and intercalated cells in the collecting duct of the kidney, suggesting that Ig-Hepta may be involved in pH-sensing or pH regulation. Wistar rats were obtained from Tokyo Laboratory Animals Science, Tokyo, Japan; restriction enzymes were from Takara, Kyoto, Japan; Expand Long Template PCR System was from Roche Molecular Biochemicals, Mannheim, Germany; pBluescript II SK−,Escherichia coli strain XL1-Blue MRF′, λZAP II, Gigapack II Gold in vitro packaging kits were from Stratagene, La Jolla, CA; mRNA purification kits and Ready-To-Go DNA labeling kit were from Amersham Pharmacia Biotech, Uppsala, Sweden; pcDNA3, pTrcHis B, and pSecTag were from Invitrogen, San Diego, CA; SequiTherm Long-read-LC cycle sequencing kit was from Epicentre Technologies, Madison, WI; [α-32P]dCTP was from Amersham Pharmacia Biotech, Buckinghamshire, UK; Superscript II reverse transcriptase and Dulbecco's modified Eagle's medium were from Life Technologies, Inc.; alkaline phosphatase-conjugated mouse anti-rabbit IgG antibody was from Sigma, Munich, Germany; immobilon polyvinylidene difluoride membrane was from Millipore, Tokyo, Japan; Protran BA85 nitrocellulose membranes were from Schleicher & Schuell, Dassel, Germany; nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate were from Wako Pure Chemicals, Osaka, Japan; BCA Protein Assay Reagent Kit was from Pierce. Based on the amino acid sequence alignment of the rat secretin receptor family, two degenerate oligonucleotides corresponding to conserved elements found in the transmembrane helices III and VII were synthesized. These oligonucleotides were used as primers in polymerase chain reaction (PCR) amplifications with a single-stranded cDNA template derived from rat hypothalamus poly(A)+ RNA. The sequence of the sense primer was 5′-AAYTAYTAYTGGATICTGGTGGARGG-3′ (where I is inosine; Y is C, T; R is A, G), and the sequence of the antisense primer was 5′-TGIACCTCICCRTTGCAGWARCARTA-3′ (I is inosine; R is A, G; W is A, T). The reactions were performed in a MJ Research thermal cycler for 35 cycles of denaturation (94 °C, 1 min), annealing (45 °C, 1 min), and extension (68 °C, 2 min) with an additional 10-min primer extension after the final cycle using Expand Long Template PCR System (Roche Molecular Biochemicals). The PCR products of expected size (about 500 base pairs) were isolated by agarose gel electrophoresis, purified, and then subcloned into the EcoRV site of pBluescript II SK− (Stratagene) and sequenced. Sequencing of about 350 individual clones led to the identification of a putative GPCR fragment whose predicted sequence was homologous to, but distinct from, corresponding sequences of all known members of the secretin receptor family. Total RNA was isolated from the lungs of 6-week-old Wistar rats by the guanidinium thiocyanate/cesium chloride method. Poly(A)+ RNA was affinity purified using an oligo(dT)-cellulose mRNA purification kit (Amersham Pharmacia Biotech) and was used as a template for first strand cDNA synthesis with Superscript II reverse transcriptase and random hexamer primers (Life Technologies, Inc.). Following second strand cDNA synthesis and EcoRI adapter ligation, double-stranded cDNA was ligated into the dephosphorylated arms of EcoRI-digested λZAP II (Stratagene). The ligated DNA was packaged into phage particles using the Gigapack II Gold packaging extract (Stratagene). Approximately 3 × 105 plaque-forming units of the library were plated on XL1-Blue MRF′ E. coli (Stratagene) to a density of approximately 3 × 104 plaques/10 × 14-cm agar plate and replicated on Protran BA85 nitrocellulose membranes (Schleicher & Schuell). Filters were prehybridized for 2 h at 42 °C in 5 × SSPE (SSPE: 0.15 m NaCl, 1 mm EDTA, and 10 mmNaH2PO4, pH 7.4), 50% formamide, 5× Denhardt's solution (0.1% each of Ficoll, polyvinylpyrrolidone, and bovine serum albumin), 0.1% SDS. The above PCR-derived rat Ig-Hepta cDNA fragment was labeled with [α-32P]dCTP (3000 Ci/mmol, Amersham Pharmacia Biotech) using a Ready-To-Go DNA labeling kit (Amersham Pharmacia Biotech), and the unincorporated nucleotides were removed by passage through a Sephadex G-50 column (Amersham Pharmacia