Sequential Cleavage and Excision of a Segment of the Thyrotropin Receptor Ectodomain
1999; Elsevier BV; Volume: 274; Issue: 1 Linguagem: Inglês
10.1074/jbc.274.1.101
ISSN1083-351X
AutoresSimon de Bernard, Micheline Misrahi, Jean‐Claude Huet, Isabelle Beau, Agnès Desroches, Hugues Loosfelt, Christophe Pichon, Jean‐Claude Pernollet, Edwin Milgröm,
Tópico(s)Blood Coagulation and Thrombosis Mechanisms
ResumoThe thyrotropin (TSH) receptor belongs to a subfamily of G protein-coupled receptors, which also includes luteinizing hormone and follicle-stimulating hormone receptors. The TSH receptor (TSHR) differs from the latter by the presence of an additional specific segment in the C-terminal part of its ectodomain. We show here that this insertion is excised in the majority of receptor molecules. Preparation of specific monoclonal antibodies to this region, microsequencing, enzyme-linked immunosorbent assay, and immunoblot studies have provided insight into the mechanisms of this excision. In the human thyroid gland, N termini of the transmembrane receptor β subunit were found to be phenylalanine 366 and leucines 370 and 378. In transfected L cells a variety of other more proximal N termini were found, probably corresponding to incomplete excisions. The most extreme N terminus was observed to lie at Ser-314. These observations suggest that after initial cleavage at Ser-314 the inserted fragment of TSHR is progressively clipped out by a series of cleavage reactions progressing up to amino acids 366–378. The impossibility of recovering the excised fragment from purified receptor, cell membranes, or culture medium supports this interpretation. The cleavage enzyme has previously been shown to be inhibited by BB-2116, an inhibitor of matrix metalloproteases. However, we show here that it is unaffected by tissue inhibitors of metalloproteases. The cleavage enzyme is very similar to TACE (tumor necrosis factor α-converting enzyme) in both these characteristics. However, incubation of the TSH receptor with the purified recombinant catalytic domain of TACE, co-transfection of cells with TACE and TSHR expression vectors, and the use of mutated Chinese hamster ovary cells in which TACE is inactive suggested that the TSHR cleavage enzyme is different from TACE. TACE and TSHR cleavage enzyme may thus possibly be related but different members of the adamalysin family of metzincin metalloproteases. The thyrotropin (TSH) receptor belongs to a subfamily of G protein-coupled receptors, which also includes luteinizing hormone and follicle-stimulating hormone receptors. The TSH receptor (TSHR) differs from the latter by the presence of an additional specific segment in the C-terminal part of its ectodomain. We show here that this insertion is excised in the majority of receptor molecules. Preparation of specific monoclonal antibodies to this region, microsequencing, enzyme-linked immunosorbent assay, and immunoblot studies have provided insight into the mechanisms of this excision. In the human thyroid gland, N termini of the transmembrane receptor β subunit were found to be phenylalanine 366 and leucines 370 and 378. In transfected L cells a variety of other more proximal N termini were found, probably corresponding to incomplete excisions. The most extreme N terminus was observed to lie at Ser-314. These observations suggest that after initial cleavage at Ser-314 the inserted fragment of TSHR is progressively clipped out by a series of cleavage reactions progressing up to amino acids 366–378. The impossibility of recovering the excised fragment from purified receptor, cell membranes, or culture medium supports this interpretation. The cleavage enzyme has previously been shown to be inhibited by BB-2116, an inhibitor of matrix metalloproteases. However, we show here that it is unaffected by tissue inhibitors of metalloproteases. The cleavage enzyme is very similar to TACE (tumor necrosis factor α-converting enzyme) in both these characteristics. However, incubation of the TSH receptor with the purified recombinant catalytic domain of TACE, co-transfection of cells with