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Is Receptor Cleavage into Two Subunits Necessary for Thyrotropin Action?
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
225, 479–484 (1996)
1198
Is Receptor Cleavage into Two Subunits Necessary for Thyrotropin Action? Gregorio D. Chazenbalk,*,1 Sandra M. McLachlan,* Yuji Nagayama,† and Basil Rapoport* *Thyroid Molecular Biology Unit, Veterans’ Administration Medical Center and the University of California, San Francisco, California 94121; and †Department of Pharmacology, Nagasaki University School of Medicine, Nagasaki 852, Japan Received June 12, 1996 Unlike the wild-type thyrotropin (TSH) receptor, the chimeric TSH-LH/CG receptor TSH-LHR-14 does not cleave into two subunits when cross-linked to [125I]TSH on the surface of intact cells. Immunoblotting of TSH-LHR-14 in whole cell homogenates demonstrated that only a single chain receptor was detected under reducing conditions. TSH-LHR-14, like the A subunit of another chimeric receptor (TSH-LHR-10) that does cleave into two subunits, was almost entirely resistant to endoglycosidase H, indicating that it contains predominantly complex carbohydrate. The fact that TSH-LHR-14 reaches the cell surface where it binds TSH with high affinity and transduces a signal indicates that receptor cleavage into two subunits is not a prerequisite for TSH action. q 1996 Academic Press, Inc.
The thyrotropin receptor (TSHR) exists in two forms. One form, originally observed by radiolabeled TSH cross-linking studies to pig thyroid tissue membranes, consists of two subunits; a Ç 50 kDa TSH-binding ‘‘A’’ subunit linked by disulfide bonding to a smaller, membrane-associated ‘‘B’’ subunit (reviewed in 1). These observations have been confirmed in human thyroid tissue extracts using mouse monoclonal antibodies to the TSHR (2). A single subunit form of the TSHR has also been observed in cultured FRTL5 rat thyroid cells (3), transfected mammalian cells expressing the recombinant TSHR (4-9) and in human thyroid membranes (10). Quantitatively, in different preparations, the proportion of single subunit TSHR relative to the two-subunit form has been found to be low (3), intermediate (4,5) or high (6,9). For example, in transfected mouse L cell homogenates, the single chain TSHR is dominant. However this form of the receptor contains mostly immature, high mannose carbohydrate and is preferentially degraded, possibly because of aborted synthesis (9). Because in L cells only a small amount of the single chain receptor acquires mature, complex carbohydrate and is then cleaved into two subunits (9), the important question has been raised as to whether or not TSHR cleavage into two subunits is necessary for cognate ligand function (11). In the present study, we demonstrate that a TSHR variant that is present entirely in single-chain form in transfected Chinese hamster ovary (CHO) cells contains predominantly mature, complex carbohydrate. The fact that this receptor does reach the cell surface where it binds TSH with high affinity and also transduces a signal clearly indicates that receptor cleavage is not a prerequisite for TSH action. MATERIAL AND METHODS Cell lines expressing TSH-LH/CG chimeric receptors. In order to enhance immunodetection by increasing the level of receptor expression in CHO cells (12), we deleted the 5* and 3* untranslated regions of the cDNA for the chimeric 1 Corresponding author. V. A. Medical Center, Thyroid Molecular Biology Unit (111T), 4150 Clement Street, San Francisco, CA 94121. Fax: (415) 752-6745.
