Substitution of the Seat-belt Region of the Thyroid-stimulating Hormone (TSH) beta -Subunit with the Corresponding Regions of Choriogonadotropin or Follitropin Confers Luteotropic but Not Follitropic Activity to Chimeric TSH*

(Received for publication, September 10, 1996, and in revised form, February 24, 1997)

Mathis Grossmann Dagger , Mariusz W. Szkudlinski , Rosemary Wong §, James A. Dias , Tae H. Ji par and Bruce D. Weintraub

From the Laboratory of Molecular Endocrinology, Department of Medicine, University of Maryland School of Medicine and the Institute of Human Virology, Medical Biotechnology Center, Baltimore, Maryland 21201, the § Molecular and Cellular Endocrinology Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, the  Wadsworth Center, New York State Department of Health, Albany, New York 12201, and the par  Department of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

The region between the 10th and 12th cysteine (Cys88-Cys105 in human thyroid-stimulating hormone beta -subunit (hTSHbeta )) of the glycoprotein hormone beta -subunits corresponds to the disulfide-linked seat-belt region. It wraps around the common alpha -subunit and has been implicated in regulating specificity between human choriogonadotropin (hCG) and human follicle-stimulating hormone (hFSH), but determinants of hTSH specificity are unknown. To characterize the role of this region for hTSH, we constructed hTSH chimeras in which the entire seat-belt region Cys88-Cys105 or individual intercysteine segments Cys88-Cys95 and Cys95-Cys105 were replaced with the corresponding sequences of hCG and hFSH or alanine cassettes. Alanine cassette mutagenesis of hTSH showed that the Cys95-Cys105 segment of the seat-belt was more important for TSH receptor binding and signal transduction than the Cys88-Cys95 determinant loop region. Replacing the entire seat-belt of hTSHbeta with the hCG sequence conferred full hCG receptor binding and activation to the hTSH chimera, whereas TSH receptor binding and activation were abolished. Conversely, introduction of the hTSHbeta seat-belt sequence into hCGbeta generated an hCG chimera that bound to and activated the TSH receptor but not the CG/lutropin (LH) receptor. In contrast, an hTSH chimera bearing hFSH seat-belt residues did not possess any follitropic activity, and its thyrotropic activity was only slightly reduced. This may in part be due to the fact that the net charge of the seat-belt is similar in hTSH and hFSH but different from hCG. However, exchanging other regions of charge heterogeneity between hTSHbeta and hFSHbeta did not confer follitropic activity to hTSH. Thus, exchanging the seat-belt region between hTSH and hCG switches hormonal specificity in a mutually exclusive fashion. In contrast, the seat-belt appears not to discriminate between the TSH and the FSH receptors, indicating for the first time that domains outside the seat-belt region contribute to glycoprotein hormone specificity.


INTRODUCTION

Thyrotropin (thyroid-stimulating hormone (TSH))1 choriogonadotropin (CG), follitropin (follicle-stimulating hormone (FSH)), and lutropin (luteinizing hormone (LH)) are structurally related heterodimers that together form the glycoprotein hormone family (1). These hormones belong to the superfamily of cystine-knot growth factors (2, 3) and activate specific G-protein-coupled receptors notable for large extracellular domains containing multiple leucine-rich motifs (4). The primary structure of the alpha -subunit, which is encoded by a single gene, is identical in these hormones. The distinct beta -subunits, despite conservation of all 12 cysteine residues and similar overall folding, are sufficiently different to confer specificity to each hormone (1-3).

The molecular mechanisms whereby glycoprotein hormones activate their receptors are largely unknown, but multiple contact points between ligand and receptor, perhaps in a stepwise fashion, appear necessary to induce conformational changes favoring receptor G-protein coupling and subsequent second messenger generation (5-9). Recently, we have described several alpha -subunit domains important for hTSH activity (10-13), but there is little information on how the hTSH beta -subunit contributes to receptor activation.

Previous studies have shown that the region between the 10th and 12th cysteine of the beta -subunit is important not only for subunit association, receptor binding, as well as activation (14-16), but also for specific receptor recognition (17-20) of hCG and hFSH. In the crystal structure of hCG (2, 3), this region corresponds to the "seat-belt" region (Cys88-Cys105 in hTSHbeta ), so-called because it wraps around the alpha -subunit and orients it in the heterodimer while remaining covalently bonded to the beta -subunit through disulfide linkages between Cys9-Cys90 and Cys26-Cys110. This seat-belt consists of two intercysteine segments, a surface-exposed hydrophilic loop between the 10th and 11th cysteine (Cys88-Cys95 in hTSHbeta ) and a carboxyl-terminal segment between the 11th and 12th cysteine (Cys95-Cys105 in hTSHbeta ), and is in close proximity to alpha -subunit domains important for the structural integrity and activity of the glycoprotein hormones (2, 3).

