Thyrotropin (thyroid-stimulating hormone, TSH) ()is a member of the glycoprotein hormone family, which also includes chorionic gonadotropin (CG), lutropin (luteinizing hormone, LH) and follitropin (follicle-stimulating hormone, FSH). These hormones are structurally related heterodimers consisting of a common subunit and a distinct subunit that confers the biological specificity for each hormone (1) . The common subunit bears two N-linked oligosaccharides, and the subunit bears either one (in TSH and LH), or two (in CG and FSH)(2, 3) . The oligosaccharides, which represent 18-35% of total weight(2, 3, 4) , have been shown to play a role in the proper folding, assembly, secretion, metabolic clearance, and biological activity of these hormones (for recent review, see Refs. 3 and 4). In the case of hCG and hFSH, enzymatic or chemical deglycosylation led to a decrease or loss of cAMP production and steroidogenesis, while high affinity binding was maintained(3, 4, 5) . Sairam et al.(6) reported that the carbohydrates of the common subunit rather than those of the subunits were important for the activity of these hormones. Using site-directed mutagenesis, Matzuk et al.(7) identified the oligosaccharide at position 52 of the subunit to be critical for the in vitro bioactivity of hCG. Subsequently, this oligosaccharide was shown to be similarly important for the stimulatory activity of hFSH(8, 9) .

In contrast to hCG and hFSH, the roles of the individual oligosaccharides for the activity of TSH or the more closely related LH are not known. Due to the limited availability of purified pituitary human TSH (phTSH), studies on the role of the carbohydrates for TSH have mostly used pituitary bovine TSH. Similar to the findings for the gonadotropins, these studies have shown by chemical or enzymatic deglycosylation that the oligosaccharides, and particularly those of the subunit, were required for full in vitro activity of pituitary bovine TSH(10, 11) . The few studies on phTSH have yielded conflicting results and the role of carbohydrates for hTSH action remained controversial(12, 13) . The recent availability of recombinant human TSH (rhTSH) (14) allowed further investigation of the role of the carbohydrates in the action of hTSH. rhTSH expressed in CHO-K1 cells contains N-linked oligosaccharides which terminate with Sia2-3Gal1-4GlcNAc1-2Man(14, 15, 16) . By comparison, phTSH, which physiologically occurs in a variety of glycosylation isoforms, terminates both in SO-4GalNAc1-4GlcNAc1-2Man and Sia2-3Gal1-4GlcNAc1-2Man as described by Green and Baenziger(2) . Interestingly, enzymatic removal of terminal sialic acid residues of rhTSH increased the in vitro bioactivity of the hormone (17) similar to findings for recombinant bovine LH(15) , but unlike for hCG, in which sequential deglycosylation resulted in a stepwise reduction of activity(18) . These findings suggested that the role of the oligosaccharides may be different for hTSH compared with hCG and hFSH.

In the present study we have used site-directed mutagenesis to study the role of individual oligosaccharides of hTSH by selectively inhibiting their cotranslational attachment. We combined site-directed mutagenesis with the expression of the selectively deglycosylated hTSH mutants in different cell lines producing hormones with distinct carbohydrate patterns(19, 20, 21) , using our recently developed and optimized transient transfection protocol(22) . This novel approach allowed us to identify unique roles for individual oligosaccharides and their terminal sialic acid residues for the in vitro as well as in vivo activity of hTSH.

EXPERIMENTAL PROCEDURES

Materials

Site-directed Mutagenesis

Transient Expression

Immunoassays

Gel Filtration

SDS-Polyacrylamide Gel Electrophoresis and Western Blotting

Enzymatic Deglycosylation

cAMP Production

Growth Assay in FRTL-5 Cells

Radioreceptor Assay of hTSH

In Vivo Bioassay

Serum Disappearance Rate

RESULTS

hTSH bears three N-linked oligosaccharide chains at positions Asn and Asn of the subunit and at Asn of the subunit(1, 2, 3) . To prevent the cotranslational attachment of individual oligosaccharides to the hTSH molecule, we disrupted the respective glycosylation recognition sequences NX(T/S), widely considered to be an absolute requirement for glycosylation to occur. In the subunit (individually and in a composite mutation) and in the TSH subunit we mutated Asn at each position to Gln, thus creating genes coding for the following mutant subunits: Q52, Q78, Q52.Q78, and TSHQ23. The conservative mutation of Asn to Gln is unlikely to affect protein conformation and has not been reported to influence the tertiary structure of the related glycoproteins hCG and hFSH, when used to generate mutants lacking individual oligosaccharide attachment sites(7, 8, 9) . Thus, the observed effects on the hTSH expression, binding, and activity should be primarily due to changes in the carbohydrate chains.