479 0006-291X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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TSH-LH/CG receptors TSH-LHR-10 (13) and TSH-LHR-14 (14). This goal was achieved by excising the 1.3 kb Sal I (blunted)- Spe I fragments from these cDNAs in the vector pSV2-NEO-ECE and using them to replace the corresponding Xho I (blunted) - Spe I fragment in pTSHR-5*3*TR-SVL (12). The latter construct contains the wild-type TSH receptor cDNA with both 5*UTR and 3*UTR deleted. Plasmid constructs were stably transfected into Chinese hamster ovary (CHO-K1) cells by a modified calcium-phosphate method (15) followed by selection in 400 mg/ml G418 (geneticin, GIBCO, Gaithersburg, MD). pSV2-NEO (2 mg) was cotransfected with pTSHR-5*3*TR-SVL (20 mg). After G418 selection, surviving clones (ú 100 per 100 mm diameter culture dish) were pooled for further study. Cells were cultured at 377C in Ham’s F-12 medium supplemented with 10% fetal calf serum in the presence of 100 units/ml penicillin, 50 mg/ml gentamicin and 2.5 mg/ml fungizone. Immunoblots of chimeric TSH-LH/CG receptor proteins. Stably transfected CHO cells (in two 100 mm diameter dishes) were rinsed three times with phosphate-buffered saline (PBS). 1.5 ml of 10 mM Tris-HCl, pH 7.4, containing the protease inhibitors phenylmethyl-sulfonyl fluoride (100 mg/ml), aprotinin (1 mg/ml), leupeptin (1 mg/ml), and pepstatin A (2 mg/ml) (all from Sigma) was added to each dish. The cells were scraped, homogenized with a Polytron homogenizer (Brinkman Instruments, Westbury, CT), centrifuged for 10 min at 500 1 g (47C), the supernatant recentrifuged for 20 min at 10,000 1 g (47C) and the pellet dissolved in 0.1 ml of the buffer described above. Samples were incubated for 30 min at 377C in Laemmli buffer (16) in the presence or absence of 5 % 2-mercaptoethanol and subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (7.5 % polyacrylamide gels, BioRad, Richmond, CA). Prestained molecular weight markers (Bio-Rad) were run in parallel with the samples. After electrophoresis, proteins were transferred (2.5 mA/cm2, 1.0 h) with a Poly Blot Transfer system (American Bionetics, Hayward, CA) to ProBlott membranes (Applied Biosystems, Foster City, CA). The membranes were rinsed three times with 50 mM Tris-HCl buffer, pH7.5, and 150 mM NaCl (TBS) and then rocked for 60 min with TBS containing 5.0 % skim milk powder. Membranes were incubated overnight (47C) with rabbit antiserum, L(1-14), to a peptide corresponding to amino acid residues 1-14 of the rat LH receptor (Dr. Mario Ascoli, University of Iowa). After rinsing, the membranes were incubated for 2 hr at 377C with alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin G (Fc fragment specific) or with alkaline phosphatase conjugated goat anti-mouse immunoglobulin G (1:400 dilution) (both from Cappel, Durham, NC). The signal was developed with nitroblue tetrazolium and 5-bromo, 4-chloro, 3-indolyl phosphate in 100mM Tris-HCl buffer, pH 9.5, containing 100 mM NaCl and 5 mM MgCl2 . Enzymatic deglycosylation of chimeric TSH-LHR proteins. The 10,000 1 g pellet from three confluent 10 cm diameter dishes was resuspended in 120 ml of buffer A (see above). Aliquots (40 ml) were treated according to the protocol of the manufacturer (N.E. Biolabs, Beverly MA). In brief, samples were denatured for 10 min at 100 C in 0.5 % SDS, 1 % b-mercaptoethanol. N-glycosidase F digestion (100 U for 2 h at 377C) was in 50 mM Na phosphate, pH 7.5, 1 % NP-40. Endoglycosidase H digestion (50 U for 2 h at 377C) was in 50 mM Na citrate, pH 5.5. Samples were then subjected to immunoblot analysis, as described above.
