Characterization of sorCS1, an Alternatively Spliced Receptor with Completely Different Cytoplasmic Domains That Mediate Different Trafficking in Cells
2003; Elsevier BV; Volume: 278; Issue: 9 Linguagem: Inglês
10.1074/jbc.m210851200
ISSN1083-351X
AutoresGuido Hermey, Sady J. Keat, Peder Madsen, Christian Jacobsen, Claus M. Petersen, Jørgen Gliemann,
Tópico(s)Chemical Synthesis and Analysis
ResumoWe previously isolated and sequenced murine sorCS1, a type 1 receptor containing a Vps10p-domain and a leucine-rich domain. We now show that human sorCS1 has three isoforms, sorCS1a–c, with completely different cytoplasmic tails and differential expression in tissues. The b tail shows high identity with that of murine sorCS1b, whereas the a and c tails have no reported counterparts. Like the Vps10p-domain receptor family members sortilin and sorLA, sorCS1 is synthesized as a proreceptor that is converted in late Golgi compartments by furin-mediated cleavage. Mature sorCS1 bound its own propeptide with low affinity but none of the ligands previously shown to interact with sortilin and sorLA. In transfected cells, about 10% of sorCS1a was expressed on the cell surface and proved capable of rapid endocytosis in complex with specific antibody, whereas sorCS1b presented a high cell surface expression but essentially no endocytosis, and sorCS1c was intermediate. This is an unusual example of an alternatively spliced single transmembrane receptor with completely different cytoplasmic domains that mediate different trafficking in cells. We previously isolated and sequenced murine sorCS1, a type 1 receptor containing a Vps10p-domain and a leucine-rich domain. We now show that human sorCS1 has three isoforms, sorCS1a–c, with completely different cytoplasmic tails and differential expression in tissues. The b tail shows high identity with that of murine sorCS1b, whereas the a and c tails have no reported counterparts. Like the Vps10p-domain receptor family members sortilin and sorLA, sorCS1 is synthesized as a proreceptor that is converted in late Golgi compartments by furin-mediated cleavage. Mature sorCS1 bound its own propeptide with low affinity but none of the ligands previously shown to interact with sortilin and sorLA. In transfected cells, about 10% of sorCS1a was expressed on the cell surface and proved capable of rapid endocytosis in complex with specific antibody, whereas sorCS1b presented a high cell surface expression but essentially no endocytosis, and sorCS1c was intermediate. This is an unusual example of an alternatively spliced single transmembrane receptor with completely different cytoplasmic domains that mediate different trafficking in cells. domain cytoplasmic domain Chinese hamster ovary chimeric receptor a-c endoglycosidase-H Golgi-localized, γ-adaptin ear containing ADP ribosylation factor-binding proteins glutathione S-transferase interleukin-2 receptor-α (Tac/CD 25) lumenal part of sorCS1 glycosidase-F receptor-associated protein SorCS1 is the first identified member of a subgroup of the mammalian Vps10p-domain (Vps10p-D)1 receptor family that comprises an N-terminal Vps10p-D (named after the yeast vacuolar protein sorting 10 protein), a leucine-rich domain, a single transmembrane domain, and a short cytoplasmic domain (cd) (1Hermey G. Riedel I.B. Hampe W. Schaller H.C. Hermans- Borgmeyer I. Biochem. Biophys. Res. Commun. 