TACE and TSHR expression vectors, and the use of mutated Chinese hamster ovary cells in which TACE is inactive suggested that the TSHR cleavage enzyme is different from TACE. TACE and TSHR cleavage enzyme may thus possibly be related but different members of the adamalysin family of metzincin metalloproteases. thyrotropin TSH receptor Endo-Ab, and Del-Ab, antibodies recognizing, respectively, receptor ectodomain, endodomain, and the putative deleted fragment matrix metalloprotease tissue inhibitor of metalloproteases tumor necrosis factor TNFα converting enzyme transforming growth factor bovine serum albumin enzyme-linked immunosorbent assay Chinese hamster ovary hemagglutinin. The thyrotropin receptor (TSHR)1 plays a key role in thyroid growth and function (reviewed in Refs. 1Vassart G. Dumont J.E. Endocr. Rev. 1992; 13: 596-611PubMed Google Scholar and 2Misrahi M. Milgrom E. Weetman A.P. Grossman A. Handbook of Experimental Pharmacology. 128. Springer-Verlag, Berlin1997: 33-73Google Scholar). This receptor is the target of stimulating or blocking autoantibodies produced in patients with autoimmune diseases (reviewed in Refs. 3Rees Smith B. McLachlan S.M. Furmaniak J. Endocr. Rev. 1988; 9: 106-121Crossref PubMed Scopus (538) Google Scholar and4Weetman A.P. McGregor A.M. Endocr. Rev. 1994; 15: 788-830PubMed Google Scholar). The TSHR was initially cloned by cross-hybridization with the luteinizing hormone receptor (5Misrahi M. Loosfelt H. Atger M. Sar S. Guiochon-Mantel A. Milgrom E. Biochem. Biophys. Res. Commun. 1990; 166: 394-403Crossref PubMed Scopus (281) Google Scholar), or by polymerase chain reaction using degenerate primers (6Libert F. Lefort A. Gerard C. Parmentier M. Perret J. Ludgate M. Dumont J.E. Vassart G. Biochem. Biophys. Res. Commun. 1989; 165: 1250-1255Crossref PubMed Scopus (398) Google Scholar, 7Nagayama Y. Kaufman K.D. Seto P. Rapoport B. Biochem. Biophys. Res. Commun. 1989; 165: 1184-1190Crossref PubMed Scopus (517) Google Scholar, 8Frazier A.L. Robbins L.S. Stork P.J. Sprengel R. Segaloff D.L. Cone R.D. Mol. Endocrinol. 1990; 4: 1264-1276Crossref PubMed Scopus (139) Google Scholar). Expression of the cloned receptor inEscherichia coli allowed its use as an immunogen to prepare monoclonal antibodies. These were used for immunoblotting and immunoprecipitation experiments which showed that the TSH receptor in human thyroid membranes underwent a post-translational cleavage event yielding two subunits: a ∼53-kDa α extracellular subunit and a ∼38-kDa broad β membrane spanning subunit. The subunits are held together by disulfide bridges (9Loosfelt 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 (176) Google Scholar). This maturation is unique among G protein-coupled receptors. In human thyroids, cleavage of the TSHR is almost complete. By contrast, in heterologous transfected cells monomeric uncleaved precursors may also be observed. They consist either of the mature ∼120-kDa uncleaved receptor present on the cell surface or of a ∼95-kDa mannose-rich precursor which can be identified by its sensitivity to specific endoglycosidases (10Misrahi 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 (109) Google Scholar). The latter form accumulates in the endoplasmic reticulum. The cleavage of the TSHR has been disputed by different authors (11Russo D. Chazenbalk G.D. Nagayama Y. Wadsworth H.L. Seto P. Rapoport B. Mol. Endocrinol. 1991; 5: 1607-1612Crossref PubMed Scopus (76) Google Scholar, 12Ban T. Kosugi S. Kohn L.D. Endocrinology. 1992; 131: 815-829Crossref PubMed Scopus (45) Google Scholar, 13Endo T. Ikeda M. Ohmori M. Anzai E. Haraguchi K. Onaya T. Biochem. Biophys. Res. Commun. 1992; 187: 887-893Crossref PubMed Scopus (18) Google Scholar, 14Harfst E. Ross M.S. Nussey S.S. Johnstone A.P. Mol. Cell. Endocrinol. 1994; 102: 77-84Crossref PubMed Scopus (21) Google Scholar, 15Potter E. Horn R. Scheumann G.F. 