RESULTS
Immunoblots of chimeric TSH-LH receptors in CHO cell homogenates. We studied the chimeric TSH-LH/CG receptor TSH-LHR-14 (14)(Fig. 1) because we had observed previously (5) that it did not cleave into two subunits when cross-linked to [125I]TSH on the cell surface. In contrast to our previous ligand cross-linking studies, we examined this receptor by immunoblotting of whole cell homogenates. As a control, we used a chimeric TSH-LH/CG receptor, TSH-LHR-10 (13), that does cleave, in large part, into two subunits when detected on the cell surface by TSH cross-linking (5). Immunoblotting was performed using rabbit antiserum L(114), which, raised to a peptide, behaves like a monoclonal antibody and recognizes only the amino termini of the chimeric receptors (Fig. 1). Under non-reducing conditions, TSH-LHR-14 and TSH-LHR-10 in cell homogenates were both detected as a broad band of Ç90-100 kDa (Fig. 2A). Upon reduction, TSHLHR-14 remained in single chain form, whereas much of TSHR-LHR-10 separated into two subunits. In the case of the cleaved receptor, the epitope is on the Ç 55 kDa A subunit. This pattern in the entire cell homogenates was, therefore, similar to that of the chimeric receptors expressed on the surface of intact cells (Fig. 2B)(5). Note that the receptor-ligand complex is larger than the unliganded receptor detected by immunoblotting. The ligand subunit contributes Ç 14 kDa. Carbohydrate composition of the uncleaved TSH-LH/CG chimeric receptor: To distinguish 480
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FIG. 1. Schematic representation of two chimeric TSH-LH/CG receptors, TSH-LHR-10 and TSH-LHR-14 (13,14). Black segments represent the human TSH receptor and clear segments represent the rat LH receptor. Cleavage of TSH-LHR-10, but not TSH-LHR-14, leads to dissociation of the former into an A subunit and a B subunit under reducing conditions. The depicted location of the disulfide bonds (represented by small horizontal bars) is hypothetical but is consistent with the cysteine-rich areas in the TSH receptor (22) as well as by the horse-shoe shape of another protein with leucine-rich repeats, ribonuclease inhibitor (23). The location of the epitope for the rabbit antibody (amino acid residues 1-14 of the LH receptor) is indicated.
between high mannose and complex carbohydrate, membranes from homogenized CHO cells expressing the chimeric TSH-LHR receptors were subjected to enzymatic deglycosylation with endoglycosidase H and N-glycosidase F followed by immunoblotting under reducing conditions. The single chain chimeric receptor TSH-LHR-14, like the A subunit of cleaved receptor TSH-LHR-10, was almost entirely resistant to endoglycosidase H, indicating that it contained, predominantly, complex carbohydrate (Fig. 3). DISCUSSION
Trimming of high mannose carbohydrate and acquisition of complex carbohydrate occurs in the Golgi apparatus after completion of nascent protein folding in the endoplasmic reticulum. Abnormal protein folding may lead to retention of a protein in the endoplasmic reticulum in a high mannose form and the failure of normal trafficking of proteins to the plasma membrane (17). There is increasing evidence that this sequence of events occurs in the case of the glycoprotein hormone receptors. Thus, expression of the truncated TSHR ectodomain in Sf9 insect cells (18), as well as in CHO cells (19), leads largely to the intracellular accumulation of incompletely glycosylated protein. A variety of mutations in the FSH holoreceptor also lead to retention of high mannose forms and failure to reach the plasma membrane (discussed in 20). In the case of the glucagon receptor, a member of another subset of G protein-coupled receptors, the membrane spanning segments are also necessary for carbohydrate maturation and receptor transport to the plasma membrane (21). The present data clearly indicate that the chimeric receptor TSH-LHR-14, that does not cleave 481
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FIG. 2. (A) Immunoblots of TSH-LH/CG chimeric receptors. Crude membrane preparations of CHO cells stably expressing TSH-LHR-10 and TSH-LHR-14 were subjected to 7.5% polyacrylamide gel electrophoresis under nonreducing or reducing conditions (Methods). After transfer to PVDF membranes, proteins were probed with a rabbit antiserum to amino acid amino acid residues 1-14 of the rat LH receptor. (B) Radiolabeled TSH crosslinking to intact CHO cells expressing TSH-LHR-10 and TSH-LHR-14 on their surface. The data are adapted from a previous publication (5), with permission, and are included to permit comparison with the immunodetection of the same chimeric receptors in cell homogenates.