1999; 266: 347-351Crossref PubMed Scopus (65) Google Scholar). Two isoforms of sorCS1, with different cds arising from differential splicing, have been identified in the mouse (2Hermey G. Schaller H.C. Biochim. Biophys. Acta. 2000; 1491: 350-354Crossref PubMed Scopus (10) Google Scholar). The two other known members of the subgroup are sorCS2 (3Nagase T. Kikuno R. Ishikawa K.I. Ohara O. DNA Res. 2000; 7: 65-73Crossref PubMed Scopus (107) Google Scholar, 4Rezgaoui M. Hermey G. Riedel I.B. Hampe W. Schaller H.C. Hermans-Borgmeyer I. Mech. Dev. 2001; 100: 335-338Crossref PubMed Scopus (47) Google Scholar) and a highly homologous receptor tentatively designated sorCS3 (5Kikuno R. Nagase T. Ishikawa K. Hirosawa M. Miyajima N. Tanaka A. Kotani H. Nomura N. Ohara O. DNA Res. 1999; 6: 197-205Crossref PubMed Scopus (175) Google Scholar). The mammalian Vps10p-D receptors also comprise the previously characterized sortilin, whose lumenal part consists of a Vps10p-D only (6Petersen C.M. Nielsen M.S. Nykjær A. Jacobsen L. Tommerup N. Rasmussen H.H. Røigaard H. Gliemann J. Madsen P. Moestrup S.K. J. Biol. Chem. 1997; 272: 3599-3605Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar), and the mosaic receptor sorLA/LR11, which, in addition to an N-terminal Vps10p-D, contains elements also found in the low density lipoprotein receptor family as well as a cluster of fibronection type III repeats (7Jacobsen L. Madsen P. Moestrup S.K. Lund A.H. Tommerup N. Nykjær A. Sottrup-Jensen L. Gliemann J. Petersen C.M. J. Biol. Chem. 1996; 271: 31379-31383Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 8Yamazaki H. Bujo H. Kusunoki J. Seimiya K. Kanaki T. Morisaki N. Schneider W.J. Saito Y. J. Biol. Chem. 1996; 271: 24761-24768Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). The N termini of all the Vps10p-D family receptors contain sequences that conform to the consensus sequence for cleavage by furin (RX(R/K)R), and recent results (9Petersen C.M. Nielsen M.S. Jacobsen C. Tauris J. Jacobsen L. Gliemann J. Moestrup S.K. Madsen P. EMBO J. 1999; 18: 595-604Crossref PubMed Scopus (174) Google Scholar, 10Jacobsen L. Madsen P. Jacobsen C. Nielsen M.S. Gliemann J. Petersen C.M. J. Biol. Chem. 2001; 276: 22788-22796Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) have shown that furin cleaves the precursor forms of sortilin and sorLA and that removal of the propeptides conditions these receptors for binding of ligands. Sortilin and sorLA are mainly found in the trans-Golgi network, and only a few percent of the receptors are on the cell surface (10Jacobsen L. Madsen P. Jacobsen C. Nielsen M.S. Gliemann J. Petersen C.M. J. Biol. Chem. 2001; 276: 22788-22796Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 11Nielsen M.S. Jacobsen C. Olivecrona G. Gliemann J. Petersen C.M. J. Biol. Chem. 1999; 274: 8832-8836Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). The lumenal domains of the fully processed receptors bind certain neuropeptides (e.g. neurotensin) (10Jacobsen L. Madsen P. Jacobsen C. Nielsen M.S. Gliemann J. Petersen C.M. J. Biol. Chem. 2001; 276: 22788-22796Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 12Mazella J. Zsürger N. Navarro V. Chabry J. Kaghad M. Caput D. Ferrara P. Vita N. Gully D. Maffrand J.-P. Vincent J.-P. J. Biol. Chem. 1998; 273: 26273-26276Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar), the endoplasmic reticulum-resident receptor associated protein (RAP) (9Petersen C.M. Nielsen M.S. Jacobsen C. Tauris J. Jacobsen L. Gliemann J. Moestrup S.K. Madsen P. EMBO J. 1999; 18: 595-604Crossref PubMed Scopus (174) Google Scholar, 10Jacobsen L. Madsen P. Jacobsen C. Nielsen M.S. Gliemann J. Petersen C.M. J. Biol. Chem. 2001; 276: 22788-22796Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), as well as lipoprotein lipase (10Jacobsen L. Madsen P. Jacobsen C. Nielsen M.S. Gliemann J. Petersen C.M. J. Biol. Chem. 2001; 276: 22788-22796Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 11Nielsen M.S. Jacobsen C. Olivecrona G. Gliemann J. Petersen C.M. J. Biol. Chem. 1999; 274: 8832-8836Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar), apolipoprotein