In contrast to the work on the gonadotropins, the role of the seat-belt for hTSH is not known. A single study using a set of overlapping synthetic peptides spanning the entire hTSH subunit (21) showed that none of the peptides encompassing the seat-belt region, hTSHbeta 81-85 or hTSHbeta 91-105, inhibited TSH receptor binding, but a peptide containing the carboxyl terminus (beta 101-112) possessed the highest TSH receptor binding activity. However, the role of the seat-belt region in the context of the intact hTSH heterodimer has not been investigated. Interestingly, recent studies on a naturally occurring hTSHbeta mutation from patients with secondary hypothyroidism have shown the importance of Cys105 (corresponding to Cys110 in hCG) for hTSH activity (22).

In the present study, using a chimeric mutagenesis approach, we demonstrate the importance of the seat-belt for hTSH action as well as specificity. Moreover, our findings reveal previously unrecognized differences in the regulation of specificity among the glycoprotein hormones.


EXPERIMENTAL PROCEDURES

Materials

The following materials were generous gifts. CHO cells stably transfected with the rhTSH receptor (clone JP09) was from Dr. G. Vassart (Brussels, Belgium) (23); the gonadotropin-responsive murine Leydig cell line MA-10 was from Dr. M. Ascoli, (Iowa City, IA) (24); and cAMP antibody was from Dr. J. L. Vaitukaitis, National Institutes of Health (Bethesda, MD). Embryonic kidney 293 cells (FSH-R/293 cells) and Y-1 cells expressing the human FSH receptor have been described previously (20, 25). Cell culture media and reagents were purchased from Life Technologies, Inc. (Gaithersburg, MD); 125I-cAMP (specific activity, 40-60 µCi/µg) and 125I-hCG (specific activity, 50-70 µCi/µg) were from Hazleton (Vienna, VA), and polymerase chain reaction (PCR) reagents were from Boehringer Mannheim and New England Biolabs (Beverly, MA).

Site-directed Mutagenesis

The chimeric hTSH were constructed with the PCR-based megaprimer method of site-directed mutagenesis (26), as described (11, 13). Individual intercysteine segments C10-C11 (hTSHbeta Cys88-Cys95) or C11-C12 (hTSHbeta Cys95-Cys105) of the hTSH beta -minigene were replaced with nucleotides coding for the respective sequence of hCG and hFSH or Ala cassettes. To replace the entire seat-belt (hTSHbeta Cys88-Cys105), chimeras with individually mutated intercysteine segments were used as templates for subsequent PCR reactions. The hTSHbeta seat-belt was introduced into the hCG beta -subunit in a single PCR reaction using a primer coding for the entire hTSHbeta seat-belt. Further hTSH/hFSH chimeras were constructed in which the carboxyl-terminal residues hTSHbeta 105-112 or amino acids hTSHbeta 44-52 were replaced with the sequence of hFSH. In addition, Asp94 of the determinant loop was replaced with Lys (TSHbeta Lys94) or Glu (TSHbeta Glu94). After subcloning into the expression vectors, the entire PCR products of all constructs were sequenced to verify the mutations and to rule out any undesired polymerase errors. Construction of the quadruple alpha -subunit mutant bearing Lys residues at positions alpha 13, 14, 16, and 20 (alpha 4K) was described previously (13).

Transient Expression

CHO-K1 cells maintained as described (10) were transiently cotransfected with the various constructs using a transient transfection protocol based on a liposome formulation (LipofectAMINE reagent, Life Technologies, Inc.) (10). After culture in CHO serum-free medium (CHO-SFM, Life Technologies, Inc.) for 48 h, conditioned media including control medium from mock transfections were harvested, concentrated with Centriprep 10 concentrators (Amicon, Beverly, MA), and stored at -70 °C to prevent neuraminidase digestion.

Immunoassays

Wild type and mutant hTSH analogs were quantified with a panel of four different hTSH immunoassays, which were described in detail previously (12). hCG immunoreactivities were measured with two different specific third-generation immunoassays without crossreactivity to other glycoprotein hormones (Nichols Institute, San Juan Capistrano, CA; ICN, Costa Mesa, CA), and hFSH immunoreactivity was measured with an hFSH-specific third-generation immunoassay (Nichols Institute).

Hormone Binding Assays

The TSH receptor-binding activity of wild type and hTSH mutants was determined by their ability to displace 125I-bTSH from a solubilized porcine thyroid membrane receptor preparation (Kronus, Dana Point, CA), as described previously (10). Binding to the CG/LH receptor was studied in MA-10 cells following a previously employed protocol (13, 24), and FSH receptor binding was analyzed using a rat testis membrane radioreceptor assay as described in detail previously (20).