Biotech). Hybridization was performed for 16 h at 42 °C in prehybridization solution by adding radiolabeled cDNA probe at about 106 cpm/ml. After hybridization, the filters were rinsed twice with 2× SSC (1× SSC: 0.15 m NaCl, 15 mm sodium citrate, pH 7.0), 0.1% SDS for 15 min at room temperature, washed in 1× SSC, 0.1% SDS for 30 min at 55 °C, and then in 0.5× SSC, 0.1% SDS for 30 min at 55 °C and exposed to Kodak X-Omat AR film for 48 h at −80 °C with an intensifying screen. Thirty-two positive clones were identified, and the clones were purified in three rounds under the same conditions as the primary screening and transformed into pBluescript II SK− byin vivo excision using ExAssist helper phage and XL1-Blue MRF′ E. coli. Nucleotide sequences were determined on both strands by the dideoxy chain termination method with an automated sequencer (LI-COR model 4000L) using a SequiTherm Long-read Cycle Sequencing Kit-LC (Epicentre Technologies). The DNA sequences were compiled and analyzed using the Genetyx-MAC computer program (Software Development). Data base searches were performed using the BLAST program of National Center for Biotechnology Information. Multiple protein sequence alignments were carried out using the program ClustalW, and final adjustments were made manually. Primary sequence motifs were identified using the PROSITE data base of ExPASy. Total RNA was isolated from various tissues of 6-week-old male Wistar rats and lungs of 3-day-old and 13-week-old Wistar rats by the acid guanidinium thiocyanate/phenol/chloroform method. For Northern analysis, 20 μg of total RNA was electrophoresed in 1.0% agarose gel containing formaldehyde and transferred to Magna MT nylon membranes (Micron Separations) by capillary blotting overnight using 10× SSC as the transfer buffer. After transfer, membranes were baked for 2 h at 80 °C and covalently immobilized on the membrane by exposure to UV light using a UV Stratalinker 2400 (Stratagene) and probed with the same 32P-labeled cDNA probe as that used for the cDNA library screening under moderate and high stringency conditions. Briefly, the membranes were prehybridized for 1 h in prehybridization buffer consisting of 50% formamide, 5× SSPE, 2× Denhardt's solution, and 0.5% SDS and hybridized at 42 (high stringency) or 37 °C (moderate stringency) for 16 h. After hybridization, the membranes were washed twice with 2× SSC and 0.1% SDS for 15 min at room temperature followed by two washes of 30 min in 0.1× SSC and 0.1% SDS at 65 °C (high stringency) or in 0.5× SSC and 0.1% SDS at 60 °C (moderate stringency) and exposed to imaging plates (Fuji Film) in a cassette for 5 h. The results were analyzed using a Fujix BAS2000 Bio-imaging analyzer (Fuji Film). A 1330-base pair fragment that encodes amino acid residues 232–675 of Ig-Hepta was subcloned into the pTrcHis B (Invitrogen), and the construct was transformed into E. coli XL1-Blue. When the A 600 reached 0.8, isopropyl-β-d-thiogalactopyranoside was added to a final concentration of 1.5 mm, and incubation was continued for an additional 6 h at 37 °C. The cells were harvested by centrifugation and resuspended in 10 mm Tris-HCl, pH 7.5, 0.1% (v/v) Triton X-100, disrupted by sonication, and centrifuged at 10,000 × g for 20 min. The pellet was solubilized by resuspending and mixing in 8 m urea, 0.1 mNaH2PO4, 0.01 m Tris-HCl, pH 8.0, for 1 h at room temperature. The mixtures were then centrifuged at 10,000 × g for 30 min to remove any insoluble material. Urea-solubilized recombinant His6-Ig-Hepta protein was purified in a denatured state using a Ni2+-nitrilotriacetic acid agarose column (Qiagen) and dialyzed against phosphate-buffered saline (PBS: 20 mmphosphate, pH 7.4, containing 140 mm NaCl). Protein concentration was determined with a BCA Protein Assay Reagent kit (Pierce). Polyclonal antibodies to Ig-Hepta were prepared in Japanese White rabbits by immunizing subcutaneously with 200 μg of purified recombinant His6-Ig-Hepta protein emulsified in complete Freund's adjuvant (1:1) at multiple sites. Rabbits were boosted three times, each time with 100 μg of the purified protein in incomplete Freund's adjuvant (1:1) at 