E (8Yamazaki H. Bujo H. Kusunoki J. Seimiya K. Kanaki T. Morisaki N. Schneider W.J. Saito Y. J. Biol. Chem. 1996; 271: 24761-24768Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar,10Jacobsen L. Madsen P. Jacobsen C. Nielsen M.S. Gliemann J. Petersen C.M. J. Biol. Chem. 2001; 276: 22788-22796Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), and notably their own propeptides, which block binding of all known ligands to the Vps10p-D (9Petersen C.M. Nielsen M.S. Jacobsen C. Tauris J. Jacobsen L. Gliemann J. Moestrup S.K. Madsen P. EMBO J. 1999; 18: 595-604Crossref PubMed Scopus (174) Google Scholar, 10Jacobsen L. Madsen P. Jacobsen C. Nielsen M.S. Gliemann J. Petersen C.M. J. Biol. Chem. 2001; 276: 22788-22796Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Both receptors provide internalization of cell surface-bound ligand (10Jacobsen L. Madsen P. Jacobsen C. Nielsen M.S. Gliemann J. Petersen C.M. J. Biol. Chem. 2001; 276: 22788-22796Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 13Nielsen M.S. Madsen P. Christensen E.I. Nykjær A. Gliemann J. Kasper D. Pohlmann R. Petersen C.M. EMBO J. 2001; 20: 2180-2190Crossref PubMed Scopus (350) Google Scholar), and the sortilin-cd has been shown to convey transport of cargo from Golgi to late endosomes and to bind GGAs (Golgi-localized γ-adaptin ear containing ADP-ribosylation factor-binding proteins) (13Nielsen M.S. Madsen P. Christensen E.I. Nykjær A. Gliemann J. Kasper D. Pohlmann R. Petersen C.M. EMBO J. 2001; 20: 2180-2190Crossref PubMed Scopus (350) Google Scholar), which are adaptor proteins believed to be involved in this type of trafficking (reviewed in Refs. 14Boman A.L. J. Cell Sci. 2001; 114: 3413-3418Crossref PubMed Google Scholar and 15Kirchhausen T. Nat. Struct. Biol. 2002; 9: 241-244Crossref PubMed Scopus (16) Google Scholar). Considering that sorLA is mainly intracellular (10Jacobsen L. Madsen P. Jacobsen C. Nielsen M.S. Gliemann J. Petersen C.M. J. Biol. Chem. 2001; 276: 22788-22796Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) and also binds GGAs (16Jacobsen L. Madsen P. Nielsen M.S. Geraerts W.P.M. Gliemann J. Smit A.B. Petersen C.M. FEBS Lett. 2002; 511: 155-158Crossref PubMed Scopus (94) Google Scholar), it seemed possible that Vps10p-D receptors other than sortilin might be targeted by similar sorting mechanisms. SorCS1 is expressed in the murine central nervous system like sortilin and sorLA, and transcripts have also been observed in kidney, liver, and heart (1Hermey G. Riedel I.B. Hampe W. Schaller H.C. Hermans- Borgmeyer I. Biochem. Biophys. Res. Commun. 1999; 266: 347-351Crossref PubMed Scopus (65) Google Scholar). The receptor prevails in neurons, and during embryonic development sorCS1 is expressed in a transient and dynamic pattern in areas of the nervous system where precursors proliferate as well as in regions where cells differentiate (17Hermey G. Schaller H.C. Hermans-Borgmeyer I. Neuroreport. 2001; 12: 29-32Crossref PubMed Scopus (13) Google Scholar). The purpose of the present work was to begin elucidating sorCS1 function. We cloned human sorCS1b, whose cd is highly similar to that of the murine orthologue, and identified two new isoforms with distinct cds and different distributions in tissues. The common lumenal domain was cleaved by furin in the late synthetic pathway demonstrating that sorCS1 is synthesized as a proprotein. Analysis of cells stably transfected with wild type and chimeric receptors showed that the three cds convey different distributions of sorCS1 in cells and have different capabilities for internalization. Neither the mature lumenal domain nor any of the cds bound any of the ligands previously shown to interact with sortilin and sorLA, demonstrating