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, 16Grossman 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). Indeed, due to the low concentration of the TSHR in thyroid tissue, nearly all studies have been performed in transfected cells where the monomeric precursors and specially the mannose-rich form have in many cases been mistaken for the mature receptor. Recently, however, a consensus has emerged and most authors now agree on the existence of a physiological cleavage of the TSH receptor (17Chazenbalk G.D. Rapoport B. J. Biol. Chem. 1994; 269: 32209-32213Abstract Full Text PDF PubMed Google Scholar, 18Graves P.N. Vlase H. Bobovnikova Y. Davies T.F. Endocrinology. 1996; 137: 3915-3920Crossref PubMed Scopus (60) Google Scholar). Very recently the group of Rapoport reported the existence of two cleavage sites in the TSHR extracellular region, suggesting the existence of a third polypeptide fragment ("C peptide" by homology with insulin) released during intramolecular cleavage of the receptor into two subunits (19Chazenbalk G.D. Tanaka K. Nagayama Y. Kakinuma A. Jaume J.C. McLachlan S.M. Rapoport B. Endocrinology. 1997; 138: 2893-2899Crossref PubMed Scopus (93) Google Scholar, 20Kakinuma 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 (19) Google Scholar, 21Tanaka 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). In this work we show that no third fragment of the TSHR is produced during its maturation, but rather that cleavage occurs initially at a first site, followed by the processive digestion and excision of a whole region of the receptor ectodomain. The region which is deleted is located in an additional segment specific to the TSHR which shows no homology with the gonadotropin receptors (5Misrahi M. Loosfelt H. Atger M. Sar S. Guiochon-Mantel A. Milgrom E. Biochem. Biophys. Res. Commun. 1990; 166: 394-403Crossref PubMed Scopus (281) Google Scholar). In addition, besides the similarities that we had previously observed between pro-TNFα (pro-tumor necrosis factor α) and TSHR convertases (22Couet J. Sar S. Jolivet A. Hai M.-T.V. Milgrom E. Misrahi M. J. Biol. Chem. 1996; 271: 4545-4552Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar), we provide here new information concerning the protease involved in the maturation of the TSHR and show that it may correspond to a novel enzyme. Phorbol 12-myristate 13-acetate was purchased from Sigma. Anti-hemagglutinin (HA) monoclonal (12CA5) antibody and the "Complete" protease inhibitor mixture tablets were obtained from Boehringer (Mannheim, Germany). Human recombinant tissue inhibitor of metalloproteases 1 (TIMP-1) was purchased from Valbiotech (Paris, France). Human recombinant tissue inhibitor of metalloproteases 2 (TIMP-2) was a gift from Dr Agnès Noël (University of Liège, Belgium). The wild-type and mutant Chinese hamster ovary (CHO) cell lines defective in the shedding of several membrane proteins (23Arribas J. Massague J. J. Cell Biol. 1995; 128: 433-441Crossref PubMed Scopus (131) Google Scholar, 24Arribas J. Coodly L. Vollmer P. Kishimoto T.K. Rose-John S. Massague J. J. Biol. Chem. 1996; 271: 11376-11382Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar) were kindly provided by Dr. Joaquin Arribas (Val d'Hebron General Hospital, Barcelona, Spain). The recombinant catalytic domain of tumor necrosis factor α enzyme (TACE) and the murine TACE expression vectors (25Black R.A. Rauch C.T. Kozlosky C.J. Peschon J.J. Slack J.L. Wolfson M.F. Castner B.J. Stocking K.L. Reddy P. Srinivasan S. Nelson N. Boiani N. Schooley K.A. Gerhart M. Davis R. Fitzner J.N. Johnson R.S. Paxton R.J. March C.J. Cerretti D.P. Nature. 1997; 385: 729-733Crossref PubMed Scopus (2761) Google Scholar) were gifts from Dr. Roy Black (Immunex Corp., Seattle, WA). BB-2116 (26Gearing A.J. Beckett P. Christodoulou M. Churchill M. Clements J. Davidson A.H. Drummond A.H. Galloway W.A. Gilbert R. Gordon J.L. Leber T.M. Mangan M. Miller K. Nayec P. Owen K. Patel S. Thomas W. Weils G. Wood L.M. Wooley K. Nature. 