2-week intervals. The rabbits were bled 10 days after the fourth immunization. Full-length Ig-Hepta cDNA was ligated into theNotI/ApaI sites of pcDNA3 (Invitrogen). COS-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere containing 5% CO2. COS-7 cells grown to 50–80% confluency in 100-mm dishes were collected in K-PBS (30.8 mm NaCl, 120.8 mm KCl, 8.1 mmNa2HPO4, 1.46 mmKH2PO4, and 5 mm MgCl2) and transiently transfected with 30 μg of pcDNA3 containing the cDNA for Ig-Hepta by electroporation at 220 V and 960 microfarads using a Genepulser (Bio-Rad). The same procedure was carried out for control cells transfected only with pcDNA3. A C-terminally truncated form, Ig-Hepta-TM1 (amino acid residues 1–1079), was similarly expressed. A soluble form of the exodomain, Ig-Hepta-ECD (residues 25–1056), was produced using the leader sequence in the pSecTag A expression vector designed for secreted proteins. At 72 h post-transfection, the COS-7 cells were rinsed in PBS to remove remaining serum, scraped from dishes, and homogenized on ice with a Dounce homogenizer in 1 ml of homogenization buffer (50 mm sodium phosphate, 25 mm NaCl, 10 mm EDTA, 2 mm phenylmethylsulfonyl fluoride, 20 μg/ml leupeptin, 10 μm pepstatin A, pH 7.5). The homogenate was centrifuged for 30 min at 10,000 × g,and the resulting pellet was extracted with 5 volumes of 1% Triton X-100 at 4 °C overnight. Insoluble material was removed by centrifugation at 10,000 × g for 60 min. Rat lungs were first cut into small pieces and disrupted using a Polytron tissue homogenizer with 5 volumes of ice-cold homogenization buffer and then further disrupted by 20 strokes in a Dounce homogenizer. Crude plasma membranes were isolated from the homogenate as described above. COS-7 cells were transfected with the Ig-Hepta-ECD/pSecTag A expression vector described above. After 5 days, culture medium was collected from 5 dishes (10-cm diameter) and dialyzed three times against 2 liters of 50 mmNaH2PO4, pH 8.0, containing 300 mmNaCl for 12 h, and the Ig-Hepta-ECD was purified by nickel chelate chromatography. Briefly, a column with 2-ml of bed volume was washed with 15 ml of wash buffer (50 mmNaH2PO4, 300 mm NaCl, 20 mm imidazole, pH 8.0), loaded with 50 ml of the dialyzed culture medium containing 20 mm imidazole, washed with 30 ml of wash buffer. Bound protein was eluted with 500 μl of elution buffer (50 mm NaH2PO4, 300 mm NaCl, 250 mm imidazole, pH 8.0). Unsecreted Ig-Hepta-ECD was recovered as Triton extracts from membrane preparations as described above. The samples (30 μg of protein/lane) were dissolved in Laemmli buffer (50 mm Tris containing 2% SDS, 10% glycerol, and 0.1% bromphenol blue, pH 6.8) in the presence or absence of 1% β-mercaptoethanol, heated at 95 °C for 5 min, electrophoresed through 7.5% SDS-polyacrylamide gel. Proteins were then transferred electrophoretically to Immobilon polyvinylidene difluoride membrane (Millipore) with a semi-dry blotting apparatus (Atto) using Bjerrum and Schafer-Nielsen buffer (48 mmTris, 39 mm glycine, 20% methanol, 0.1% SDS) for 75 min at ∼1.2 mA/cm2. The membrane was blocked with 5% nonfat dry milk, 0.05% Tween 20, in Tris-buffered saline (TBS, 150 mm NaCl, 10 mm Tris-HCl, pH 7.6) (T-TBS) for 1 h at room temperature. Membrane was washed 3 times for 10 min each with T-TBS and subsequently treated for 1 h at 25 °C with the primary antibody (anti-Ig-Hepta diluted 1:1000) in T-TBS. After washing the blots 3 times for 10 min each with T-TBS, the membrane was incubated for 1 h at 25 °C with an alkaline phosphatase-conjugated mouse anti-rabbit IgG antibody (Sigma) diluted 1:5000 in T-TBS. The blots were washed 3 times for 10 min each with T-TBS. The membrane was then developed with 0.4 mm5-bromo-4-chloro-3-indolyl phosphate and 0.4 mm nitro blue tetrazolium chloride (Wako Pure Chemicals) in 0.1 mTris-HCl, pH 9.5, containing 50 mm MgCl2 and 150 mm NaCl. Rat lung membrane proteins were solubilized with 1% Triton X-100, and the extracts (20 μg of protein) were boiled for 3 min in 20 μl of 0.5% SDS (w/v), 0.1m β-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 glycopeptidase F (EC 3.5.1.52, Takara), 50 milliunits