that sorCS1 is functionally different from the previously characterized Vps10p-D family receptors. Murine sorCS1b cDNA (GenBankTM accession number AF195056) was radiolabeled and used for screening a human brain cDNA library in the lambda ZAP vector (Stratagene, La Jolla, CA). Five positive clones were purified and rescued into the pBK-CMV vector, and sequencing revealed three different overlapping clones representing bp 462–3504 of human sorCS1b cDNA in addition to a 3′ untranslated region. The 5′ part of the open reading frame was obtained by reverse transcription PCR. The first strand cDNA was synthesized from 1 μg human fetal brain RNA (Clontech, Palo Alto, CA) using the primer 5′-AGGTGAAGGTGTAGTGAGCAATAGGG representing bp 1549–1574. RNA was denatured in the presence of 40 pmol primer for 10 min at 94 °C and transferred to prewarmed reaction mixture (50 mm Tris, pH 8.3, 75 mm KCl, 3 mm MgCl2, 0.5 mm deoxynucleoside triphosphates, 200 mmdithiothreitol). Following addition of reverse transcriptase (SuperScript II, Invitrogen), first strand reaction was performed for 55 min at 42 °C. The reaction was terminated by 15 min heating to 70 °C followed by 5 min on ice and incubation for 20 min at 37 °C with RNase-H (AP Biotech, Little Chalfont, UK). A two-step PCR reaction was performed using Advantage-GC polymerase (Clontech). The same reverse primer was used as for the first strand reaction, and the forward primer (5′-CTCCCGCGATGGGAAAAGTTGGC) was obtained from a sequence in the human chromosome 10 derived bacterial artificial chromosome RP11-557K21 (GenBank™ accession number AL356439) homologous to the 5′ end of murine sorCS1. The 1.5-kb product was subcloned into the pGem-T Easy Vector (Promega, Madison, WI) and sequenced. The full-length cDNA was obtained by ligation of overlapping fragments using thePstI (bp 1522), SapI (bp 1753), and BamHI (bp 3128) sites and transferred into pBluescript (Stratagene). Chromosomal localization and organization were determined using the program tblastn (18Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (71456) Google Scholar) at the NCBI (National Institutes of Health, Bethesda, MD). Exonic sequences were determined by aligning cDNA and genomic sequences, and exon/intron boundaries were in agreement with consensus splice sites (19Goldstrohm A.C. Greenleaf A.L. Garcia-Blanco M.A. Gene. 2001; 277: 31-47Crossref PubMed Scopus (144) Google Scholar). Primers corresponding to the 3′ ends of putative terminal exons of sorCS1a and sorCS1c (5′-TTAATAGAAACCATCACTGCTATG and 5′-TGGTGGCTACTGGGAATCATTTAC) were used for first strand synthesis on 1 μg of total RNA from human fetal brain followed by two-step PCR reactions using the forward primer for generation of full-length sorCS1b. A multiple tissue blot (AP Biotech) was hybridized with a [32P]dCTP randomly labeled probe of sorCS1 (bp 463–1924) and washed under high stringency conditions followed by autoradiography. For identification of sorCS1 isoforms, reverse transcription PCR reactions were performed on 1 μg of total human RNA from fetal liver, fetal brain, adult cerebellum, and adult total brain (Clontech) using the reverse primer 5′-GCCTGTAGCCTTTGGGGGTTTTCC for sorCS1b as well as the reverse primers designed for isolation of sorCS1a and -c cDNA. The forward primer (5′-TACCCACCACTGCTGAACTCTTTG) common to all isoforms was derived from exon 23. The PCR products were verified by sequencing. For generation of wild type receptor constructs, the sorCS1 cDNA was cut out of pBluescript using the KpnI site of the polylinker preceding the 5′ end of the cDNA and the natural BamHI site at bp 3128. This was combined either with a BamHI/ApaI 3′ fragment and cloned into pcDNA4/Myc-HisA (Invitrogen) for expression of the