1994; 370: 555-557Crossref PubMed Scopus (1118) Google Scholar) was a gift from British Biotech Co. (Oxford, United Kingdom). The synthetic peptides used for the localization of Del-Ab (antibodies recognizing the putative deleted fragment of TSHR ectodomain) epitope(s) and the study of the action of TACE were purchased from the microchemistry laboratory of Institut Gustave Roussy (Villejuif, France). The preparation of Ecto-Ab (T5–317, an antibody recognizing the TSHR ectodomain) and Endo-Ab (T3–365, an antibody recognizing the TSHR endodomain) has been previously described (9Loosfelt 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 (176) Google Scholar). For the preparation of Del-Ab, a cDNA fragment encoding amino acids 19–389 of the human TSHR was introduced into the polylinker of the vector pNMHUB. A fusion protein of TSHR with ubiquitin and polyhistidine was produced in E. coli. The protein was purified from inclusion bodies using chelate chromatography on nickel-agarose in denaturing conditions. Immunization of BALB/c mice, preparation and screening of hybridomas, production of ascites, and purification of antibodies were performed as described previously (9Loosfelt 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 (176) Google Scholar). The L cell line stably expressing the human TSH receptor has been previously described (10Misrahi 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 (109) Google Scholar). The TSH receptor was immunopurified and Western blotting performed as described (10Misrahi 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 (109) Google Scholar), except that the secondary antibody used during the Western blots was a mouse anti-Ig coupled to horseradish peroxidase. Revelation was performed with the ECL detection reagent (Amersham Corp., Buckinghamshire, United Kingdom). Ecto-Ab, Endo-Ab, or Del-Ab were used for immunomatrix preparation and Western blot detection. For the immunopurification of the shed α subunit from the cell culture medium, the medium was first concentrated approximately 15-fold using a Minitan apparatus (Millipore, Bedford, MA) equipped with a filter having a 1,000 Da molecular mass cut-off. Enzyme-linked immunosorbent assays (ELISA) were performed as described previously (9Loosfelt 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 (176) Google Scholar). The concentration of the TSH receptor molecules was measured by reference to known concentrations of TSHR fragments expressed in E. coli (hTSHR 19–389 when Ecto-Ab or Del-Ab were used as primary antibodies, hTSHR 640–764 when Endo-Ab was used as primary antibody). The primary antibodies were used at saturating concentrations (5 μg/ml for Ecto-Ab or Endo-Ab, 1 μg/ml for Del-Ab) in order to bypass differences in antibody affinities. It was also verified that the secondary polyclonal antibody had the same affinity for the three primary antibodies. The immunoradiometric assay of the TSH receptor and of its α subunit have been previously described (22Couet J. Sar S. Jolivet A. Hai M.-T.V. Milgrom E. Misrahi M. J. Biol. Chem. 1996; 271: 4545-4552Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). For the assay of the α subunit, the cell culture medium of wild-type and mutant CHO cells transfected with TSHR cDNA was concentrated 13-fold using Centriprep-10 concentrators (Amicon, Beverly, MA). L cells expressing the human TSH receptor were incubated for 2 h at 4 °C with 5 ml/g of phosphate-buffered saline, 100 mm dithiothreitol, and protease inhibitors. This procedure provokes the reduction of disulfide bonds and the release of α subunits (22Couet J. Sar S. Jolivet A. Hai M.-T.V. Milgrom E. Misrahi M. J. Biol. Chem. 1996; 271: 4545-4552Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). The cells were washed three times with phosphate-buffered saline. TSHR was then extracted as described (10Misrahi 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 (109) Google Scholar) and immunopurified on an immunomatrix containing Ecto-Ab. This procedure leads to the purification of uncleaved monomeric TSHR since α subunits have previously been released. The recombinant catalytic domain of TACE (10,000 units) was preincubated for 30 min at 37 °C in the presence or absence of the BB-2116 inhibitor. The TSH receptor enriched in monomeric forms was then added and incubation was continued for 2 h at 37 °C. The samples were then subjected to