of neuraminidase (sialidase A) (EC 3.2.1.18, Roche Molecular Biochemicals), and 2 milliunits of O-glycosidase (EC3.2.1.97, Roche Molecular Biochemicals) were added, and the reaction mixture was incubated at 37 °C for 18 h. A control incubation was carried out in which 50 mm sodium phosphate buffer was added in place of the enzyme(s). The proteins were separated on SDS-PAGE and transferred to a polyvinylidene difluoride membrane and were detected by immunoblotting as described above. Pieces of rat lungs and kidneys were fixed in methyl-Carnoy fixative overnight and embedded in paraffin. Sections of the paraffin-embedded tissues were dewaxed in xylene, hydrated through graded ethanol, and incubated with the antibody, normal rabbit serum, or anti-rat H+-ATPase antibody (18Yamamoto T. Sasaki S. Fushimi K. Kawasaki K. Yaoita E. Oota K. Hirata Y. Marumo F. Kihara I. Exp. Nephrol. 1995; 3: 193-201PubMed Google Scholar) (1:1000 dilution) overnight. After washing in PBS, the sections were incubated with peroxidase- and goat anti-rabbit IgG-conjugated dextran polymer (EnVision, Dako Japan, Kyoto, Japan) for 1 h and colored with diaminobenzidine and hydroperoxide. The clone to be described was obtained as an unexpected by-product of the following attempt to isolate a receptor selective for PTHrP. The presence of such a PTHrP receptor has been suggested in the rat supraoptic nucleus of the hypothalamus (19Yamamoto S. Morimoto I. Yanagihara N. Zeki K. Fujihira T. Izumi F. Yamashita H. Eto S. Endocrinology. 1997; 138: 2066-2072Crossref PubMed Google Scholar, 20Yamamoto S. Morimoto I. Zeki K. Ueta Y. Yamashita H. Kannan H. Eto S. Endocrinology. 1998; 139: 383-388Crossref PubMed Google Scholar). We therefore isolated poly(A)+ RNA from the rat supraoptic nucleus, synthesized cDNA, and amplified PTH receptor-related sequences by PCR. Analysis of the PCR products generated by a combination of primers revealed a total of 183 clones possessing features of the class II GPCR family: 182 clones identical to the known sequences and a single clone with a novel sequence. The known species included the receptors for PTH/PTHrP (66 clones), PTH (56 clones), pituitary adenylate cyclase-activating peptide (26 clones), calcitonin (22 clones), VIP (3 clones), calcitonin gene-related peptide (3 clones), glucagon (3 clones), glucagon-like peptide 1 (2 clones), secretin (1 clone). We therefore decided to isolate full-length cDNA corresponding to the novel clone. A preliminary search, using the PCR product as a probe, for the tissues that contain relatively large amounts of the message indicated that the rat lung is such a tissue and is suitable for use as a source of cDNA library construction. A full-length cDNA clone was obtained by screening 3 × 105 plaques of a rat lung cDNA library with the above novel PCR product as a probe under stringent conditions. The nucleotide sequence of the clone (5 kb) has been deposited in the DDBJ/EMBL/GenBankTM data base (accession number AB019120), and the amino acid sequence deduced from its open reading frame is shown in Fig. 1 A together with the structural features (Fig. 1, B and C). The novel sequence codes for a protein of 1389 amino acid residues. Hydropathy and motif analyses indicated that the protein is a member of the class II GPCR family and is composed of two major domains as follows: a large N-terminal domain containing two immunoglobulin-like repeats and a seven-transmembrane domain. We therefore named the protein Ig-Hepta. At the N terminus, there is a typical signal peptide sequence, indicating that the large N-terminal region is located extracellularly. In addition to the two immunoglobulin-like repeats, the exodomain contains a Cys-rich domain of ∼50 amino acid residues (residues 107–160) and multiple potential N-linked andO-linked glycosylation sites (Fig. 1 C). The hepta-helical domain in the C-terminal region is distantly related to the secretin family members including the PTH receptor (Figs.2 and 3). Two cysteine residues that are located in exoloops 1 and 2 and are known to be highly conserved among GPCRs are also present in Ig-Hepta: Cys-1129 in exoloop 1 between transmembrane segments 2 and 3 and Cys-1205 in exoloop 2 connecting transmembrane segments 4 and 5. These conserved cysteine residues are expected to be disulfide-linked as has been generally considered for other members of the GPCR family based on the structural analyses of rhodopsin (21Kamik S.S. Khorana H.G. J. Biol. Chem. 1990; 265: 17520-17524PubMed Google Scholar) and the receptors for thyrotropin-releasing hormone (22Cook J.V. McGregor A. Lee T. Milligan G. Eidne K.A. Endocrinology. 