lumenal part of sorCS1 (l-sorCS1) or withBamHI/NotI 3′ fragments and cloned into pcDNA3.1/Zeo(+) (Invitrogen) for expression of full-length receptor isoforms. ApaI and NotI sites were introduced by PCR to the 3′ ends using modified primers corresponding to nucleotides 2991–3308 or to segments following the stop codons. Alternatively, sorCS1a–c cDNA was transferred via NheI andNotI restriction sites from pcDNA3.1/Zeo(+) to pcDNA3.1/Hygro(−) (Invitrogen) to generate doubly transfected cells. To produce l-sorCS1 mutated in the furin cleavage site (74RRRR to 74GRGR) we performed a two-step PCR using Advantage-GC polymerase. Overlapping 5′ and 3′ fragments were amplified from the original expression construct using the primers 5′-ATTAATACGACTCACTATAGGGAG, 5′-GATCCGCTCCGCTCCGTCCCCTCCCGC, 5′-CCGGCGGGAGGGGACGGAGCGGAG, and 5′-TTGTTCCATAATCGGTTGACCTCC. The resulting PCR fragment was digested with KpnI and SacI and used to replace the 5′ end of l-sorCS1 cDNA in the pcDNA4/Myc-HisA vector. To produce chimeric constructs covering the lumenal and transmembrane parts of the interleukin-2 receptor-α (IL2R, Tac/CD25) and the sorCS1 cytoplasmic tails (chi-a, -b, and -c), cDNA encoding the three tails was amplified by standard PCR technique using primers generating a 5′ HindIII site and a 3′ XhoI site. The primers for the a, b, and c tail constructs were: 5′-TCGTAAGCTTAAGTTTAAAAGGTGCG and 5′-CCTCTCGAGTTAATAGAAACCATCACTGCTATG, 5′-CGTCAAGCTTAAGTTTAAAAGGAGAGTAGCTTTACCC and 5′-GGGGCTCGAGTTAAATTGCATACTGTGCCCCAGCAGATCC, and 5′-CAAGCTTAAGTTTAAAAGGAA GATC and 5′-TACCTCGAGTCATTTACCTATGAGC. The HindIII/XhoI fragments were ligated into pcDNA3.1/Zeo(+) together with a NheI/HindIII fragment representing the lumenal and transmembrane parts of IL2R cut out of pCMV-IL2R/CD25/Tac (20LaFlamme S.E. Thomas L.A. Yamada S.S. Yamada K.M. J. Cell Biol. 1994; 126: 1287-1298Crossref PubMed Scopus (207) Google Scholar). CHO-K1 cells were cultured in HyQ-CCM5 (HyClone, Logan, UT) and transfected using FuGENE 6 (Roche Molecular Biochemicals). Stable transfectants were selected in medium containing 300 μg/ml Zeocin (Invitrogen) and identified by Western blotting or immunocytochemistry. Double transfectants were generated by transfecting CHO cells expressing IL2R/sorCS1 chimeras with pcDNA3.1/Hygro(−) wild type receptor constructs followed by additional selection using 500 μg/ml Hygromycin (Invitrogen). Secreted His6-taggedl-sorCS1 was purified by affinity chromatography on Talon Metal Affinity Resin (Clontech). Prewashed resin (3 ml) was recirculated for 16 h at 4 °C with about 90 ml of culture medium followed by washings in 50 mmNa2HPO4, 300 mm NaCl, 0.1% Tween 20, pH 7.0, and elution in 50 mm NaAc, 300 mmNaCl, pH 5.0. The propeptide sequence was amplified from sorCS1 cDNA using a 5′ primer (5′-TCTGGATCCGGCGGCTCCTGCTGC) introducing aBamHI site to the propeptide N-terminal sequence and a 3′ primer (5′-ATCCTCGAGTCACCGTCTCCTCCG) introducing a stop codon and aXhoI site after the furin cleavage site 72RRRR. Following two-step PCR, the product was cloned viaBamHI/XhoI into pGEX-4T-1 (AP Biotech). For expression of the leucine-rich domain (Glu942-Ile1023) the nativeEcoRI and EcoRV sites were used to generate a construct in pGEX-4T-1. Both constructs were expressed in the bacterial strain BL21 (DE3), and the resulting GST-tagged proteins were purified using glutathion-Sepharose beads. EcoRI andXhoI restriction sites were introduced into cDNA of the three sorCS1 cytoplasmic tails via PCR followed by insertion into the pLexA vector to generate bait strains. A Matchmaker LexA two-hybrid system (Clontech) was used as described before (13Nielsen M.S. Madsen P. Christensen E.I. Nykjær A. Gliemann J. Kasper D. Pohlmann R. Petersen C.M. EMBO J. 2001; 20: 