Hormone Activity Assays

The ability of the various chimeras to induce cAMP production was studied at the TSH receptor using JP09 cells (23), at the CG/LH receptor using MA-10 cells (24), and at the FSH receptor using FSH-R/293 cells (25). Briefly, confluent cells in 96-well tissue culture plates were incubated in a modified Krebs Ringer buffer for 2 h at 37 °C, 5% CO2 with serial dilutions of wild type and mutant hTSH, as well as control medium from mock transfections. The amount of cAMP released into the medium was assayed by radioimmunoassay (10). Progesterone production at the CG/LH or FSH receptor was determined using a commercially available progesterone radioimmunoassay kit (ICN) after incubation of the chimeric constructs with MA-10 cells or Y-1 cells, respectively, as detailed previously (20, 24).


RESULTS

Construction of Chimeric Glycoprotein Hormone Mutants

Replacing individual intercysteine segments C10-C11 of the hTSH beta -subunit (the determinant loop, hTSHbeta Cys88-Cys95) or C11-C12 (the carboxyl-terminal segment, hTSHbeta Cys95-Cys105) (Fig. 1) with Ala cassettes or with the corresponding sequences of hCG and hFSH generated hTSHbeta constructs designated 89Ala94, 96Ala104, 89CG94, 96CG104, 89FSH94 and 96FSH104, and exchange of the entire hTSH beta  seat-belt chimeras 89CG104 and 89FSH104. The hCG/hTSH chimera 94TSH109 was obtained by introduction of the hTSHbeta seat-belt residues into the hCG beta -subunit (Fig. 2). In addition, hTSHbeta Asp94, which is conserved in all known beta -subunits and essential for hCG activity (27), was replaced with Lys (hTSHbeta Lys94) or with Glu (hTSHbeta Glu94). Finally, sequences of the hTSH beta -subunit outside the seat-belt were replaced with the corresponding sequence of hFSH to create 44FSH52 and 105FSH112. These regions were chosen because they display the greatest charge heterogeneity among these beta -subunits, based on the proposed role of variable charges for glycoprotein hormone specificity (17) (and see below). Receptor binding and biological properties of these analogs, described in detail below, are summarized in Table I. A comparison of the receptor specificity of glycoprotein hormone seat-belt chimeras from this and other studies (18-20) is given in Table II.


Fig. 1. Domains of hTSH important for activity. The schematic drawing of hTSH is based on a molecular homology model of hTSH (13) and built on a template of an hCG model derived from crystallographic coordinates (2). The alpha -subunit is shown in gray, and the beta -subunit is black. The seat-belt region between the 10th (Cys88) and 12th (Cys105) Cys is depicted by an interrupted line; the N-terminal determinant loop (Cys88-Cys95 in hTSHbeta ) corresponds to the dotted line (bullet bullet bullet bullet ), and the carboxyl-terminal segment (Cys95-Cys105 in hTSHbeta ) corresponds to the dashed line (- - - -). Since the carboxyl terminus beyond hCGbeta 111 was not traceable in the original electron density map, hTSHbeta is only drawn to the corresponding residue 106. Domains of the alpha -subunit of known importance for hTSH activity are boxed. Because of hydrogen fluoride treatment of hCG prior to crystallization, the oligosaccharides, including that linked to alpha Asn52, are not shown. The alpha Asn52 carbohydrate is predicted to project into the proposed receptor binding domain, which also includes the alpha 40-46 helix and the alpha -carboxyl terminus alpha 88-92 (2, 3). In contrast, the alpha 11-20 domain is not located in proximity to the beta  seat-belt (see "Discussion").
[View Larger Version of this Image (23K GIF file)]


Fig. 2. Mutant hTSH and hCG beta -subunit seat-belt constructs. Shown are the native amino acid sequences of the seat-belt region between the 10th and 12th Cys for hTSH, hCG, and hFSH beta -subunits in the 1-letter code. For comparison, the Cys numbering is maintained according to its position in the respective subunit. Also depicted are the mutant seat-belt hTSH constructs and the hCG chimera prepared by site-directed mutagenesis, with the mutated segments underlined. In the Ala cassette constructs, either the determinant loop (between the 10th and 11th Cys, 89TSHbeta 94) or the carboxyl-terminal segment (between the 11th and 12th Cys, 96TSHbeta 104) were replaced with Ala residues. In the hTSH/hCG and hTSH/hFSH chimeras, individual intercysteine segments or the entire seat-belt of the hTSH beta -subunit were replaced with the respective gonadotropin residues. In contrast, in 94TSH109, the hTSHbeta seat-belt was introduced into the hCG beta -subunit.
[View Larger Version of this Image (47K GIF file)]

Table I. Receptor binding and biological activity of chimeric analogs at the TSH, CG, and FSH receptor

