Subunit Structure of Thyrotropin Receptors Expressed on the Cell Surface
1999; Elsevier BV; Volume: 274; Issue: 48 Linguagem: Inglês
10.1074/jbc.274.48.33979
ISSN1083-351X
AutoresKunihiko Tanaka, Gregorio D. Chazenbalk, Sandra M. McLachlan, Basil Rapoport,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoWe studied cell surface thyrotropin receptor (TSHR) by biotinylating proteins on the surface of metabolically labeled, intact cells. In addition to TSHR cleaved into A and B subunits, mature single-chain receptors with complex carbohydrate were also present on the cell surface. A low A/B subunit ratio indicated partial shedding of extracellular A subunits from transmembrane B subunits. TSHR cleavage at upstream site 1 (within amino acid residues 305–316) would generate a B subunit of 51–52 kDa. However, only smaller B subunits (40–46 kDa) were detected, corresponding to N termini from residues ∼370 (site 2) extending downstream to the region of B subunit insertion into the plasma membrane. The intervening C peptide region between sites 1 and 2 could not be purified from TSHR epitope-tagged (c-myc) within this region. However, the small proportion of B subunits recovered with a c-myc antibody were larger (45–52 kDa) than the majority of B subunits recovered with a C-terminal antibody. In conclusion, our study provides the first characterization of cell surface TSHR including their A and B subunits. Single-chain, mature TSHR do exist on the cell surface. The C peptide lost during intramolecular cleavage disintegrates rapidly following cleavage at upstream site 1 of the single-chain TSHR into A and B subunits. N-terminal disintegration of the B subunit pauses at site 2, but then progresses downstream to the vicinity of the plasma membrane, revealing a novel mechanism for A subunit shedding. We studied cell surface thyrotropin receptor (TSHR) by biotinylating proteins on the surface of metabolically labeled, intact cells. In addition to TSHR cleaved into A and B subunits, mature single-chain receptors with complex carbohydrate were also present on the cell surface. A low A/B subunit ratio indicated partial shedding of extracellular A subunits from transmembrane B subunits. TSHR cleavage at upstream site 1 (within amino acid residues 305–316) would generate a B subunit of 51–52 kDa. However, only smaller B subunits (40–46 kDa) were detected, corresponding to N termini from residues ∼370 (site 2) extending downstream to the region of B subunit insertion into the plasma membrane. The intervening C peptide region between sites 1 and 2 could not be purified from TSHR epitope-tagged (c-myc) within this region. However, the small proportion of B subunits recovered with a c-myc antibody were larger (45–52 kDa) than the majority of B subunits recovered with a C-terminal antibody. In conclusion, our study provides the first characterization of cell surface TSHR including their A and B subunits. Single-chain, mature TSHR do exist on the cell surface. The C peptide lost during intramolecular cleavage disintegrates rapidly following cleavage at upstream site 1 of the single-chain TSHR into A and B subunits. N-terminal disintegration of the B subunit pauses at site 2, but then progresses downstream to the vicinity of the plasma membrane, revealing a novel mechanism for A subunit shedding. thyrotropin thyrotropin receptor Chinese hamster ovary phosphate-buffered saline monoclonal antibody fetal calf serum Graves' disease, one of the most common autoimmune diseases affecting humans, is caused by autoantibodies that activate the thyrotropin (TSH)1 receptor (TSHR) (reviewed in Ref. 1Rapoport B. Chazenbalk G.D. Jaime J.C. McLachlan S.M. Endocr. Rev. 1998; 19: 673-716Crossref PubMed Scopus (503) Google