2180-2190Crossref PubMed Scopus (350) Google Scholar). Antisera were raised in rabbits against GST-sorCS1-(942–1023)(anti-Leu sorCS1), l-sorCS1, and GST-sorCS1-(1–77) propeptide (DAKO, Glostrup, Denmark). Monoclonal mouse anti-IL2Rα (anti-Tac) was from Roche. Recombinant RAP, GST-sortilin and −sorLA propeptides, as well as the lumenal domains of sortilin (l-sortilin) and sorLA, were produced as described (9Petersen C.M. Nielsen M.S. Jacobsen C. Tauris J. Jacobsen L. Gliemann J. Moestrup S.K. Madsen P. EMBO J. 1999; 18: 595-604Crossref PubMed Scopus (174) Google Scholar, 10Jacobsen L. Madsen P. Jacobsen C. Nielsen M.S. Gliemann J. Petersen C.M. J. Biol. Chem. 2001; 276: 22788-22796Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Neurotensin was from Sigma, recombinant apolipoprotein E3 fromCalbiochem, and bovine lipoprotein lipase was a gift from Dr. G. Olivecrona, Umeå University, Sweden. CHO-K1 cells stably transfected withl-sorCS1 were grown to 80% confluency and biolabeled essentially as described previously (9Petersen C.M. Nielsen M.S. Jacobsen C. Tauris J. Jacobsen L. Gliemann J. Moestrup S.K. Madsen P. EMBO J. 1999; 18: 595-604Crossref PubMed Scopus (174) Google Scholar, 10Jacobsen L. Madsen P. Jacobsen C. Nielsen M.S. Gliemann J. Petersen C.M. J. Biol. Chem. 2001; 276: 22788-22796Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) using 200 μCi/ml35S-labeled cysteine and methionine (Pro-mix, AP Biotech). The medium was harvested, and washed cells were lysed in 1% Triton X-100, 20 mm Tris-HCl, 10 mm EDTA, pH 8.0, supplemented with proteinase inhibitor (CompleteMini, Roche Molecular Biochemicals). Labeled l-sorCS1 was precipitated from lysate (200 μl + 600 μl of non-labeled medium) and medium (600 μl + 200 μl of non-labeled lysate) via its His6 tag using Talon beads. For treatment with PNGase-F (Roche Molecular Biochemicals), the beads were washed, heated in 10 μl 1% SDS (3 min, 95 °C), heated again after addition of 90 μl 20 mmNaH2PO4, 10 mm EDTA, 10 mm Na-azide, 0.5% Triton X-100, pH 7.2, and cooled before the addition of 0.5 units PNGase-F and incubation for 16 h at 30 °C. Alternatively, washed beads were treated with endoglycosidase-H (Endo-H, Roche) as described (9Petersen C.M. Nielsen M.S. Jacobsen C. Tauris J. Jacobsen L. Gliemann J. Moestrup S.K. Madsen P. EMBO J. 1999; 18: 595-604Crossref PubMed Scopus (174) Google Scholar). For furin cleavage, beads with bound l-sorCS1 were incubated in 100 μl of 100 mm Hepes, 1 mm CaCl2, 1 mm 2-mercaptoethanol, 0.5% Triton X-100, pH 7.6, and 4 units of furin (Alexis Biochemicals) followed by successive incubations for 2 h at 30 °C and 2 h at 37 °C. For immunoblotting, medium or cell lysates were subjected to reducing SDS-PAGE and blotted following standard procedures. For detection of sorCS1 in blots of human kidney and spleen (Chemicon), anti-l-sorCS1 was used, followed by horseradish peroxidase conjugated swine anti-rabbit Ig (DAKO), and visualization by ECL (AP Biotech). Measurements were performed on a BIAcore 2000 instrument using CM5 sensor chips activated as described (9Petersen C.M. Nielsen M.S. Jacobsen C. Tauris J. Jacobsen L. Gliemann J. Moestrup S.K. Madsen P. EMBO J. 1999; 18: 595-604Crossref PubMed Scopus (174) Google Scholar). l-sorCS1 and the lumenal domains of sortilin and sorLA were immobilized to an estimated density of ∼60 fmol/mm2, and samples for binding (40 μl, 25 °C) were injected at 5 μl/min in 10 mm Hepes, 150 mmNaCl, 1.5 mm CaCl2, 1 mm EGTA, 0.005% Tween 20, pH 7.4. Binding was expressed in units as the response obtained with immobilized receptor minus the response with an activated but uncoupled chip. The chips were regenerated as described, and kinetic parameters were determined using BIAevaluation 3.0 software as