Thyrotropic, luteotropic, or follitropic activities of the chimeric analogs constructed by PCR-based mutagenesis and obtained by expression in CHO cells were tested in a variety of bioassays as described under "Experimental Procedures." Due to limitations of the amounts provided by transient transfection and due to the considerably reduced activity of certain analogs, it was not possible to calculate the half-maximum binding inhibition or stimulation in all cases. Therefore, the relative values of this table do not compare Kd or EC50. Rather, for the radioreceptor assays (RRA), values represent relative binding inhibition of an analog at the concentration of the appropriate wild type hormone resulting in half-maximal inhibition. Similarly, for cAMP and progesterone assays, the relative stimulation by hTSH mutants at the EC50 of the wild type hormone is given. *, no significant relative receptor binding or biological activity was observed at half-maximal concentration of wild-type hormone. Shown are mean values from individual experiments performed in triplicate and repeated at least twice. The S.E. were usually <10% (see also Figs. 3, 4, 5, 6, 7, 8, 9 for representative experiments). Prog., progesterone.

TSH-RRA TSH-cAMP CG-RRA CG-cAMP CG-Prog. FSH-RRA FSH-cAMP FSH Prog.

hTSH-wt 1.0 1.0 <0.01* <0.01* <0.01* <0.01* <0.01* <0.01*
89Ala94 0.22 0.24 <0.01* <0.01*
96Ala104 0.08 0.13 <0.01* <0.01*
D94K <0.01* <0.01*
D94E 1.1 0.96
94TSH109 0.13 0.11 <0.01* <0.01* <0.01*
hCG-wt <0.01* <0.01* 1.0 1.0 1.0 <0.01* <0.01* <0.01*
89CG94 0.21 0.20 0.15 0.05 0.1
 alpha 4K/89CG94 0.3 0.2 0.3
96CG104 <0.01* <0.01* 0.1 <0.01* 0.1
89CG104 <0.01* <0.01* 0.93 0.4 0.97
hFSH-wt <0.01* <0.01* <0.01* <0.01* <0.01* 1.0 1.0 1.0
89FSH94 0.77 0.75 <0.01* <0.01* <0.01*
96FSH104 0.62 0.72 <0.01* <0.01* <0.01*
89FSH104 0.54 0.33 <0.01* <0.01* <0.01*
105FSH112 0.28 <0.01* <0.01* <0.01*
44FSH52 1.1 <0.01* <0.01* <0.01*

Table II. Glycoprotein hormone seat-belt chimeras and their receptor specificity

Results are from this paper unless specified otherwise. Receptor specificity was determined either by receptor binding and/or activity assays (see text for details). For most chimeras, binding affinity and activation correlated closely. hTSH bearing the entire hCG seat-belt was 10-fold more potent for progesterone compared with cAMP stimulation (see text). The region between the 10th and the 11th Cys (C10-C11) corresponds to the determinant loop, and the region between the 11th and the 12th Cys (C11-C12) to the carboxyl-terminal segment of the seat-belt. +++, specificity equivalent to native hormone (80-100%); ++, 20-80%; +, 5-20%; -, <5% of native hormone. The known cross-reactivity of natural glycoprotein hormones, such as thyrotropic activity of hCG, by comparison, occurs with much higher amounts and requires a >1000-fold molar excess relative to the native hormone in the assays employed (11, 31). R, receptor.

Hormone Seat-belt sequence
Receptor specificity
Ref. no.
C10-C11 C11-C12 TSH-R CG-R FSH-R

hTSH hCG + +
hCG  - +
hCG hCG  - +++
hFSH ++  -
hFSH ++  -
hFSH hFSH ++  -
hCG hTSH hTSH ++  -
hFSH +  - (18, 19)
hFSH ++ ++ (18, 19)
hFSH hFSH  - +++ (18, 19)
hFSH hCG ++ +++ (20)
hCG  - + (20)

Secretion of Chimeric Mutants

All chimeric hTSH heterodimers were secreted from the transfected CHO cells, and relative secretion, compared with hTSH-wt, ranged from 35.3 ± 7.6% in the case of 89Ala94 to 100.2 ± 14.7% in the case of 96CG104. As evidence for accurate quantitation, hTSH immunoreactivity of each chimera was comparable in four different hTSH immunoassays, which recognize different epitopes of the hTSH molecule (10-13) (data not shown). Similarly, secretion of 94TSH109 bearing the hTSH seat-belt sequence within the hCG beta -subunit was 94.3 ± 17.7% that of hCG-wt, as determined by two different hCG immunoassays. Therefore, in accord with previous studies on hCG and hFSH (18-20), intercysteine loops appear to be interchangeable between the different beta -subunits without major changes in subunit assembly and heterodimer secretion of hTSH or global conformational changes of the heterodimer. Secretion of 44FSH52 and 105FSH112 with replacements outside the seat-belt was less than 25% for both chimeras. The reasons for the reduced secretion of these mutants are unclear but could be related to decreased stability of messenger RNA, improper folding of the mutant subunit, or decreased efficiency of subunit assembly.