1996; 137: 2851-2858Crossref PubMed Scopus (30) Google Scholar), thromboxane (23Chiang N. Kan W.M. Tai H.H. Arch. Biochem. Biophys. 1996; 334: 9-17Crossref PubMed Scopus (56) Google Scholar), and gonadotropin-releasing hormone (24Cook J.V. Eidne K.A. Endocrinology. 1997; 138: 2800-2806Crossref PubMed Scopus (95) Google Scholar). There are two consensus sequences for phosphorylation by protein kinase C as follows: TQK (residues 1169–1171) in cytoloop 2 and SRR (residues 1353–1355) in the cytoplasmic C-terminal tail. Another structural feature is the presence of a relatively long stretch of Ser/Thr-rich sequence at the C terminus; more than 40% of the C-terminal 66 residues are Ser or Thr (Fig. 1, A and C). Northern analysis of 10 rat tissues revealed a very restricted expression pattern of Ig-Hepta (Fig.4 A). Ig-Hepta mRNA was highly expressed, as a 7.8-kb transcript, in the lung and to a much lesser extent in the kidney and heart. The expression in the lung was so abundant as to be easily detectable using total RNA preparations. During the postnatal development of rat, expression of Ig-Hepta mRNA in the lung increased markedly (Fig. 4 B). Control hybridization of the same blot, used for the above determination of the developmental expression of Ig-Hepta mRNA, with a glyceraldehyde-3-phosphate dehydrogenase probe revealed no significant difference in the signal intensity (Fig. 4 B). A weak band at 4.8 kb in Fig. 4 A is likely to represent a homologous mRNA species since its relative intensity increased greatly under moderate stringency conditions (Fig. 4 B), and total Southern blot analysis of the rat genomic DNA indicated the presence of a closely related gene (data not shown). For characterization of biochemical properties of Ig-Hepta, we raised an antiserum and used it as an analytical reagent for detecting the Ig-Hepta molecule. The antiserum stained a single band of ∼160 kDa on Western blots of detergent extracts of rat lung membrane preparations, demonstrating its specificity (Fig.5 B, lane 1). Under nonreducing conditions, however, significant amounts of Ig-Hepta migrated as an ∼260-kDa species on SDS-PAGE (Fig. 5 C,lane 1). Similarly, recombinant Ig-Hepta expressed in COS-7 cells behaved as an ∼130- and ∼260-kDa species under reducing and nonreducing conditions, respectively (Fig. 5, B andC, lane 2). These results suggest that considerable amounts of Ig-Hepta exist as a dimer linked by disulfide bond(s). We further determined whether the large exodomain of Ig-Hepta has by itself the ability to form the disulfide-linked dimer or not. The exodomain (Fig. 5 A, Ig-Hepta ECD) was expressed as a His6-tagged soluble protein in COS-7 cells, purified from culture medium by metal chelate chromatography on a Ni2+ column, and analyzed by SDS-PAGE. The exodomain existed as an ∼125-kDa species even under nonreducing conditions (Fig. 5 C, lane 6). If we assume that the truncated exodomain has the tertiary structure identical to that of the exodomain of the wild type receptor, this result indicates that the exodomain lacks the ability to dimerize by forming" @default.
• W2068903566 created "2016-06-24" @default.
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• W2068903566 creator A5024136589 @default.
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• W2068903566 date "1999-07-01" @default.
• W2068903566 modified "2023-09-30" @default.
• W2068903566 title "Ig-Hepta, a Novel Member of the G Protein-coupled Hepta-helical Receptor (GPCR) Family That Has Immunoglobulin-like Repeats in a Long N-terminal Extracellular Domain and Defines a New Subfamily of GPCRs" @default.
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