described (9Petersen C.M. Nielsen M.S. Jacobsen C. Tauris J. Jacobsen L. Gliemann J. Moestrup S.K. Madsen P. EMBO J. 1999; 18: 595-604Crossref PubMed Scopus (174) Google Scholar). Cells were surface-labeled with the impermeable reagent sulfo-N-hydroxysuccinimidobiotin (Pierce), washed and lysed as described previously (10Jacobsen L. Madsen P. Jacobsen C. Nielsen M.S. Gliemann J. Petersen C.M. J. Biol. Chem. 2001; 276: 22788-22796Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 11Nielsen M.S. Jacobsen C. Olivecrona G. Gliemann J. Petersen C.M. J. Biol. Chem. 1999; 274: 8832-8836Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar), and biotinylated proteins were precipitated with streptavidin-coupled Sepharose (Zymed Laboratories Inc., San Francisco, CA). The fractions of streptavidin-bound and unbound IL2R/sorCS1 chimera or sorCS1 isoforms in cell lysates were detected by Western blotting and quantified using a FUJIFILM LAS-1000 luminescence image analyzer. Transfected or control cells were washed in 10 mm phosphate, 150 mm NaCl, pH 7.3, fixed in the same buffer with 4% paraformaldehyde, and finally washed in buffer containing 0.05% Triton X-100 followed by incubation with primary (anti-Tac or anti-l-sorCS1) and secondary antibodies (fluorescein isothiocyanate-conjugated rabbit anti-mouse, DAKO, Alexa 488-conjugated goat anti-rabbit or Alexa 568-conjugated goat anti-mouse, Molecular Probes, Leiden, The Netherlands). To visualize internalization, cells were surface-labeled with primary antibody at 4 °C for 2 h followed by incubation at 37 °C for various times. Fluorescence microscopy was performed using a laser scanning confocal unit (LSM510, Zeiss). Alternatively, cells transfected with chimeras were incubated at 4 °C with 125I-labeled monoclonal anti-Tac (3 × 104 cpm/ml) for 2 h at 4 °C, washed, and reincubated at 37 °C for 0–60 min. Incubations were stopped by the addition of ice cold acetic acid, 150 mm NaCl, pH 2.5. After 5 min, the supernatant was recovered, the cells lysed in 1 m NaOH, and radioactivity determined in the two fractions was defined as surface-associated and internalized antibody, respectively. The human sorCS1b cDNA (GenBankTM accession number AF284756) encodes a 33 amino acid signal peptide followed by a 1135 amino acid type 1 receptor (Fig. 1 A) with 92% sequence identity to the murine sorCS1b protein. The N-terminal part contains the sequence (74RRRR77) corresponding to the optimal multibasic motif (RXR/KR) for cleavage by furin and the sequence (93RSPR96) corresponding to the minimal requirements (RXXR) for cleavage. The human sorCS1b gene maps at 10q23.3, and analysis of genomic sequences indicated the presence of 26 exons. As two isoforms (sorCS1a and -b) differing only in their cds have been identified in the mouse (2Hermey G. Schaller H.C. Biochim. Biophys. Acta. 2000; 1491: 350-354Crossref PubMed Scopus (10) Google Scholar), and only the sorCS1b variant was found by human cDNA library screening, we searched in human genomic databases for sequences corresponding to the 3′ end of murine sorCS1a. However, no such sequence was found, and genomic sequences between exon 25 (encoding the transmembrane domain and four residues of the cytoplasmic tail) and exon 26 (encoding the remaining sorCS1b tail) were therefore analyzed to identify possible splice variants with little or no homology to murine sorCS1a. Putative exon sequences identified as open reading frames followed by polyadenylation signals were analyzed using primers 3′ to potential stop codons and a 5′ primer corresponding to exon 23 encoding part of the leucine-rich domain (cf. Fig. 1 B). This approach revealed two new splice variants in human fetal brain RNA, and cDNA clones encoding the complete proteins