Scholar). Remarkably, functional autoantibodies do not arise to the other, closely related members of the glycoprotein hormone receptor family. A potential reason for this difference is the unique subunit structure of the TSH receptor. Thus, unlike the other glycoprotein hormone receptors, a variable proportion of TSHR on the cell surface cleaves into an extracellular A subunit and a largely transmembrane B subunit that remain linked by disulfide bonds (2Furmaniak J. Hashim F.A. Buckland P.R. Petersen V.B. Beever K. Howells R.D. Rees Smith B. FEBS Lett. 1987; 215: 316-322Crossref PubMed Scopus (38) Google Scholar, 3Russo D. Chazenbalk G.D. Nagayama Y. Wadsworth H.L. Seto P. Rapoport B. Mol. Endocrinol. 1991; 5: 1607-1612Crossref PubMed Scopus (75) Google Scholar, 4Loosfelt H. Pichon C. Jolivet A. Misrahi M. Caillou B. Jamous M. Vannier B. Milgrom E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3765-3769Crossref PubMed Scopus (175) Google Scholar). This cleavage introduces the potential for antigenic stimulation of the immune system, for two reasons. First, TSHR cleavage into A and B subunits results in the deletion of an intervening "C peptide" region (5Chazenbalk G.D. Tanaka K. Nagayama Y. Kakinuma A. Jaume J.C. McLachlan S.M. Rapoport B. Endocrinology. 1997; 138: 2893-2899Crossref PubMed Scopus (91) Google Scholar, 6de Bernard S. Misrahi M. Huet J.-C. Beau I. Desroches A. Loosfelt H. Pichon C. Pernollet J.-C. Milgrom E. J. Biol. Chem. 1999; 274: 101-107Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Extracellular release of this polypeptide, either intact or in fragments, could initiate or propagate an immune response to the TSHR. Second, there is the propensity for the A subunit itself to be shed, at least in vitro (7Couet J. Sokhavut S. Jolivet A. Vu Hai M.-T. Milgrom E. Misrahi M. J. Biol. Chem. 1996; 271: 4545-4552Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 8Tanaka K. Chazenbalk G.D. McLachlan S.M. Rapoport B. Mol. Cell. Endocrinol. 1999; 150: 113-119Crossref PubMed Scopus (33) Google Scholar). Determination of the molecular basis for TSH receptor cleavage and shedding is, therefore, of potential clinical importance in understanding the pathogenesis of Graves' disease.Many uncertainties remain regarding the process of TSHR cleavage and shedding, including the enzyme(s) involved (6de Bernard S. Misrahi M. Huet J.-C. Beau I. Desroches A. Loosfelt H. Pichon C. Pernollet J.-C. Milgrom E. J. Biol. Chem. 1999; 274: 101-107Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 7Couet J. Sokhavut S. Jolivet A. Vu Hai M.-T. Milgrom E. Misrahi M. J. Biol. Chem. 1996; 271: 4545-4552Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 9Couet J. de Bernard S. Loosfelt H. Saunier B. Milgrom E. Misrahi M. Biochemistry. 1996; 35: 14800-14805Crossref PubMed Scopus (112) Google Scholar) and the properties of the shed A subunits (7Couet J. Sokhavut S. Jolivet A. Vu Hai M.-T. Milgrom E. Misrahi M. J. Biol. Chem. 1996; 271: 4545-4552Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 8Tanaka K. Chazenbalk G.D. McLachlan S.M. Rapoport B. Mol. Cell. Endocrinol. 1999; 150: 113-119Crossref PubMed Scopus (33) Google Scholar). The location and number of cleavage sites has also been controversial. Initial evidence for a cleavage site closely upstream of amino acid residue 317, obtained by TSH cross-linking to a TSHR deletion mutant (3Russo D. Chazenbalk G.D. Nagayama Y. Wadsworth H.L. Seto P. Rapoport B. Mol. Endocrinol. 1991; 5: 1607-1612Crossref PubMed Scopus (75) Google Scholar), was contradicted by reports that amino acid residues 352–366 lie within the A subunit (10Ban T. Kosugi S. Kohn L.D. Endocrinology. 1992; 131: 815-829Crossref PubMed Scopus (45) Google Scholar,11Graves P.N. Vlase H. Bobovnikova Y. Davies T.F. Endocrinology. 