into two subunits and that is bound by TSH on the cell surface (5), is expressed in CHO cells almost entirely as a mature protein with complex carbohydrate. Because so much of the receptors present in CHO cell homogenates contain mature carbohydrate, the intracellular pool of precursor receptors may be small. These data differ from those reported for the wild-type TSHR expressed in mouse L cells, in which the majority of single chain receptors contain immature, high-mannose carbohydrate (9). This difference may reflect the use of different cells, as well as a difference between a chimeric TSH-LH/CG receptor and the wild-type TSH receptor. Most important, the present data support the concept that a single chain receptor with mature, complex carbohydrate can reach the cell surface, bind TSH with high affinity (14) and transduce an intracellular signal (14). These data clearly indicate that receptor cleavage into two subunits is not a prerequisite for TSH binding and TSH-induced signal transduction. Further, our observations raise the possibility that cleavage of the TSH receptor into two subunits relates to post-ligand binding processes, such as receptor degradation, rather than to ligand action. 482
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FIG. 3. Enzymatic deglycosylation of TSH-LH/CG chimeric receptors. Crude membrane preparations of CHO cells stably expressing TSH-LHR-10 and TSH-LHR-14 were incubated with N-glycosidase F (Endo F) or endoglycosidase H (Endo H)(Methods). Control (CON) aliquots were not subjected to enzymatic deglycosylation. Samples were then electrophoresed on 7.5% polyacrylamide gels under reducing conditions. After transfer to PVDF membranes, proteins were probed with the rabbit antiserum to amino acid amino acid residues 1-14 of the rat LH receptor. Similar findings were observed in three separate experiments.
ACKNOWLEDGMENTS This research was supported by NIH Grants DK 19289 and DK48216. We thank Dr. Mario Ascoli, University of Iowa, for providing us with the antibody to the LH receptor.
REFERENCES 1. Rees Smith, B., McLachlan, S. M., and Furmaniak, J. (1988) Endocr. Rev. 9, 106–121. 2. Loosfelt, H., Pichon, C., Jolivet, A., Misrahi, M., Caillou, B., Jamous, M., Vannier, B., and Milgrom, E. (1992) Proc. Natl. Acad. Sci. USA 895, 3765–3769. 3. Furmaniak, J., Hashim, F. A., Buckland, P. R., Petersen, V. B., Beever, K., Howells, R. D., and Rees Smith, B. (1987) FEBS Letters 215, 316–322. 4. Russo, D., Chazenbalk, G. D., Nagayama, Y., Wadsworth, H. L., Seto, P., and Rapoport, B. (1991) Mol. Endocrinol. 5, 1607–1612. 5. Russo, D., Nagayama, Y., Chazenbalk, G. D., Wadsworth, H. L., and Rapoport, B. (1992) Endocrinology 130, 2135–2138. 6. Endo, T., Ikeda, M., Ohmori, M., Anzai, E., Haraguchi, K., and Onaya, T. (1992) Biochem. Biophys. Res. Comm. 187, 887–893. 7. Ban, T., Kosugi, S., and Kohn, L. D. (1992) Endocrinology 131, 815–829. 8. Harfst, E., Ross, M. S., Nussey, S. S., and Johnstone, A. P. (1994) Molec. Cell. Endocrinol. 