electrophoresis and Western blots were performed using various monoclonal antibodies. COS-7 cells (∼106 cells per Petri dish) were seeded 24 h before transfection. TACE expression vector (5 μg) was then transfected with 5 μg of either human TSHR expression vector or herring sperm DNA (Promega, Madison, WI). The Superfect reagent (Qiagen Inc., Hilden, Germany) was used according to the manufacturer's protocol. The human TSH receptor was immunopurified with either Endo-Ab or Del-Ab. Endo-Ab immunopurification and microsequencing were performed twice using two different euthyroid goiters (yielding ∼1 pmol of TSHR/g of tissue) or four times with different pools of stably transfected L cells (yielding ∼50 pmol of TSHR/g of cells). Del-Ab was used to enrich receptor preparation in incompletely processed β subunits present in lower amounts in transfected L cells. A microsequencing experiment was performed on this material. After immunopurification, the TSH receptor (∼200–500 pmol) was electrophoresed and transferred onto a polyvinylidene difluoride membrane (ProBlott, Applied Biosystems, Foster City, CA). Proteins were colored by Coomassie Blue. Bands of interest were localized by reference to immunoblots, separately cut and sequenced following Edman's method using a multi-sample chemical microsequencer (Procise 494–610A, Applied Biosystems). The sequences observed were at least 12 amino acids long and the yield of the different fragments varied from 0.5 to 8 pmol. In repeated experiments identical results were obtained. These cell lines were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 600 μg/ml geneticin (all reagents were from Life Technologies, Inc., Grand Island, NY). The DEAE-dextran method was used to transfect the PSG5-TSHR expression vector. Exponentially growing wild-type and mutant CHO cells (∼2 × 106 cells) expressing HA-tagged pro-TGFα (pro-transforming growth factor α) were incubated for 30 min at 37 °C with labeling medium (cysteine-free modified Eagle's medium, 20 mm Hepes pH 7.5, 1 mg/ml bovine serum albumin (BSA)). [35S]Cysteine (500 μCi/ml) (NEN Life Science Products, Boston, MA) was then added to the labeling medium and cells were further incubated for 1 h at 37 °C. The cells were then washed twice in Dulbecco's modified Eagle's medium without NaHCO3, 20 mm Hepes pH 7.5, 2 mg/ml BSA and incubated for 45 min in chase buffer (Dulbecco's modified Eagle's medium without NaHCO3, 20 mm Hepes pH 7.5, 10 mg/ml BSA) with or without 1 μm phorbol 12-myristate 13-acetate. Protease inhibitors were added to the collected cell culture medium. The cells were washed twice with cold chase buffer and once with cold phosphate-buffered saline. The cells were then gently scraped and harvested in lysis buffer (20 mm Tris pH 7.5, 150 mm NaCl, 1% Triton X-100, 5 mm EDTA, 0.2% BSA, and protease inhibitors). The cells were lysed for 30 min at 4 °C on a rotating wheel. The lysed cells were centrifugated for 30 min at 100,000 × g at 4 °C and the supernatant collected. Anti-HA monoclonal antibody was added to the cell culture media and the cell lysates for 1 h. The immune complexes were incubated with protein A-Sepharose (Pharmacia Biotech, Uppsala, Sweden) for 1 h at 4 °C, washed twice with lysis buffer, twice with 20 mm Tris pH 7.5, 150 mm NaCl, 0.5% Triton X-100, 5 mm EDTA, 0.1% sodium dodecyl sulfate, 0.2% BSA, twice with 20 mm Tris pH 7.5, 500 mm NaCl, 0.5% Triton X-100, 0.2% BSA and twice with 50 mm Tris pH 7.5. The samples were then boiled for 10 min in Laemmli buffer and analyzed on a 14% polyacrylamide gel for the lysate samples and on a 16% polyacrylamide gel for the medium samples. A TSH receptor fragment (amino acids 19–389) was expressed in E. coli as a fusion protein with ubiquitin. Immunization of mice with this antigen allowed the preparation of several monoclonal antibodies. The study of one group of these monoclonal antibodies (called here Del-Ab) gave puzzling results. When used for Western blot analysis of TSHR from human thyroids they reacted neither with the α nor with the β subunits but recognized a group of proteins (∼39–44 kDa) larger