The shift in the elution profiles of the hTSH mutants observed using gel filtration (Fig. 1A) and the differences in gel migration using Western blotting (Fig. 1B) were consistent with the absence of individual oligosaccharides. After N-glycanase digestion, wild type hTSH, Q52/TSH, and Q78/TSH had similar profiles (Fig. 1, A and B), further demonstrating that the differences prior to N-glycanase treatment were due to differences in glycosylation.

Figure 1: A, Superdex 75 elution profiles of the hTSH mutants before (open symbols) and after (filled symbols, +) N-glycanase digestion. Conditioned media from transfected CHO-K1 cells were chromatographed using hCG as an internal standard (the peak of hCG immunoreactivity corresponds to fraction 0 in all cases). Fraction size was 0.175 ml. hTSH immunoreactivity was monitored by immunoassay (Nichols Institute). B, Western blotting analysis of ConA-Sepharose-fractionated hTSH deglycosylation mutants before(-) and after (+) N-glycanase digestion (see ``Experimental Procedures''), using an antibody against the hTSH subunit. rhTSH (Genzyme Corp.) served as the internal standard. In the case of N-glycanase-treated wild type and Q52/TSH, presumably due to incomplete enzymatic digestion, two bands were visible, representing hTSH subunit lacking one or two oligosaccharides, respectively.

Quantitation of Wild Type and Mutant hTSH

hTSH Expression

In Vitro Bioactivity: cAMP Induction

Figure 2: cAMP induction in CHO cells expressing the rhTSH receptor (JP09) by the hTSH mutants produced in CHO-K1 cells. Increasing concentrations of wild type or mutant hTSH were incubated with JP09 cells, and the cAMP concentration in the resulting supernatants was assayed by radioimmunoassay. The amount of cAMP released from the cells in the presence of concentrated medium from mock-transfected cells was not different from base-line levels (buffer only). A representative experiment, repeated at least twice, is shown. Values from triplicate determinations are depicted as mean ± S.E.

In FRTL-5 cells expressing endogenous rat TSH receptor(27) , we observed, relative to wild type (EC = 12.6 ± 2.1 ng/ml), a similar 6-fold decrease in the EC of Q52/TSH (EC = 2.0 ± 0.7 ng/ml, p < 0.001) (Fig. 3).

Figure 3: cAMP induction in FRTL-5 cells. Wild type or Q52/TSH, expressed in CHO-K1 cells, was incubated with FRTL-5 cells expressing the endogenous rat TSH receptor. A representative experiment, repeated at least twice, is shown. Values from triplicate determinations are depicted as mean ± S.E.

We next expressed mutants Q52/TSH, Q78/TSH, and /TSHQ23 in 293 cells. In contrast to CHO-K1 cells, 293 cells express N-acetylgalactosaminyl-transferase and GalNAc1,4GlcNAc1,2Man4-sulfotransferase and produce oligosaccharides terminating in >70% in sulfated N-linked carbohydrate moieties(19) . Since we previously found that 293 cells produce small amounts of free subunit(29) , we initially cotransfected the hTSH minigene alone to assess whether significant amounts of wild type hTSH would be produced. This, however, was not the case, indicating that significant contamination of the mutants with wild type hTSH did not occur. Wild type hTSH, expressed in 293 cells had increased cAMP inducing activity, evidenced by a 5.6-fold left shift in the EC compared with wild type expressed in CHO-K1 cells (Fig. 4, Table 2) (p < 0.001). However, in relation to the increased wild type activity, there was no difference in activity between wild type and the selectively deglycosylated hTSH mutants (Fig. 4, Table 2).

Figure 4: cAMP induction in JP09 cells by the hTSH mutants produced in 293 cells. Experiments were carried out as described in the legend to Fig. 2and under ``Experimental Procedures.'' For comparison, cAMP induction of wild type expressed in CHO-K1 cells assayed in the same experiment is also shown. A representative experiment, repeated at least twice, is shown. Values from triplicate determinations are depicted as mean ± S.E.