Ala Cassette hTSH Mutants

TSH receptor binding as well as thyrotropic activity of both 89Ala94 and 96Ala104 was substantially reduced (Fig. 3, A and B), showing that the native seat-belt sequence is important for hTSH activity. Interestingly, maximal cAMP stimulation of the 96Ala104 mutant was significantly lower than that of the 89Ala94 mutant (28.3 ± 3.2 versus 63.0 ± 4.6% of hTSH-wt, respectively), indicating that the carboxyl-terminal segment of the seat-belt is more important for hTSH activity than the determinant loop.


Fig. 3. Thyrotropic activity of Ala cassette hTSH mutants. A, TSH receptor binding. Increasing doses of wild type or mutant hTSH were incubated with porcine membranes in the presence of a constant amount of 125I-bTSH. 125I-bTSH bound to membranes was precipitated and quantitated in a gamma  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 twice. B, cAMP induction at the TSH receptor. cAMP induction by the Ala cassette mutants was assessed in CHO cells expressing the rhTSH receptor (JP09). 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. Basal cAMP concentrations ranged from 8-19 pmol/ml. 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 this and the following figures, S.E. values are shown for all data points. In certain cases, the S.E. values are smaller than the size of the symbol.
[View Larger Version of this Image (15K GIF file)]

Role of Asp94 for hTSH Activity

A single mutation of the conserved Asp94 to Lys (hTSHbeta Lys94) completely abolished TSH receptor binding and activation, whereas preserving the negative charge at this position by mutating Asp94 to Glu (hTSHbeta Glu94) did not have a significant effect on TSH receptor binding or activation (Table I). This confirmed the importance of a negative charge in this particular position for glycoprotein hormone activity (27).

hTSH/hCG Chimeras

Replacement of the hTSH seat-belt segments with the respective sequences of hCG either substantially decreased (89CG94) or abolished measurable TSH receptor binding and activation (96CG104, 89CG104) of the chimeras (Fig. 4, A and B). hTSH/hCG chimeras 89CG94 and 96CG104 showed only very little CG/LH receptor binding and activation, both with cAMP stimulation as well as progesterone production. Thus, at the highest doses possible within the limitations of the transient transfection system, between 100-200 ng/ml, cAMP or progesterone production was only 10-18% that of hCG-wt (Fig. 5, A-C). To more conclusively test whether the Cys88-Cys95 determinant loop was important for differential hormonal activity, we constructed a hTSH mutant alpha 4K/89CG94. In alpha 4K/89CG94, residues alpha 13, 14, 16, and 20 were substituted with Lys residues in addition to the replacement of Cys88-Cys95 with the respective hCG residues. We had previously shown that introduction of positive charges into this alpha 11-20 domain led to substantial increases of glycoprotein hormone receptor binding affinity (13). We therefore expected that increasing the binding affinity of 89CG94 (Fig. 5A) should accentuate its gonadotropic properties. Maximal cAMP and progesterone production with this alpha 4K/89CG94 combination chimera increased to 35% and 50% of hCG-wt levels, respectively (Fig. 5, B and C). At the same time, the thyrotropic activity of alpha 4K/89CG94 remained unchanged (data not shown).


Fig. 4. Thyrotropic activity of hTSH/hCG seat-belt chimeras. A, TSH receptor binding. TSH receptor binding for the hTSH/hCG chimeras was performed as described in Fig. 3B. B, cAMP induction at the TSH receptor. The ability of the chimeras to induce cAMP production at the hTSH receptor was assessed in JP09 cells as described in Fig. 3B. Consistent with previous observations (11), hCG-wt did not bind to or activate the TSH receptor in the concentration range employed. A representative experiment, repeated at least twice, is shown.
[View Larger Version of this Image (18K GIF file)]


Fig. 5. Luteotropic activity of the hTSH/hCG seat-belt chimeras. CG/LH receptor binding. A, receptor binding affinity of the hTSH/hCG chimeras was performed in confluent MA-10 cells incubated in 96-well plates with serial dilutions of recombinant hormones in assay medium and 125I-hCG (70,000-100,000 counts/well) for 20 h at room temperature. After repeated washing, cells were dissolved in 1 N NaOH and counted in a gamma  counter. B, cAMP production at the CG/LH receptor. cAMP induction by the hTSH/hCG chimeras was determined in MA-10 cells according to the protocol described in Fig. 4B and under "Experimental Procedures." Basal cAMP levels were < 1.5 pmol/ml. C, progesterone production in MA-10 cells. Cells were incubated with hCG-wt or hTSH/hCG constructs for 6 h at 37 °C, 5% CO2. Progesterone concentrations were determined in the supernatant using a commercially available progesterone radioimmunoassay kit (ICN). A representative experiment, repeated at least twice, is shown.
[View Larger Version of this Image (27K GIF file)]