were identified using a primer corresponding to the 5′ untranslated region and the primers corresponding to sequences 3′ to the stop codons. Fig. 1 B shows the generation of human sorCS1 isoforms. Like in the mouse, human sorCS1a (GenBank™ accession numberAY099453) is generated by using exon 25 as the terminal exon (stop codon a), but the amino acid sequence encoded by the part of exon 25 that is specific for the sorCS1a-cd is completely different from that of the murine receptor (Fig. 1 C and Ref. 2Hermey G. Schaller H.C. Biochim. Biophys. Acta. 2000; 1491: 350-354Crossref PubMed Scopus (10) Google Scholar). When the 5′ splice site within exon 25 is active, sorCS1b is generated by skipping the 3′ part of this exon and using exon 26 as the terminal exon. Comparison of the amino acid encoded by exon 26 of human sorCS1b showed 89% sequence identity with the corresponding segment of the mouse sorCS1b. Finally, human sorCS1c (GenBank™ accession numberAY099452) is generated by skipping the middle part of exon 25 by using both splice sites within the exon. Because a murine counterpart was expected, we subsequently cloned mouse sorCS1c (GenBank™ accession number AF284755), 2G. Hermey, unpublished result. which revealed a cd with 87% amino acid sequence identity to the human sorCS1c-cd. Searches in databases indicated that the cds of human sorCS1a and -b show little similarity to other receptor cds, whereas the sorCS1c-cd exhibits about 50% identity to the cds of sorCS2 and -3. Northern blotting of human tissues using a probe common to all three isoforms showed high levels of transcripts in adult kidney and comparatively moderate levels in brain, heart, and small intestine (Fig.2 A). To identify the sorCS1 protein, we performed immunoblots on two human tissue homogenates and on extracts of CHO cells mock-transfected or stably transfected with sorCS1a cDNA. A band of ∼130 kDa was detected in the sorCS1a-transfected CHO cells and in kidney, but not in non-transfected CHO cells and spleen (Fig. 2 B), in agreement with the Northern blots. We next examined expression of the three splice variants in human adult brain, adult cerebellum, fetal brain, and fetal liver by reverse transcription PCR. As shown in Fig. 2 C, all three splice variants were detected in the brain samples (lanes 2–4), whereas only sorCS1c was found in human fetal liver (lane 1), demonstrating that sorCS1 isoforms are differentially expressed among tissues. To determine whether sorCS1 processing includes propeptide cleavage, CHO cells were stably transfected with the His6-tagged lumenal part (amino acid 1–1067) of sorCS1 (l-sorCS1). The transfectants were biolabeled, and l-sorCS1 secreted into the medium or present in cell lysates was recovered on Talon beads. Fig. 3 A shows that labeledl-sorCS1 in the medium (lane 1) has a higher apparent molecular size than that obtained from lysates (lane 2), whereas, after deglycosylation with PNGase-F, the cellular form (lane 4) is larger than the secreted form (lane 3), demonstrating cleavage of l-sorCS1. This occurred within cells because labeled l-sorCS1 isolated from cell lysates was unchanged by incubation in conditioned CHO medium (not shown). Fig. 3 further shows that the cellular form of the receptor was cleaved upon incubation with furin (panel B, lane 4 versus lane 3), whereas the secreted form was unaffected (lane 2 versus lane 1), and that only the uncleaved cellular form was sensitive to Endo-H (panel C, lane 4 versus lane 3), demonstrating that cleavage occurs in the furin-containing distal synthetic pathway. Western blotting was then performed, and antibody against sorCS1-(1–77) propeptide reacted only with the cellular form of the r
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