To further understand which carbohydrate components may be associated with the increase of bioactivity upon expressing the hTSH mutants in CHO-K1 cells, we also transiently transfected CHO-LEC2 and CHO-LEC1 cells. These are CHO glycosylation mutant cell lines selected from the wild type CHO-K1 cell line for resistance to toxic plant lectins(23) . CHO-LEC2 cells have a defect in the CMP-sialic acid translocation into the Golgi resulting in the synthesis of glycoproteins with a > 90% reduction in sialic acid(20) , whereas CHO-LEC1 cells lack N-acetylgalactosaminyl-transferase I and hence give rise to oligosaccharides bearing ManGlcNAc intermediates (21) . The reduction of sialic acid on the CHO-LEC2-expressed hTSH was evidenced by a decrease in L. flavus agglutinin binding, a lectin from the slug L. flavus that recognizes terminal sialic acids (30) (53 ± 8.3% compared with 94.5 ± 2.7% in the case of CHO-K1-expressed hTSH) as well as by an accelerated elution profile from L. flavus agglutinin minicolumns. Further, neuraminidase digestion decreased L. flavus agglutinin binding of hTSH from CHO-K1 cells to levels similar to nondigested CHO-LEC2-expressed hTSH. As expected, neuraminidase digestion did not further decrease L. flavus agglutinin binding of hTSH from CHO-LEC2 cells (data not shown).

Wild type hTSH expressed in both CHO-LEC2 and CHO-LEC1 was 7-8-fold more active than wild type from CHO-K1 cells (p < 0.001) (Fig. 5A, Table 2). Compared with this increased wild type activity, there was no difference in the EC of cAMP induction for the various selectively deglycosylated mutants expressed in these cells.

Figure 5: A, cAMP induction in JP09 cells by the hTSH mutants produced in CHO-LEC2 cells. Experiments were carried out as described in the legend to Fig. 2. For comparison, cAMP induction of the wild type expressed in CHO-K1 cells from the same experiment is depicted. A representative experiment, repeated at least twice, is shown. Similar results were obtained when wild type and Q52/TSH were expressed in CHO-LEC1 cells (Table 2). B, the effects of neuraminidase digestion on hTSH mutants produced in CHO-K1 cells (filled symbols, +). For comparison, nondigested wild type and mutants from the same transfection are shown, as well as wild type hTSH from CHO-LEC2 cells (open symbols). As expected, neuraminidase digestion did not further increase activity of hTSH from CHO-LEC2 cells (data not shown). Values from triplicate determinations are depicted as mean ± S.E.

Direct evidence for the inhibitory roles of terminal sialic acids was obtained by testing for cAMP stimulation of wild type and mutant hTSH after neuraminidase digestion (Fig. 5B). As expected, neuraminidase digestion of wild type hTSH from CHO-K1 cells increased its in vitro activity to an EC similar to CHO-LEC2-expressed hTSH. Furthermore, digestion of Q78/TSH, which retains the sialylated Asn oligosaccharide also increased in vitro activity to similar levels. Conversely, the already maximally increased in vitro activity of Q52/TSH, in which the Asn sialic acid moieties are absent, was not further increased. As expected, neuraminidase digestion did not further increase the activity of hTSH from CHO-LEC2 cells (data not shown).

In Vitro Bioactivity: Growth Induction of FRTL-5 Cells

Figure 6: Induction of cell growth by Q52/TSH from CHO-K1 cells. Increasing concentrations of wild type or mutant hTSH were incubated with FRTL-5 cells, which were previously grown in the absence of TSH. After 48 h, [H]thymidine was added, and after an additional 24 h, radioactivity incorporated into the DNA was measured. Values are shown as the mean of triplicate observations ± S.E. The [H]thymidine incorporation in the presence of concentrated medium from mock-transfected cells was not different from base-line levels.

Receptor Binding

Figure 7: Inhibition of I-bTSH binding by wild type and Q52/TSH expressed in CHO-K1, CHO-LEC2, and 293 cells. Increasing doses of wild type or mutant hTSH were incubated with porcine membranes in the presence of a constant amount of I-bTSH. I-bTSH bound to membranes was precipitated and quantitated in a counter, and radioactivity precipitated in the presence of concentrated medium from mock transfections was defined as 100%. Values are shown as mean ± S.E. of triplicate determinations. Experiments were repeated two times. Binding of wild type and Q52/TSH expressed in CHO-LEC1 was not different from that expressed in CHO-LEC2 cells.