Remarkably, replacement of the entire seat-belt of hTSHbeta with the hCG sequence (89CG104) resulted in a chimera with hCG receptor binding comparable to hCG-wt, suggesting that the two individual intercysteine segments confer hCG specificity in a synergistic fashion (Fig. 5A). Further, the 89CG104 chimera was able to induce biological responses in MA-10 cells expressing the CG/LH receptor (Fig. 5, B and C). Whereas potency and efficacy of progesterone production as well as efficacy of cAMP induction were similar to hCG-wt, the potency of 89CG104 for cAMP production was 10-fold less than that of hCG-wt. These differences may stem in part from differences in the cAMP and progesterone assay conditions in MA-10 cells (see "Experimental Procedures"). Moreover, such generation of full hormonal responses at submaximal cAMP levels, termed "the cAMP superfluity concept," has been well recognized in studies on structure-function relationships of glycoprotein hormones (8, 28). Analogous findings for recombinant analogs with substantially higher progesterone-inducing than cAMP-inducing ability have been described by others (29). Interestingly, hTSH/hCG specificity appeared mutually exclusive since the 89CG104 chimera did not possess significant thyrotropic activity (Fig. 4, A and B).

hCG/hTSH Chimera

Conversely, the reciprocal chimera 94TSH109, which bears the hTSHbeta seat-belt in the context of the hCG-beta subunit, bound to the TSH receptor and was able to activate cAMP production in JP09 cells, with an EC50 that was 26.7 ± 4.7-fold higher than that of hTSH-wt (Fig. 6, A and B). At the same time, 94TSH109 did not bind to the CG/LH receptor, nor did it stimulate cAMP or progesterone production in MA-10 cells at concentrations up to 1000 ng/ml (Table I).


Fig. 6. Thyrotropic activity of the hCG/hTSH seat-belt chimera 94TSH109. This chimera bears the hTSHbeta seat-belt introduced into the hCG beta -subunit. A, TSH receptor binding. TSH receptor binding experiments were carried out as described in Fig. 3B. B, cAMP induction at the TSH receptor. cAMP production at the hTSH receptor was determined in JP09 cells as described in Fig. 3B (see legend to Fig. 3). 94TSH109 did not possess significant gonadotropic properties (Table I). A representative experiment, repeated at least twice, is shown.
[View Larger Version of this Image (15K GIF file)]

hTSH/hFSH Chimeras

Analogous replacement of individual intercysteine segments of hTSHbeta with the corresponding hFSH residues only slightly reduced TSH receptor binding or activation of these hTSH/hFSH chimeras (Fig. 7, A and B). Further, in contrast to 89CG104, the 89FSH104 construct bearing the entire hFSH seat-belt sequence was able to significantly bind to the TSH receptor and induce 50% of maximal hTSH-wt cAMP production in JP09 cells. Interestingly, none of the three hTSH/hFSH chimeras showed significant follitropic activity. Unlike hFSH-wt, the chimeras did not stimulate cAMP production at the hFSH receptor expressed in 293 cells (Fig. 8). Further, they did not show significant binding in a rat testis FSH radioreceptor assay and did not stimulate progesterone production in Y-1 cells expressing the hFSH receptor (Table I).


Fig. 7. Thyrotropic activity of hTSH/hFSH seat-belt chimeras. A, TSH receptor binding. TSH receptor binding experiments for the hTSH/hFSH chimeras were carried out as described in Fig. 3B. B, cAMP induction at the TSH receptor. The ability of the chimeras to induce cAMP production at the hTSH receptor was assessed in JP09 cells as described in Fig. 3B. A representative experiment, repeated at least twice, is shown.
[View Larger Version of this Image (18K GIF file)]


Fig. 8. Follitropic activity of hTSH/hFSH seat-belt chimeras. hFSH-wt or hTSH/hFSH chimeras were incubated with FSH-R/293 cells following the protocol described for JP09 cells in Fig. 3B. Basal cAMP levels were < 2 pmol/ml. The hTSH/hFSH chimeras did not show specific binding in a rat testis radioreceptor assay. Further, there was no significant progesterone production in Y-1 cells (Table I). A representative experiment, repeated at least twice, is shown.
[View Larger Version of this Image (19K GIF file)]

Since charge heterogeneity could play a role for hTSH/hFSH specificity (17), we replaced candidate regions hTSHbeta 44-52 and the carboxyl-terminal residues 105-112 with hFSH sequences. Thus, hTSHbeta 44KYALSQDVC52 has a net charge of 0, and the corresponding hFSHbeta sequence ARPKIQKTC has a charge of +3. hTSHbeta 105CTKPQKSY112 has a net charge of +2, and the corresponding hFSHbeta sequence CSFGEMKE has a charge of -1. The hTSHbeta 44-52 region corresponds to the carboxyl-terminal part of the long beta 2 loop identified by Keutmann et al. (30), which forms a wedge-shaped and partly surface-exposed extrusion in proximity to the determinant loop (2). However, none of the resulting hTSH/hFSH chimeras showed any follitropic activity in the three different assay systems (Table I). Interestingly, the thyrotropic activity of 105FSH112, but not that of 44FSH52 was significantly reduced (Fig. 9), in accord with previous studies on hTSH structure-function using a synthetic peptide approach (21).