1996; 137: 3915-3920Crossref PubMed Scopus (60) Google Scholar). However, residues 352–366 cannot be part of the A subunit because they are part of a C peptide region excised from the TSHR during intramolecular cleavage (5Chazenbalk G.D. Tanaka K. Nagayama Y. Kakinuma A. Jaume J.C. McLachlan S.M. Rapoport B. Endocrinology. 1997; 138: 2893-2899Crossref PubMed Scopus (91) Google Scholar), an observation recently confirmed (6de Bernard S. Misrahi M. Huet J.-C. Beau I. Desroches A. Loosfelt H. Pichon C. Pernollet J.-C. Milgrom E. J. Biol. Chem. 1999; 274: 101-107Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Further support for an upstream cleavage site in the TSHR in the region of residues 305–316 was obtained in studies examining the effect of trypsin on subunit structure (12Tanaka K. Chazenbalk G.D. McLachlan S.M. Rapoport B. J. Biol. Chem. 1998; 273: 1959-1963Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), as well as by the identification in transfected L cells of a minor B subunit component with an N terminus at residue 314 (6de Bernard S. Misrahi M. Huet J.-C. Beau I. Desroches A. Loosfelt H. Pichon C. Pernollet J.-C. Milgrom E. J. Biol. Chem. 1999; 274: 101-107Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar).Less clear, however, is the process by which the C peptide region between the A and B subunits is lost. The observed deletion of this segment (5Chazenbalk G.D. Tanaka K. Nagayama Y. Kakinuma A. Jaume J.C. McLachlan S.M. Rapoport B. Endocrinology. 1997; 138: 2893-2899Crossref PubMed Scopus (91) Google Scholar), taken together with previous TSH cross-linking studies to TSH-luteinizing hormone/chorionic gonadotropin chimeric receptors (13Russo D. Nagayama Y. Chazenbalk G.D. Wadsworth H.L. Rapoport B. Endocrinology. 1992; 130: 2135-2138PubMed Google Scholar), provided evidence for two separate TSHR cleavage sites, the second (downstream) site being in the vicinity of amino acid residue 370 (14Kakinuma A. Chazenbalk G.D. Tanaka K. Nagayama Y. McLachlan S.M. Rapoport B. J. Biol. Chem. 1997; 272: 28296-28300Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). A recent refinement of this concept is that, rather than there being two distinct cleavage sites, cleavage at upstream site 1 (approximately residue 314) is primary and is followed by the sequential excision of the C peptide region, terminating in the region of site 2 (6de Bernard S. Misrahi M. Huet J.-C. Beau I. Desroches A. Loosfelt H. Pichon C. Pernollet J.-C. Milgrom E. J. Biol. Chem. 1999; 274: 101-107Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). However, in this report, there is a discrepancy between the small size of the B subunit polypeptide chains observed and their predicted size based on their N-terminal residues.Overall, a major problem in evaluating this somewhat confusing compendium of data is that previous studies on the immunodetection or immunopurification of TSHR subunits have been performed on thyroid tissue or cell homogenates that include TSHR products in the degradation and synthetic pathways as well as TSHR on the cell surface (4Loosfelt H. Pichon C. Jolivet A. Misrahi M. Caillou B. Jamous M. Vannier B. Milgrom E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3765-3769Crossref PubMed Scopus (175) Google Scholar, 5Chazenbalk G.D. Tanaka K. Nagayama Y. Kakinuma A. Jaume J.C. McLachlan S.M. Rapoport B. Endocrinology. 1997; 138: 2893-2899Crossref PubMed Scopus (91) Google Scholar, 6de Bernard S. Misrahi M. Huet J.-C. Beau I. Desroches A. Loosfelt H. Pichon C. Pernollet J.-C. Milgrom E. J. Biol. Chem. 1999; 274: 101-107Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 10Ban T. Kosugi S. Kohn L.D. Endocrinology. 