102, 77–84. 9. Misrahi, M., Ghinea, N., Sar, S., Saunier, B., Jolivet, A., Loosfelt, H., Cerutti, M., Devauchelle, G., and Milgrom, E. (1994) Eur. J. Biochem. 222, 711–719. 10. Grossman, R. F., Ban, T., Duh, Q. Y., Tezelman, S., Jossart, G., Soh, E. Y., Clark, O. H., and Siperstein, A. E. (1995) Thyroid 5, 101–105. 11. Couet, J., Sokhavut, S., Jolivet, A., Vu Hai, M.-T., Milgrom, E., and Misrahi, M. (1996) J. Biol. Chem. 271, 4545–4552. 12. Kakinuma, A., Chazenbalk, G., Filetti, S., McLachlan, S., and Rapoport, B. (1995) Endocrinology, in press. 13. Nagayama, Y., Wadsworth, H. L., Chazenbalk, G. D., Russo, D., Seto, P., and Rapoport, B. (1991) Proc. Natl. Acad. Sci. USA 88, 902–905. 14. Nagayama, Y., Russo, D., Chazenbalk, G. D., Wadsworth, H. L., and Rapoport, B. (1990) Biochem. Biophys. Res. Comm. 173, 1150–1156. 15. Chen, C., and Okayama, H. (1987) Mol. Cell. Biol. 7, 2745–2752. 16. Laemmli, U. K. (1970) Nature 227, 680–685. 483
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17. Fiedler, K. l., and Simons, K. (1995) Cell 81, 309–312. 18. Chazenbalk, G. D., and Rapoport, B. (1995) J. Biol. Chem. 270, 1543–1549. 19. Rapoport, B., McLachlan, S. M., Kakinuma, A., and Chazenbalk, G. D. (1996) J. Clin. Endocrinol. Metab. (in press). 20. Rozell, T. G., Wang, H., Liu, X., and Segaloff, D. L. (1995) Mol. Endocrinol. 9, 1727–1736. 21. Unson, C. G., Cypess, A. M., Kim, H. N., Goldsmith, P. K., Carruthers, C. J. L., Merrifield, R. B., and Sakmar, T. P. (1995) J. Biol. Chem. 270, 27720–27727. 22. Nagayama, Y., Kaufman, K. D., Seto, P., and Rapoport, B. (1989) Biochem. Biophys. Res. Comm. 165, 1184– 1190. 23. Kobe, B., and Deisenhofer, J. (1993) Nature 366, 751–756.
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Is Receptor Cleavage into Two Subunits Necessary for Thyrotropin Action? Gregorio D. Chazenbalk,*,1 Sandra M. McLachlan,* Yuji Nagayama,† and Basil Rapoport* *Thyroid Molecular Biology Unit, Veterans’ Administration Medical Center and the University of California, San Francisco, California 94121; and †Department of Pharmacology, Nagasaki University School of Medicine, Nagasaki 852, Japan Received June 12, 1996 Unlike the wild-type thyrotropin (TSH) receptor, the chimeric TSH-LH/CG receptor TSH-LHR-14 does not cleave into two subunits when cross-linked to [125I]TSH on the surface of intact cells. Immunoblotting of TSH-LHR-14 in whole cell homogenates demonstrated that only a single chain receptor was detected under reducing conditions. TSH-LHR-14, like the A subunit of another chimeric receptor (TSH-LHR-10) that does cleave into two subunits, was almost entirely resistant to endoglycosidase H, indicating that it contains predominantly complex carbohydrate. The fact that TSH-LHR-14 reaches the cell surface where it binds TSH with high affinity and transduces a signal indicates that receptor cleavage into two subunits is not a prerequisite for TSH action. q 1996 Academic Press, Inc.