Fig. 9. Thyrotropic activity of hTSH/hFSH chimeras 44FSH52 and 105FSH112. The ability of the chimeras to induce cAMP production at the hTSH receptor was assessed in JP09 cells as described in Fig. 3B. In contrast, these chimeras did not posses follitropic activity (Table I). A representative experiment, repeated at least twice, is shown.
[View Larger Version of this Image (18K GIF file)]


DISCUSSION

Previous studies on glycoprotein hormone specificity had focused on analogs that bound either to the CG/LH or the FSH receptor. These studies had suggested that domains within the seat-belt region of the beta -subunit are involved in directing gonadotropin specificity (18-20). It is unknown, however, how hTSH specificity is achieved and whether the seat-belt is critical for interaction with the TSH receptor. We directly compared the effects of systematically replacing the hTSHbeta seat-belt and its individual intercysteine segments with the corresponding regions of two different hormones, hCG and hFSH in parallel. Conversely, the hTSHbeta seat-belt residues were introduced into hCG. This strategy allowed us to characterize the role of the seat-belt for hTSH activity as well as specificity and to reveal divergent principles of specificity regulation among the members of the glycoprotein hormone family (see Table II).

Ala cassette mutations showed that the primary sequence of the seat-belt is essential for hTSH receptor binding and activation. Of central importance was the negatively charged Asp94 of the determinant loop since a single mutation of this residue to Lys, but not to Glu, abolished hTSH receptor binding and activity. The critical role of the negative charge of Asp94, which is conserved in all known glycoprotein hormone beta -subunits and forms a non-bonded interaction with alpha Thr54 (2, 3), was first identified in hCG by Chen et al. (27), suggesting that this residue is universally important for the members of the glycoprotein hormone family.

Our chimeric studies demonstrated that the seat-belt region of the hTSH beta -subunit, if placed into the context of the hCG beta -subunit, confers thyrotropic activity although the seat-belt alone was not sufficient for full thyrotropic activity. This suggests that additional hTSHbeta domains beside the seat-belt may contribute to hTSH specificity or that the hCG beta -subunit contains segments that restrict interaction with the TSH receptor. In this respect, it had been shown that removal of the C-terminal extension peptide of hCG (31) as well as of the N-linked carbohydrate side chain at alpha Asn52 increased the weak inherent thyrotropic activity of hCG (11). This thyrotropic activity of native hCG however, unlike the chimera described here, requires 1000-fold higher concentrations than TSH itself in most systems (11, 31). In contrast to the results with the hCG/hTSH chimera, the hCGbeta seat-belt, in the context of the hTSH beta -subunit, was sufficient for full CG/LH receptor binding and secretory response. This reciprocal exchange of hCG/hTSH receptor specificity was mutually exclusive as both chimeras possessed no significant residual activity at their native receptor.

Remarkably, introduction of the hFSH seat-belt into the hTSH beta -subunit did not result in FSH receptor binding or follitropic activity, and the hTSH/hFSH chimera retained most of its thyrotropic activity. In contrast to this finding, hCG could be converted to hFSH by placing the hFSHbeta seat-belt into the hCG beta -subunit (18, 19), and hFSH adopted partial CG/LH receptor binding after exchange of its determinant loop with the corresponding hCG sequence (20) (see Table II). Hence, the role of the seat-belt in conferring specificity appears to depend on the particular subunit into which it is introduced. These findings are best reconciled by considering the concept of "negative specificity," which proposes that specificity of glycoprotein hormones evolved independently from signal transduction by the introduction of domains that block inappropriate ligand-receptor interactions (9, 19). In this respect, it is interesting to note that the net charge of the determinant loop, the N-terminal part of the seat-belt region, is similar in hTSH (-2) and hFSH (-3) but different from that in hCG (+1). Thus, it is conceivable that a net positive charge of the determinant loop, in conjunction with the carboxyl-terminal segment of the seat-belt, interferes with hormone binding to the TSH as well as FSH receptor; whereas a net negative charge, again in conjunction with carboxyl-terminal residues reduces interaction with the CG/LH receptor. From an evolutionary standpoint, it is justifiable to assume that diversification and ligand selectivity did not evolve by development of new mechanisms of receptor activation but rather by the emergence of inhibitory domains that impose steric hindrances, thus allowing only the intended ligand to interact with the common activation domain. This concept of negative specificity is not without precedent among cystine-knot growth factors. Thus, binding specificity among members of the neurotrophin family is achieved by the cooperation of distinct active and inhibitory binding determinants that restrict ligand-receptor interactions, enabling the creation of analogs with multiple specificities (32).