1992; 131: 815-829Crossref PubMed Scopus (45) Google Scholar, 15Endo T. Ikeda M. Ohmori M. Anzai E. Haraguchi K. Onaya T. Biochem. Biophys. Res. Commun. 1992; 187: 887-893Crossref PubMed Scopus (18) Google Scholar, 16Harfst E. Ross M.S. Nussey S.S. Johnstone A.P. Mol. Cell. Endocrinol. 1994; 102: 77-84Crossref PubMed Scopus (21) Google Scholar, 17Misrahi M. Ghinea N. Sar S. Saunier B. Jolivet A. Loosfelt H. Cerutti M. Devauchelle G. Milgrom E. Eur. J. Biochem. 1994; 222: 711-719Crossref PubMed Scopus (108) Google Scholar, 18Potter E. Horn R. Scheumann G.F.W. Dralle H. Costagliola S. Ludgate M. Vassart G. Dumont J.E. Brabant G. Biochem. Biophys. Res. Commun. 1994; 205: 361-367Crossref PubMed Scopus (12) Google Scholar, 19Grossman R.F. Ban T. Duh Q.Y. Tezelman S. Jossart G. Soh E.Y. Clark O.H. Siperstein A.E. Thyroid. 1995; 5: 101-105Crossref PubMed Scopus (12) Google Scholar, 20Chazenbalk G.D. Kakinuma A. Jaume J.C. McLachlan S.M. Rapoport B. Endocrinology. 1996; 137: 4586-4591Crossref PubMed Scopus (77) Google Scholar). Although in one study, surface-radiolabeled TSHR were examined, subunit forms of the receptor, in particular the B subunit, could not be clearly identified (16Harfst E. Ross M.S. Nussey S.S. Johnstone A.P. Mol. Cell. Endocrinol. 1994; 102: 77-84Crossref PubMed Scopus (21) Google Scholar). In the present study, we have succeeded in using a cell surface biotinylation approach to examine the properties of both TSHR subunits expressed exclusively on the cell surface.MATERIALS AND METHODSCell Lines and CultureTSHR-10,000 is a Chinese hamster ovary (CHO) cell line overexpressing the human TSHR (∼2 × 106receptors/cell) (20Chazenbalk G.D. Kakinuma A. Jaume J.C. McLachlan S.M. Rapoport B. Endocrinology. 1996; 137: 4586-4591Crossref PubMed Scopus (77) Google Scholar). Overexpression was attained by transgenome amplification using a dihydrofolate reductase minigene approach. The TSHRmyc-10,000 CHO cell line was generated by the same method. To accomplish this, we transferred the cDNA for an epitope-tagged TSHR (TSHR amino acids 338–349 replaced with the human c-myc peptide EEQKLISEEDLL) (21Tanaka K. Nagayama Y. Yamasaki H. Hayashi H. Namba H. Yamashita S. Niwa M. Biochem. Biophys. Res. Commun. 1996; 228: 21-28Crossref PubMed Scopus (10) Google Scholar) into the plasmid pSV2-DHFR-ECE (22Kaufman K.D. Foti D. Seto P. Rapoport B. Mol. Cell. Endocrinol. 1991; 78: 107-114Crossref PubMed Scopus (41) Google Scholar). Cells were propagated in Ham's F-12 medium supplemented with 10% fetal calf serum (FCS), penicillin (100 units/ml), gentamicin (50 μg/ml), and amphotericin B (2.5 μg/ml).Biotinylation and Extraction of Metabolically Labeled TSHR on the Cell SurfaceTSHR-10,000 or TSHRmyc-10,000 cells in 60-mm diameter culture dishes were pulsed (1 h at 37 °C) with 0.2 mCi/ml [35S]methionine/cysteine in Dulbecco's modified Eagle's, high glucose (4500 mg/l), methionine- and cysteine-free medium containing 5% heat-inactivated FCS, as described previously (20Chazenbalk G.D. Kakinuma A. Jaume J.C. McLachlan S.M. Rapoport B. Endocrinology. 1996; 137: 4586-4591Crossref PubMed Scopus (77) Google Scholar). The cell monolayers were washed twice with ice-cold PBS, pH 8.0, and the biotin cross-linker sulfosuccinimidyl-2-(biotinamide) ethyl-1,3′-dithiopropionate (Pierce; 0.5 mg/ml in PBS, pH 8.0) was added for 20 min on ice. The solution was removed and the cross-linking procedure was repeated once. After aspiration, remaining reactive sulfosuccinimidyl-2-(biotinamide) ethyl-1,3′-dithiopropionate was blocked by addition of 50 mm NH4Cl in PBS, pH 8.0, for 10 min on ice with occasional agitation.Extraction of TSHR was as described previously (20Chazenbalk G.D. Kakinuma A. Jaume J.C. McLachlan S.M. Rapoport B. Endocrinology. 