Figure 8: Inhibition of I-bTSH binding by the hTSH mutants expressed in CHO-K1 cells. The binding assay was performed as described in the legend to Fig. 7. Values of a representative experiment, performed at least three times, are shown as mean ± S.E. of triplicate determinations.

Thyrotropic Activity of Site-specifically Deglycosylated hCG

Figure 9: Thyrotropic activity of CHO-K1-expressed, site-specifically -deglycosylated hCG in JPO9 cells. For experimental details, see the legend to Fig. 2and ``Experimental Procedures.'' Values of a representative experiment, performed at least three times, are depicted as mean ± S.E. of triplicate determinations.

In Vivo Bioactivity

Figure 10: In vivo activity of site-specifically hTSH mutants from CHO-K1 cells in T-suppressed mice. T values of nonsuppressed mice ranged from 6 to 8 µg/ml. Mock injection (n = 3) did not significantly increase T values of suppressed mice (control, n = 3). For each concentration, 100, 200, and 400 ng, a total number of five mice were injected intraperitoneally with wild type as well as with each hTSH mutant. After 6 h, blood was obtained from the orbital sinus for T determinations. Data were compiled from individual injections. *, p < 0.05 compared with an equal dose of wild type.

Serum Disappearance Rate

Figure 11: Serum disappearance rate of hTSH mutants from CHO-K1 cells in male rats. After bolus injection of 200-300 ng of wild type or mutant hTSH into the femoral vein, blood for hTSH determinations was obtained over 120 min at equal time points. An IRMA without cross-reactivity to rat TSH (Nichols Institute), was used. Immunoreactivity was expressed as percentage remaining, and serum concentration at 0 min was defined as 100%. Wild type, n = 4; Q52/TSH, n = 4; Q78/TSH, n = 3. Data were compiled from individual experiments.

DISCUSSION

The glycoprotein hormones hTSH, hCG, hLH, and hFSH share a common signal transduction pathway involving interaction with a hormone-specific G protein-coupled receptor and the generation of cAMP as the initial event in the signal transduction cascade(3, 4) . Previous studies on hCG (7) and hFSH (8, 9) had indicated a site-specific requirement of the oligosaccharide at Asn for their in vitro activity, but similar studies on hTSH and the more closely related hLH have not been performed. Moreover, previous studies using recombinant DNA techniques to study oligosaccharides in glycoprotein hormones had been limited to using either site-directed mutagenesis of glycosylation attachment sites or glycosylation mutant cell lines to synthesize these hormones. Therefore, in a novel approach to study the role of carbohydrates for hTSH, we combined site-specific deletion of individual oligosaccharides with the expression of recombinant hormones in a variety of cell lines that differentially process the carbohydrate moieties.

In this study we provide evidence for a different role of the oligosaccharides for hTSH bioactivity, distinct from their role for hCG and hFSH. Site-specific disruption of each of the three individual carbohydrate attachment sites led to an increase in the in vitro activity of hTSH expressed in CHO-K1 cells producing sialylated carbohydrates(14, 15, 16) . Our findings using a sialic acid-binding lectin (30) as well as enzymatic desialylation show that the hTSH transiently expressed in CHO-K1 cells was indeed sialylated. Further, its in vitro bioactivity as well as its circulatory half-life was indistinguishable from that of rhTSH (Genzyme Corp.), ()which contains 1.8-2.2 sialic acid residues/chain(14, 16) . Interestingly, this increase of in vitro activity upon site-specific deglycosylation was most pronounced upon deletion of the carbohydrate at Asn. In contrast, this Asn oligosaccharide had previously been shown to be essential for receptor stimulation of hCG and hFSH, which were also expressed in CHO-K1 cells (7, 8, 9) . The increase of cAMP-inducing activity in our study occurred to a similar degree with the recombinant hTSH receptor and the endogenous rat receptor, indicating that these effects were neither system-dependent nor species-specific. Furthermore, in addition to the immediate induction of cAMP synthesis, site-specific deglycosylation of hTSH also affected long term effects, including DNA synthesis by and growth of target cells in a similar fashion.