The thyrotropin receptor (TSHR) exists in two forms. One form, originally observed by radiolabeled TSH cross-linking studies to pig thyroid tissue membranes, consists of two subunits; a Ç 50 kDa TSH-binding ‘‘A’’ subunit linked by disulfide bonding to a smaller, membrane-associated ‘‘B’’ subunit (reviewed in 1). These observations have been confirmed in human thyroid tissue extracts using mouse monoclonal antibodies to the TSHR (2). A single subunit form of the TSHR has also been observed in cultured FRTL5 rat thyroid cells (3), transfected mammalian cells expressing the recombinant TSHR (4-9) and in human thyroid membranes (10). Quantitatively, in different preparations, the proportion of single subunit TSHR relative to the two-subunit form has been found to be low (3), intermediate (4,5) or high (6,9). For example, in transfected mouse L cell homogenates, the single chain TSHR is dominant. However this form of the receptor contains mostly immature, high mannose carbohydrate and is preferentially degraded, possibly because of aborted synthesis (9). Because in L cells only a small amount of the single chain receptor acquires mature, complex carbohydrate and is then cleaved into two subunits (9), the important question has been raised as to whether or not TSHR cleavage into two subunits is necessary for cognate ligand function (11). In the present study, we demonstrate that a TSHR variant that is present entirely in single-chain form in transfected Chinese hamster ovary (CHO) cells contains predominantly mature, complex carbohydrate. The fact that this receptor does reach the cell surface where it binds TSH with high affinity and also transduces a signal clearly indicates that receptor cleavage is not a prerequisite for TSH action. MATERIAL AND METHODS Cell lines expressing TSH-LH/CG chimeric receptors. In order to enhance immunodetection by increasing the level of receptor expression in CHO cells (12), we deleted the 5* and 3* untranslated regions of the cDNA for the chimeric 1 Corresponding author. V. A. Medical Center, Thyroid Molecular Biology Unit (111T), 4150 Clement Street, San Francisco, CA 94121. Fax: (415) 752-6745.
479 0006-291X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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TSH-LH/CG receptors TSH-LHR-10 (13) and TSH-LHR-14 (14). This goal was achieved by excising the 1.3 kb Sal I (blunted)- Spe I fragments from these cDNAs in the vector pSV2-NEO-ECE and using them to replace the corresponding Xho I (blunted) - Spe I fragment in pTSHR-5*3*TR-SVL (12). The latter construct contains the wild-type TSH receptor cDNA with both 5*UTR and 3*UTR deleted. Plasmid constructs were stably transfected into Chinese hamster ovary (CHO-K1) cells by a modified calcium-phosphate method (15) followed by selection in 400 mg/ml G418 (geneticin, GIBCO, Gaithersburg, MD). pSV2-NEO (2 mg) was cotransfected with pTSHR-5*3*TR-SVL (20 mg). After G418 selection, surviving clones (ú 100 per 100 mm diameter culture dish) were pooled for further study. Cells were cultured at 377C in Ham’s F-12 medium supplemented with 10% fetal calf serum in the presence of 100 units/ml penicillin, 50 mg/ml gentamicin and 2.5 mg/ml fungizone. Immunoblots of chimeric TSH-LH/CG receptor proteins. Stably transfected CHO cells (in two 100 mm diameter dishes) were rinsed three times with phosphate-buffered saline (PBS). 1.5 ml of 10 mM Tris-HCl, pH 7.4, containing the protease inhibitors phenylmethyl-sulfonyl fluoride (100 mg/ml), aprotinin (1 mg/ml), leupeptin (1 mg/ml), and pepstatin A (2 mg/ml) (all from Sigma) was added to each dish. The cells were scraped, homogenized with a Polytron homogenizer (Brinkman Instruments, Westbury, CT), centrifuged for 10 min at 500 1 g (47C), the supernatant recentrifuged for 20 min at 10,000 1 g (47C) and the pellet dissolved in 0.1 ml of the buffer described above. Samples were incubated for 30 min at 377C in Laemmli buffer (16) in the presence or absence of 5 % 2-mercaptoethanol and subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (7.5 % polyacrylamide gels, BioRad, Richmond, CA). Prestained molecular weight markers (Bio-Rad) were run in parallel with the samples. After electrophoresis, proteins were transferred (2.5 mA/cm2, 1.0 h) with a Poly Blot Transfer system (American Bionetics, Hayward, CA) to ProBlott membranes (Applied Biosystems, Foster City, CA). The membranes were rinsed three times with 50 mM Tris-HCl buffer, pH7.5, and 150 mM NaCl (TBS) and then rocked for 60 min with TBS containing 5.0 % skim milk powder. Membranes were incubated overnight (47C) with rabbit antiserum, L(1-14), to a peptide corresponding to amino acid residues 1-14 of the rat LH receptor (Dr. Mario Ascoli, University of Iowa). After rinsing, the membranes were incubated for 2 hr at 377C with alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin G (Fc fragment specific) or with alkaline phosphatase conjugated goat anti-mouse immunoglobulin G (1:400 dilution) (both from Cappel, Durham, NC). The signal was developed with nitroblue tetrazolium and 5-bromo, 4-chloro, 3-indolyl phosphate in 100mM Tris-HCl buffer, pH 9.5, containing 100 mM NaCl and 5 mM MgCl2 . Enzymatic deglycosylation of chimeric TSH-LHR proteins. The 10,000 1 g pellet from three confluent 10 cm diameter dishes was resuspended in 120 ml of buffer A (see above). Aliquots (40 ml) were treated according to the protocol of the manufacturer (N.E. Biolabs, Beverly MA). In brief, samples were denatured for 10 min at 100 C in 0.5 % SDS, 1 % b-mercaptoethanol. N-glycosidase F digestion (100 U for 2 h at 377C) was in 50 mM Na phosphate, pH 7.5, 1 % NP-40. Endoglycosidase H digestion (50 U for 2 h at 377C) was in 50 mM Na citrate, pH 5.5. Samples were then subjected to immunoblot analysis, as described above.
RESULTS
Immunoblots of chimeric TSH-LH receptors in CHO cell homogenates. We studied the chimeric TSH-LH/CG receptor TSH-LHR-14 (14)(Fig. 1) because we had observed previously (5) that it did not cleave into two subunits when cross-linked to [125I]TSH on the cell surface. In contrast to our previous ligand cross-linking studies, we examined this receptor by immunoblotting of whole cell homogenates. As a control, we used a chimeric TSH-LH/CG receptor, TSH-LHR-10 (13), that does cleave, in large part, into two subunits when detected on the cell surface by TSH cross-linking (5). Immunoblotting was performed using rabbit antiserum L(114), which, raised to a peptide, behaves like a monoclonal antibody and recognizes only the amino termini of the chimeric receptors (Fig. 1). Under non-reducing conditions, TSH-LHR-14 and TSH-LHR-10 in cell homogenates were both detected as a broad band of Ç90-100 kDa (Fig. 2A). Upon reduction, TSHLHR-14 remained in single chain form, whereas much of TSHR-LHR-10 separated into two subunits. In the case of the cleaved receptor, the epitope is on the Ç 55 kDa A subunit. This pattern in the entire cell homogenates was, therefore, similar to that of the chimeric receptors expressed on the surface of intact cells (Fig. 2B)(5). Note that the receptor-ligand complex is larger than the unliganded receptor detected by immunoblotting. The ligand subunit contributes Ç 14 kDa. Carbohydrate composition of the uncleaved TSH-LH/CG chimeric receptor: To distinguish 480
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FIG. 1. Schematic representation of two chimeric TSH-LH/CG receptors, TSH-LHR-10 and TSH-LHR-14 (13,14). Black segments represent the human TSH receptor and clear segments represent the rat LH receptor. Cleavage of TSH-LHR-10, but not TSH-LHR-14, leads to dissociation of the former into an A subunit and a B subunit under reducing conditions. The depicted location of the disulfide bonds (represented by small horizontal bars) is hypothetical but is consistent with the cysteine-rich areas in the TSH receptor (22) as well as by the horse-shoe shape of another protein with leucine-rich repeats, ribonuclease inhibitor (23). The location of the epitope for the rabbit antibody (amino acid residues 1-14 of the LH receptor) is indicated.