1996; 137: 4586-4591Crossref PubMed Scopus (77) Google Scholar). In brief, cells were washed twice with PBS and scraped into 1 ml of ice-cold buffer A (20 mm Hepes, pH 7.2, 150 mm NaCl, and 100 μg/ml phenylmethylsulfonyl fluoride and 1 μg/ml leupeptin (both from Sigma)). The cells were pelleted, rinsed, and resuspended in buffer A containing 1% Triton X-100. After 2 h at 4 °C with occasional vortexing, the mixture was centrifuged for 1 h at 100,000 × g and the supernatant was retained for immunoprecipitations. Where indicated, in an alternative procedure (23Kakinuma A. Chazenbalk G.D. Jaume J.C. Rapoport B. McLachlan S.M. J. Clin. Endocrinol. Metab. 1997; 82: 2129-2134PubMed Google Scholar), 1.2 ml of buffer containing 10 mm Tris, pH 7.4, 50 mm NaCl, 1% Triton X-100, and 0.5% bovine serum albumin was added directly to the cell monolayer without scraping. After rocking for 2 h at 4 °C, the supernatant was recovered and centrifuged as described above.Immunoprecipitation of TSHR and Separation in Biotinylated and Non-biotinylated FractionsThe immunoprecipitation procedure used has been described previously in detail (20Chazenbalk G.D. Kakinuma A. Jaume J.C. McLachlan S.M. Rapoport B. Endocrinology. 1996; 137: 4586-4591Crossref PubMed Scopus (77) Google Scholar). Briefly, solubilized cell proteins were diluted 1:1 in buffer B (20 mm HEPES, pH 7.2, 300 mm NaCl, 0.1% SDS, 0.5% Nonidet P-40, and the protease inhibitors described above) and were precleared with normal mouse serum IgG prebound to 25 μl of packed and washed protein A-agarose (Sigma). Mouse monoclonal antibodies were added (16 h at 4 °C), and immune complexes were recovered using protein A-agarose. The following antibodies were used: (i) to TSHR A subunit amino acid residues 147–229 (A9; Dr. Paul Banga, King's College, London, United Kingdom; 1:1,500 final) (24Nicholson L.B. Vlase H. Graves P. Nilsson M. Molne J. Huang G.C. Morgenthaler N.G. Davies T.F. McGregor A.M. Banga J.P. J. Mol. Endocrinol. 1996; 16: 159-170Crossref PubMed Scopus (68) Google Scholar), (ii) to the TSHR B subunit (T3–365; Dr. Edwin Milgrom, Hôpital de Bicetre, Le Kremlin Bicetre, France; 2 μg/ml) (4Loosfelt H. Pichon C. Jolivet A. Misrahi M. Caillou B. Jamous M. Vannier B. Milgrom E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3765-3769Crossref PubMed Scopus (175) Google Scholar), or (iii) to the c-myc epitope introduced into the TSHR at residues 338–349 (21Tanaka K. Nagayama Y. Yamasaki H. Hayashi H. Namba H. Yamashita S. Niwa M. Biochem. Biophys. Res. Commun. 1996; 228: 21-28Crossref PubMed Scopus (10) Google Scholar) (9E10; American Type Tissue Culture; 1:500).After extensive washing in buffer B, immunoprecipitated TSHR were removed from the protein A by adding 3 μl of 10% SDS and 150 μl of buffer B (30 min at 50 °C). After centrifugation for 3 min at 10,000 × g at 4 °C, the supernatant was diluted with 150 μl of buffer B, and incubated for 2 h with 25 μl of pre-washed streptavidin-agarose (Pierce) and centrifuged. The supernatant containing non-biotinylated TSHR was retained. The beads were washed four times with buffer B and once with PBS and resuspended in Laemmli sample buffer with 0.7 m β-mercaptoethanol (30 min at 50 °C) to release the bound, biotinylated TSHR. Aliquots of both biotinylated and non-biotinylated TSHR were electrophoresed on 10% SDS-polyacrylamide gels (Bio-Rad). Prestained molecular weight markers (Bio-Rad) were included in parallel lanes. We precalibrated these markers against more accurate unstained markers (Bio-Rad) to obtain the molecular weights indicated in the text. Radiolabeled proteins were visualized by autography on Biomax MS x-ray film (Eastman Kodak Co.).Enzymatic Deglycosylation of TSHR ProteinBiotinylated TSHR bound to streptavidin-agarose or TSHR not bound to the