Wild type hTSH expressed in 293, CHO-LEC2, or CHO-LEC1 cells had a higher in vitro activity than wild type from CHO-K1 cells, and this increased activity was not further augmented by site-specific deglycosylation. CHO-LEC mutant cell lines lack a defined glycosylation activity and accumulate specifically truncated carbohydrates typical of intermediates in the biosynthetic pathway(23) . In contrast to CHO-K1 cells, which produce terminally sialylated oligosaccharides, Golgi vesicle membranes from CHO-LEC2 cells translocate CMP-sialic acid at only 2% of the rate of vesicles from CHO-K1 cells and thus produce carbohydrates with a greater than 90% decrease in sialic acid content(20) . CHO-LEC1 cells lack N-acetylgalactosaminyl-transferase I and hence terminate in ManGlcNAc intermediates(21) . 293 cells express Nacetylgalactosaminyl-transferase and GalNAc1,4GlcNAc1, 2Man4-sulfotransferase and produce N-linked carbohydrate moieties terminating in sulfate in more than 70% of the chains(19) . The fact that the increase of the in vitro activity upon site-specific deglycosylation was confined to hTSH expressed in CHO-K1 cells therefore indicates that the terminal sialic acid residues attenuate signal transduction of hTSH. This was further supported by our present findings after neuraminidase digestion and is in accord with recent reports on sequential deglycosylation of rhTSH (17) and recombinant bovine LH(15) . Importantly, our current approach allowed us to identify site-specific differences in the role of the terminal sialic acid residues and showed that the residues at the Asn oligosaccharide attenuated TSH receptor activation to a much higher degree than those at the Asn or Asn carbohydrate chain. Further, our data demonstrate that hTSH expressing ManGlcNAc intermediates retained full receptor coupling. In contrast to our findings for hTSH, Keene et al.(32) showed that the activity of wild type hCG expressed in these CHO glycosylation mutant cells was lower compared with hCG from CHO-K1 cells. The same group reported that the in vitro activity of hFSH did not change upon expression in these cell lines(33) . These studies, however, did not assess the effects of site-specific deglycosylation on the activity of hCG or hFSH expressed in these cells. Taken together, these findings indicate that the terminal sialic acid residues can differently modulate glycoprotein hormone activity in a hormone-dependent manner.

Our study has further demonstrated a unique role of the carbohydrate at Asn for hTSH receptor binding. In previous reports for hCG and hFSH, deletion of the oligosaccharide at Asn had no effect (7, 8) or led to only a minor increase in receptor binding(9) . The nonessential role of oligosaccharides Asn and Asn in hTSH receptor binding observed in this study is similar to that for the gonadotropins(7, 8, 9) . In contrast to the findings on bioactivity, the increased binding of Q52/TSH was independent of the carbohydrate pattern, suggesting that the increase of activity of Q52/TSH from CHO-K1 cells was not a direct result of the increased binding of this mutant. The molecular mechanism by which carbohydrates activate the receptor is unknown, but in accord with our present findings, is believed to occur at a postreceptor binding step(3, 4) . An indirect mechanism involving a conformational change of the hormone (35) appears more likely than a direct interaction of the oligosaccharide with the receptor, since a lectin-like component identified in the hCG receptor (34) is not present in the hTSH receptor. Recent reports on the crystal structure of hydrofluoric acid-treated hCG (36, 37) located the oligosaccharide at position Asn to be in a putative receptor binding region. Since the hormone-specific subunit influences the conformation of the subunit in a hormone-specific manner(38) , differences in spatial orientation of this oligosaccharide may contribute to its differential role in hTSH compared with hCG or hFSH. This speculation, however, awaits confirmation by structural analysis, since, among the glycoprotein hormones, only hCG has been crystallized to date.

It had previously been reported that the weak thyrotropic activity of hCG (for review, see (39) ), which has recently been linked to a direct interaction with the rhTSH receptor(31, 40, 41) , increased upon desialylation(42) , in contrast to a decrease at its native receptor (18) . When we tested the thyrotropic activity of CHO-K1-expressed hCG site-specifically deglycosylated at the subunit, we observed an increase of activity of Q52/hCG but not of Q78/hCG. This leads to the conclusion that the deletion of the oligosaccharide at Asn can have opposite effects on hCG activity, depending on the receptor with which the hormone interacts. Therefore, these findings point to the intriguing possibility that the observed differences in the role of individual oligosaccharides for glycoprotein hormone action may be related, at least in part, to differences in glycoprotein hormone receptor structure and/or to receptordependent differences in receptor-ligand interaction.