between high mannose and complex carbohydrate, membranes from homogenized CHO cells expressing the chimeric TSH-LHR receptors were subjected to enzymatic deglycosylation with endoglycosidase H and N-glycosidase F followed by immunoblotting under reducing conditions. The single chain chimeric receptor TSH-LHR-14, like the A subunit of cleaved receptor TSH-LHR-10, was almost entirely resistant to endoglycosidase H, indicating that it contained, predominantly, complex carbohydrate (Fig. 3). DISCUSSION
Trimming of high mannose carbohydrate and acquisition of complex carbohydrate occurs in the Golgi apparatus after completion of nascent protein folding in the endoplasmic reticulum. Abnormal protein folding may lead to retention of a protein in the endoplasmic reticulum in a high mannose form and the failure of normal trafficking of proteins to the plasma membrane (17). There is increasing evidence that this sequence of events occurs in the case of the glycoprotein hormone receptors. Thus, expression of the truncated TSHR ectodomain in Sf9 insect cells (18), as well as in CHO cells (19), leads largely to the intracellular accumulation of incompletely glycosylated protein. A variety of mutations in the FSH holoreceptor also lead to retention of high mannose forms and failure to reach the plasma membrane (discussed in 20). In the case of the glucagon receptor, a member of another subset of G protein-coupled receptors, the membrane spanning segments are also necessary for carbohydrate maturation and receptor transport to the plasma membrane (21). The present data clearly indicate that the chimeric receptor TSH-LHR-14, that does not cleave 481
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FIG. 2. (A) Immunoblots of TSH-LH/CG chimeric receptors. Crude membrane preparations of CHO cells stably expressing TSH-LHR-10 and TSH-LHR-14 were subjected to 7.5% polyacrylamide gel electrophoresis under nonreducing or reducing conditions (Methods). After transfer to PVDF membranes, proteins were probed with a rabbit antiserum to amino acid amino acid residues 1-14 of the rat LH receptor. (B) Radiolabeled TSH crosslinking to intact CHO cells expressing TSH-LHR-10 and TSH-LHR-14 on their surface. The data are adapted from a previous publication (5), with permission, and are included to permit comparison with the immunodetection of the same chimeric receptors in cell homogenates.
into two subunits and that is bound by TSH on the cell surface (5), is expressed in CHO cells almost entirely as a mature protein with complex carbohydrate. Because so much of the receptors present in CHO cell homogenates contain mature carbohydrate, the intracellular pool of precursor receptors may be small. These data differ from those reported for the wild-type TSHR expressed in mouse L cells, in which the majority of single chain receptors contain immature, high-mannose carbohydrate (9). This difference may reflect the use of different cells, as well as a difference between a chimeric TSH-LH/CG receptor and the wild-type TSH receptor. Most important, the present data support the concept that a single chain receptor with mature, complex carbohydrate can reach the cell surface, bind TSH with high affinity (14) and transduce an intracellular signal (14). These data clearly indicate that receptor cleavage into two subunits is not a prerequisite for TSH binding and TSH-induced signal transduction. Further, our observations raise the possibility that cleavage of the TSH receptor into two subunits relates to post-ligand binding processes, such as receptor degradation, rather than to ligand action. 482
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FIG. 3. Enzymatic deglycosylation of TSH-LH/CG chimeric receptors. Crude membrane preparations of CHO cells stably expressing TSH-LHR-10 and TSH-LHR-14 were incubated with N-glycosidase F (Endo F) or endoglycosidase H (Endo H)(Methods). Control (CON) aliquots were not subjected to enzymatic deglycosylation. Samples were then electrophoresed on 7.5% polyacrylamide gels under reducing conditions. After transfer to PVDF membranes, proteins were probed with the rabbit antiserum to amino acid amino acid residues 1-14 of the rat LH receptor. Similar findings were observed in three separate experiments.
ACKNOWLEDGMENTS This research was supported by NIH Grants DK 19289 and DK48216. We thank Dr. Mario Ascoli, University of Iowa, for providing us with the antibody to the LH receptor.
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