streptavidin-agarose beads were incubated (10 min, 100 °C) in denaturing buffer containing 0.5% SDS, 1% β-mercaptoethanol. Enzymatic deglycosylation was performed according to the protocol of the manufacturer (New England Biolabs).N-Glycosidase F digestion (100 units for 2 h at 37 °C) was in 50 mm sodium phosphate, pH 7.5, 1% Nonidet P-40. Endoglycosidase H digestion (50 units for 2 h at 37 °C) was in 50 mm sodium citrate, pH 5.5. Samples were then subjected to SDS-polyacrylamide gel electrophoresis as described above.Attempts to Purify the TSHR C Peptide Region Released into the Culture MediumMetabolic Labeling and ImmunoprecipitationTSHRmyc-10,000 cells near confluence in 60-mm diameter culture dishes were metabolically labeled as described above, with the following modifications. Cells were pulsed for 3 h at 37 °C in 2 ml of Earle's balanced salt solution with 5% dialyzed FCS and 5 μCi/mll-[U-14C]amino acid mixture (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom). After rinsing, cells were cultured in standard F12 medium with 10% FCS. The medium was harvested after 16 h, and 9E10 mouse monoclonal antibody to the c-myc epitope was added, as described above.Affinity Purification23 mg of purified 9E10 IgG was added to 2 g of pre-washed CNBr-Sepharose 4B (Amersham Pharmacia Biotech) and tumbled for 2 h at room temperature. The beads were then blocked with 0.1 m Tris-HCl, rinsed successively with 0.1 m acetate buffer, pH 4.0, 0.5 m NaCl and with 0.1 m Tris-HCl, pH 8.0, 0.5 m NaCl. F-12 medium with 10% FCS (1200 ml) harvested from 10-cm diameter dishes of TSHRmyc-10,000 cells was applied (1.5 ml/min) to the 9E10-agarose affinity column. After rinsing the column with 0.1 mammonium acetate, pH 7.5, protein was eluted with 1 macetic acid, pH 2.2. The fractions were evaporated to dryness under vacuum, resuspended in Laemmli sample buffer with 0.7 mβ-mercaptoethanol (30 min at 50 °C), electrophoresed on 16.5% SDS-polyacrylamide gels, and electrophoretically transferred to polyvinylidene difluoride membranes (Bio-Rad). Membranes were incubated overnight (4 °C) with 9E10 (final dilution;1:500). After rinsing, the membranes were incubated for 1 h at room temperature with alkaline phosphatase-conjugated goat anti-mouse IgG (1:400) (Cappel, Durham, NC). The signal was developed with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate in 100 mm Tris-HCl buffer, pH 9.5, containing 100 mm NaCl and 5 mmMgCl2.DISCUSSIONThe present data are the first to examine the properties of TSHR, including their A and B subunits, expressed on the surface of intact cells. Analysis of TSHR carbohydrate moieties establishes the efficacy of the cell surface biotinylation approach that we used to distinguish between cell surface and intracellular receptors. Previous studies on TSH cross-linking to TSHR on the surface of intact cells (for example, Refs. 2Furmaniak J. Hashim F.A. Buckland P.R. Petersen V.B. Beever K. Howells R.D. Rees Smith B. FEBS Lett. 1987; 215: 316-322Crossref PubMed Scopus (38) Google Scholar and 3Russo D. Chazenbalk G.D. Nagayama Y. Wadsworth H.L. Seto P. Rapoport B. Mol. Endocrinol. 1991; 5: 1607-1612Crossref PubMed Scopus (75) Google Scholar) could distinguish between single-chain receptors and the A subunits of cleaved receptors, but the ligand-receptor complexes were not amenable to fine resolution of their sizes and the non-ligand binding B subunit could not be detected. Immunopurification of TSHR does provide more detailed information (for example, Ref. 4Loosfelt H. Pichon C. Jolivet A. Misrahi M. Caillou B. Jamous M. Vannier B. Milgrom E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3765-3769Crossref PubMed Scopus (175) Google Scholar). However, such studies have been performed on cell and thyroid homogenates, which include mixtures of cell surface receptors and intracellular receptors at different stages of the synthetic and degradative pathways. Intracellular products are reported to be particularly abundant in transfected cells expressing the recombinant TSHR (17Misrahi M. Ghinea N. Sar S. Saunier B. Jolivet A. Loosfelt H. Cerutti M. Devauchelle G. Milgrom E. Eur. J. Biochem. 