than the most abundant β subunit (∼38 kDa) (Fig.1 A). The same proteins were recognized by anti-endodomain antibodies (Endo-Ab), as seen after overexposure of the corresponding immunoblot (Fig. 1 A). These proteins were thus extended β subunits. The Del-Ab gave no reaction with the α subunit nor with any protein larger than this subunit. Traces of the mannose-rich precursor of TSHR present in human thyroid glands and observed in overexposed immunoblots (10Misrahi 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 (109) Google Scholar) were also detected by these antibodies. The same results were obtained when five different thyroid samples were studied. These results are compatible with the possibility that the epitope recognized by Del-Ab antibodies was cleaved-off during maturation of the TSHR in human thyroids. However, this epitope remained covalently attached to a minority of β subunits ("extended" β subunits). There were no extended α subunits carrying the epitope. We undertook the localization of the epitope recognized by this group of antibodies. Using bacterially expressed proteins encoding various segments of the TSHR ectodomain (amino acids 19–389, 19–246, and 246–389) and chemically synthesized peptides corresponding to residues 332–369, 332–356, and 357–369 we localized the epitope to amino acids 357–369. We also observed that the antibodies we had prepared, and which gave the pattern described in Fig. 1, were all non-additive and thus probably recognized the same epitope or very closely positioned epitopes (data not shown). Western blot experiments thus suggested that only a minority of human thyroid TSHR molecules had conserved the epitope recognized by Del-Ab. We used a quantitative method to measure this population of receptors more precisely. ELISA tests were performed with purified human thyroid TSHR, using an antibody which recognizes either the receptor endodomain (Endo-Ab) or the segment which was cleaved-off (Del-Ab). As shown in Fig. 1 B, about 75% of receptor molecules were devoid of this segment. Since human thyroid tissue is difficult to obtain, and since many studies of the TSH receptor are performed using transfected cells, we repeated the immunoblot studies using TSHR purified from L cells stably expressing the receptor. Previous studies (10Misrahi 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 (109) Google Scholar) have shown that in transfected cells there is accumulation of a mannose-rich intracellular precursor (∼95 kDa) and incomplete receptor cleavage with persistence of various amounts of uncleaved full-length mature receptor (∼120 kDa). Furthermore, in transfected cells the β subunit has been shown to be heterogeneous (10Misrahi 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 (109) Google Scholar). In the receptor prepared from transfected L cells, Del-Ab antibody reacted as expected with the 2 uncleaved forms of the receptor (Fig. 2). It also reacted with the largest forms of the β subunit (extended β subunits). Again no reaction was seen with the α subunit and there was no indication of larger forms of the α subunit carrying the Del-Ab epitope. The preceding experiments raised the question of the fate of the excised receptor fragment. Theoretically, it could have remained bound to the receptor by non-covalent interactions (constituting a third subunit of the receptor), or it could have remained attached to the cell membrane, or it could have been released from the cells. We examined the latter possibility by searching for the fragment in the cell culture medium of L cells expressing the TSH receptor. As shown in Fig. 3 no antigen able to bind Del-Ab could be detected in the cell culture medium, whereas the shed α subunit (27Couet J. de Bernard S. Loosfelt H. Saunier B. Milgrom E. Misrahi M. Biochemistry. 