The carbohydrate moieties are known to be important for the circulatory half-life and hence the in vivo bioactivity of the glycoprotein hormones (for review, see (43) ). However, the role of individual oligosaccharides for the in vivo activity of hTSH has not, to our knowledge, been investigated previously. Since the availability of a hTSH superagonist may have potential clinical applications(44) , we were interested to determine whether the increased in vitro activity of sialylated Q52/TSH from CHO-K1 cells could be maintained in vivo, despite the known protective effect of terminal sialic acid for the plasma half-life of rhTSH(16) . However, we found a significant decrease in the in vivo activity of Q52/TSH at the highest dose tested, as well as a slightly increased serum disappearance rate. By comparison, the significantly greater relative loss in the in vivo activity of the mutant Q78/TSH was correlated with a significant increase in its serum disappearance rate. This significantly greater decrease in circulatory half-life upon deletion of the oligosaccharide at Asn compared with Asn may be related to its peripheral, surface-exposed location, whereas the oligosaccharide at Asn appears to be buried at the dimer interface(36, 37) . These results are comparable with recent findings on the site-specific role of the carbohydrates for the in vivo activity of hFSH(45) . The lack of correlation between the in vitro and in vivo activities illustrates the importance of carbohydrates in determining hTSH activity in the whole organism. Further, our findings emphasize that the circulatory half-life, and not the in vitro activity, appears to be the primary determinant of the in vivo bioactivity of these hormones(16, 45) . It will be interesting to study whether further modifications of the Q52/TSH mutant, e.g. fusion of the hCG carboxyl-terminal peptide to the TSH carboxyl terminus (24, 46) , can compensate for the increased clearance rate and hence help to generate hTSH mutants with increased in vivo bioactivity.

In conclusion, we have demonstrated that the roles of the terminal sialic acids as well as of individual oligosaccharides are different for hTSH compared with hCG and hFSH. Consistent with our recent observations of the unique importance of the carboxyl-terminal Ser for hTSH action(22) , these findings indicate that conserved structures within the context of a given ligand receptor complex may contribute to signal transduction in different ways. In conjunction with previous findings on the glycoprotein hormones(15, 16, 17, 18, 32, 33) , we conclude that in hCG, which is exclusively sialylated, sialic acid is required for full expression of in vitro activity. In hFSH, which is predominantly sialylated, sialic acid residues appear to be dispensable for in vitro activity. However, in the case of bLH or phTSH, which bear sialic acids and are predominantly sulfated, expression of terminally sialylated oligosaccharides attenuates effective in vitro receptor activation. In particular, whereas the oligosaccharide at Asn is necessary for hCG and hFSH action, the same chain, and specifically its terminal sialic acid residues markedly attenuate TSH receptor binding and activation. As posttranslational modifications of carbohydrates regulate glycoprotein hormone activity in normal physiology, modulation of terminal sialylation of the Asn oligosaccharide, which appears more heterogeneous than other side chains(47) , may hence be important in regulating activity in a hormone-specific manner. Our observation that Q52/CG also had an increased activity at the hTSH receptor, opposite to the effect at its native receptor, points to differences in receptor-dependent ligand receptor interactions as a possible explanation for these distinct roles that may have evolved during evolution to maintain the specificity of receptor ligand interactions within the glycoprotein hormone family.

FOOTNOTES

*

Preliminary portions of these results were presented at the Clinical Research Meeting, San Diego, CA(1995). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Recipient of Grant DFG Gr 1316/1-1 from the Deutsche Forschungsgemeinschaft. To whom all correspondence and requests for reprints should be addressed. Molecular and Cellular Endocrinology Branch, NIDDK, National Institutes of Health, Bldg. 10, Room 8 D14, Bethesda, MD 20892-1758. Tel.: 301-496-3406; Fax: 301-496-1649.

()

The abbreviations used are: hTSH, human thyroid-stimulating hormone; TSH, thyroid-stimulating hormone subunit; CG, chorionic gonadotropin; hCG, human CG; FSH, follitropin; LH, lutropin; rhTSH, recombinant human thyroid-stimulating hormone; phTSH, pituitary human thyroid-stimulating hormone; CHO, chinese hamster ovary cell line; FRTL-5, Fischer rat thyroid cell line; 293, 293 human embryonic kidney cell line; T, 3,5,3`-triiodo-L-thyronine; T, 3,5,3`,5`-tetraiodo-L-thyronine.

()

M. Grossmann, M. W. Szkudlinski, J. E. Tropea, and B. D. Weintraub, unpublished observations.

ACKNOWLEDGEMENTS

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