1994; 222: 711-719Crossref PubMed Scopus (108) Google Scholar).The data on cell surface TSHR expression clarify a number of puzzling or controversial issues. One issue that has been the subject of debate for many years is whether or not mature, single-chain TSHR exist on the cell surface. This possibility was raised by the detection of single-chain TSHR on TSH cross-linking to intact cells (2Furmaniak J. Hashim F.A. Buckland P.R. Petersen V.B. Beever K. Howells R.D. Rees Smith B. FEBS Lett. 1987; 215: 316-322Crossref PubMed Scopus (38) Google Scholar, 3Russo D. Chazenbalk G.D. Nagayama Y. Wadsworth H.L. Seto P. Rapoport B. Mol. Endocrinol. 1991; 5: 1607-1612Crossref PubMed Scopus (75) Google Scholar). Disulfide-linked, two-subunit receptors were well recognized (4Loosfelt H. Pichon C. Jolivet A. Misrahi M. Caillou B. Jamous M. Vannier B. Milgrom E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3765-3769Crossref PubMed Scopus (175) Google Scholar, 26Buckland P.R. Rickards C.R. Howells R.D. Jones E.D. Rees Smith B. FEBS Lett. 1982; 145: 245-249Crossref Scopus (50) Google Scholar). However, high affinity TSH binding to the single-chain TSHR comparable to the cleaved receptor led to the suggestion that the former were physiological (3Russo D. Chazenbalk G.D. Nagayama Y. Wadsworth H.L. Seto P. Rapoport B. Mol. Endocrinol. 1991; 5: 1607-1612Crossref PubMed Scopus (75) Google Scholar). This concept has been vigorously disputed, and these observations have been considered an artifact of transfected cells (4Loosfelt H. Pichon C. Jolivet A. Misrahi M. Caillou B. Jamous M. Vannier B. Milgrom E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3765-3769Crossref PubMed Scopus (175) Google Scholar,6de Bernard S. Misrahi M. Huet J.-C. Beau I. Desroches A. Loosfelt H. Pichon C. Pernollet J.-C. Milgrom E. J. Biol. Chem. 1999; 274: 101-107Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 17Misrahi M. Ghinea N. Sar S. Saunier B. Jolivet A. Loosfelt H. Cerutti M. Devauchelle G. Milgrom E. Eur. J. Biochem. 1994; 222: 711-719Crossref PubMed Scopus (108) Google Scholar, 27Misrahi M. Milgrom E. Eur. J. Endocrinol. 1997; 137: 599-602Crossref PubMed Scopus (33) Google Scholar). It is proposed that single-chain TSHR precursors with immature carbohydrate have been "mistaken for the mature receptor" (6de Bernard S. Misrahi M. Huet J.-C. Beau I. Desroches A. Loosfelt H. Pichon C. Pernollet J.-C. Milgrom E. J. Biol. Chem. 1999; 274: 101-107Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) and that such precursors are readily detected in transfected cells because of their abnormal abundance consequent to TSHR overexpression overwhelming the mechanism for glycan maturation and TSHR processing (17Misrahi M. Ghinea N. Sar S. Saunier B. Jolivet A. Loosfelt H. Cerutti M. Devauchelle G. Milgrom E. Eur. J. Biochem. 1994; 222: 711-719Crossref PubMed Scopus (108) Google Scholar, 27Misrahi M. Milgrom E. Eur. J. Endocrinol. 1997; 137: 599-602Crossref PubMed Scopus (33) Google Scholar). The present data clearly indicate that single-chain TSHR on the cell surface have mature, complex carbohydrate and are consistent with the previous observation of high affinity TSH binding to single-chain TSHR on the cell surface (3Russo D. Chazenbalk G.D. Nagayama Y. Wadsworth H.L. Seto P. Rapoport B. Mol. Endocrinol. 