1996; 35: 14800-14805Crossref PubMed Scopus (113) Google Scholar) was detected by anti-ectodomain antibodies. We then prepared a human thyroid membrane Triton X-100 extract and subjected it to chromatography on a Del-Ab immunomatrix. Immunoblots (Fig. 4 A) with Del-Ab only showed the extended forms of β subunit previously described (see Fig.1 A). There was no evidence of a fragment of ∼6.5 kDa which could correspond to the excised fragment (not shown). This experiment thus suggested that the receptor fragment which was cleaved-off did not remain attached to the membrane either in a free form or bound to the receptor. Another line of evidence also showed that the excised fragment did not constitute a third subunit of the receptor. Indeed, if this had been the case immunopurification using Del-Ab would have yielded all 3 fragments of the receptor. This was not the case: no normal (non-extended) β subunit of ∼38 kDa was purified. Only the larger β subunits of ∼39–44 kDa, to which the epitope for Del-Ab remains covalently attached due to incomplete cleavage, were purified. The existence of these extended β subunits to which α subunits remain bound would explain why some of the latter are also retained during chromatography on the Del-Ab immunomatrix. Furthermore, when the human thyroid membrane extract was chromatographed on two successive immunomatrices, first Del-Ab then Endo-Ab, the latter retained the majority of β subunits (of ∼38 kDa) and the majority of α subunits (not shown). This experiment shows that there are two populations of receptors: a majority of α-β dimers devoid of the Del-Ab epitope and a minority of α-"extended β" dimers. The latter carry the Del-Ab epitope. We also used the Del-Ab immunomatrix to purify a Triton X-100 extract from membranes of L cells expressing the TSHR (Fig. 4 B). The results were similar to those obtained with human thyroid TSHR: absence of a third fragment and purification of only the extended, largest, forms of the β subunit. The inability to recover the excised fragment from the receptor, membrane extracts, and cell culture medium suggested that it was either immediately destroyed or initially excised in small pieces. Microsequencing experiments suggested that the latter explanation was the most probable. The TSHR was immunopurified from human thyroid glands using an anti-endodomain antibody. After electrophoresis and transfer to a polyvinylidene difluoride membrane the region corresponding to the β subunits was excised in successive slices and submitted to microsequencing (Fig. 5). Three N-terminal TSHR amino acid sequences were observed in approximately equimolar amounts, originating at amino acids phenylalanine 366, leucine 370, and leucine 378. We have previously observed that the cleavage of TSHR is incomplete in transfected cells (10Misrahi 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 (109) Google Scholar). Furthermore, we have shown here that immunochromatography with Del-Ab allows receptor preparations to be enriched with large incompletely cleaved β subunits (see Fig. 4). We thus immunopurified the receptor from TSHR-expressing L cells, using either Endo-Ab or Del-Ab. Microsequencing of β subunits from such preparations showed a variety of species extending upstream from those observed in the human thyroid, up to Ser-314 (Fig. 5). The most abundant species started at leucine 370 and threonine 388. These observations suggested that the excised fragment of the receptor could not be isolated because it was not produced as a single polypeptide, but that multiple cleavages occurred which yielded fragments too small to be detected. Furthermore, the initial cleavage probably occurred in the N terminus of this region around Ser-314. Successive cleavages would then progress toward the C terminus up to phenylalanine 366 and leucines 370 and 378. This conclusion is based on the fact that the epitope recognized by Del-Ab remains in some molecules associated with the β subunit, but never in α subunits. Furthermore, whereas the β subunit is known to be of heterogeneous size, especially in transfected cells, the α subunit has a discrete size (more conveniently observed after deglycosylation (10Misrahi M. Ghinea N. Sar S. Saunier B. Jolivet A. Loosfelt H. Cerutti M. Devauchelle G. Milgrom E. Eur. J. Biochem. 1994; 222: 711-7
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