1991; 5: 1607-1612Crossref PubMed Scopus (75) Google Scholar). Moreover, single-chain receptors capable of TSH binding are not confined to transfected cells, but are also observed in a well differentiated rat thyroid cell line (2Furmaniak J. Hashim F.A. Buckland P.R. Petersen V.B. Beever K. Howells R.D. Rees Smith B. FEBS Lett. 1987; 215: 316-322Crossref PubMed Scopus (38) Google Scholar). Finally, the same proportion of single-chain versuscleaved TSHR on the cell surface is observed over a 100-fold range in the level of expression in transfected cells (20Chazenbalk G.D. Kakinuma A. Jaume J.C. McLachlan S.M. Rapoport B. Endocrinology. 1996; 137: 4586-4591Crossref PubMed Scopus (77) Google Scholar), excluding the suggested limitation in synthetic capacity.Turning to the process of TSHR cleavage, the impressive accomplishment of de Bernard et al. (6de Bernard S. Misrahi M. Huet J.-C. Beau I. Desroches A. Loosfelt H. Pichon C. Pernollet J.-C. Milgrom E. J. Biol. Chem. 1999; 274: 101-107Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) in affinity-purifying large quantities of TSHR B subunits from thyroid tissue and from transfected mouse L cells confirms previous findings that an epitope within a C peptide segment is excised from the TSHR (5Chazenbalk G.D. Tanaka K. Nagayama Y. Kakinuma A. Jaume J.C. McLachlan S.M. Rapoport B. Endocrinology. 1997; 138: 2893-2899Crossref PubMed Scopus (91) Google Scholar) with an upstream cleavage site in the vicinity of amino acid residues 305–316 (3Russo D. Chazenbalk G.D. Nagayama Y. Wadsworth H.L. Seto P. Rapoport B. Mol. Endocrinol. 1991; 5: 1607-1612Crossref PubMed Scopus (75) Google Scholar, 12Tanaka K. Chazenbalk G.D. McLachlan S.M. Rapoport B. J. Biol. Chem. 1998; 273: 1959-1963Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Our inability to purify an intact C peptide, similar to the experience of this group (6de Bernard S. Misrahi M. Huet J.-C. Beau I. Desroches A. Loosfelt H. Pichon C. Pernollet J.-C. Milgrom E. J. Biol. Chem. 1999; 274: 101-107Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), supports the concept that the C peptide region is removed through a process of progressive cleavage or degradation. Regarding the region downstream of the excised C peptide segment, B subunits purified from homogenates of thyroid tissue and transfected mouse L cells reveal multiple N termini, the dominant ones being between amino acid residues 366 and 378 (6de Bernard S. Misrahi M. Huet J.-C. Beau I. Desroches A. Loosfelt H. Pichon C. Pernollet J.-C. Milgrom E. J. Biol. Chem. 1999; 274: 101-107Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), consistent with previous mutagenesis data suggesting the approximate location of cleavage "site 2" (14Kakinuma A. Chazenbalk G.D. Tanaka K. Nagayama Y. McLachlan S.M. Rapoport B. J. Biol. Chem. 1997; 272: 28296-28300Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Taken together, these data suggest that, in contrast to our previous interpretation of available evidence, C peptide excision may not occur by means of two distinct cleavage sites. Rather, as reported by de Bernard et al. (6de Bernard S. Misrahi M. Huet J.-C. Beau I. Desroches A. Loosfelt H. Pichon C. Pernollet J.-C. Milgrom E. J. Biol. Chem. 1999; 274: 101-107Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), cleavage is likely to occur initially upstream at site 1 and is followed by progressive cleavage or N-terminal degradation of the B subunit downstream to the region of site 2. Regardless of whether there are one or two primary cleavage sites, the end result is the same, namely removal of a C peptide region between sites 1 and 2.Our present data provide new insight into other aspects of TSHR cleavage that have been diffi
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