Our findings extend the original observation of Moore et al. (17), who proposed that the variable charge of this loop may act as a determinant of hormone specificity. However, our data show that the carboxyl-terminal segment of the seat-belt is of similar importance for specificity, and charge differences of the determinant loop per se are, therefore, not sufficient for the switch of hormonal activity. Indeed, conversion of hTSH to a full hCG analog required concomitant replacement of the determinant loop as well as the carboxyl-terminal segment of the seat-belt, which displays a high degree of sequence but not charge heterogeneity among the glycoprotein hormones. Interestingly, the luteotropic activity of the hTSH/hCG determinant loop chimera could be increased by concomitant introduction of a cluster of Lys residues into the spatially unrelated alpha 11-20 domain, previously shown to increase receptor binding of hTSH as well as hCG (13).

In an attempt to identify domains determining hTSH/hFSH specificity outside the seat-belt, we focused on regions hTSHbeta 44-52, which correspond to the carboxyl-terminal part of the long beta 2 loop identified by Keutmann et al. (30), and the beta  carboxyl terminus 105-112. These domains were chosen because they display the greatest degree of charge heterogeneity between their beta -subunits. The decrease of thyrotropic activity of the 105FSH112 chimera confirmed the importance of the hTSHbeta carboxyl terminus, which was identified with a synthetic peptide approach (21), for hTSH activity. However, introduction of hFSH residues into these regions of the hTSH beta -subunit did not confer FSH receptor binding or follitropic activity to any of these chimeras. This was in agreement with the findings of Campbell et al., who showed that the long beta 2 loop was not important for hCG versus hFSH specificity (18). Thus, charged residues appear to play a lesser role in determining hTSH/hFSH specificity. It is possible that hTSH/hFSH specificity is not located within distinct segments of the beta -subunit but is mediated by a combination of topically related domains although the present study was not designed to systematically test this possibility.

Our study cannot define the molecular mechanisms whereby the seat-belt determines glycoprotein hormone specificity as this will require complete elucidation of the structure of hormone-receptor complexes. In this respect, the seat-belt could either directly contact the receptor or influence the conformation of functionally important but unrelated portions of the hormone. An indirect effect of the seat-belt on the conformation of the alpha -subunit would be consistent with antibody binding studies showing that the conformation of the alpha -subunit could change depending upon with which beta -subunit it associated (33), as well as with a recent model of glycoprotein hormone-receptor interaction predicting that the seat-belt does not directly contact the receptor (6). It could also explain the lack of TSH receptor binding of hTSHbeta peptides spanning the seat-belt region (21), as well as observations that mutations of identical alpha -subunit residues have hormone-dependent effects on activity (10-13, 25, 34). In this respect, we have shown that identical mutations in the alpha 33-44 domain, which includes a positively charged alpha -helix at alpha 40-46, truncation of the alpha -carboxyl terminus, and deletion of the carbohydrate consensus sequence at alpha Asn52, all affected hTSH subunit association or activity differently than in the analogous hCG and hFSH mutants (10-12). Intriguingly, these alpha -subunit domains are in close proximity to the seat-belt in the crystal structure of hCG and an hTSH homology model (5, 13). It has thus been proposed that they may form a composite receptor-binding domain (2, 3). In contrast, a peripheral alpha -subunit receptor binding domain located at the tip of the alpha 1 loop, alpha 11-20, appears to be important for all glycoprotein hormones (13). It is tempting to speculate that the lack of specificity of the alpha 11-20 domain is related to its distance from the seat-belt region. On the other hand, the alpha -subunit domains in close proximity to the beta -subunit seat-belt may be spatially oriented by the seat-belt to contact the appropriate receptor in a hormone-dependent fashion. In this respect, a direct cooperation between the complementary charged residues Lys91 of the alpha -subunit and Asp397 of the CG/LH receptor has recently been demonstrated (35).

Thus our findings suggest that, during the evolutionary divergence of the glycoprotein hormones from a common ancestor gene (36), determinants of ligand specificity have evolved independently and in different ways. The seat-belt region appears to be critical to direct glycoprotein hormone binding to either the CG/LH receptor or to the TSH and FSH receptor. Determinants mediating discrimination between the TSH and FSH receptor remain to be elucidated.


FOOTNOTES

*   Preliminary portions of these results were presented at the 10th International Congress of Endocrinology, San Francisco, CA (1996).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom all correspondence and requests for reprints should be addressed: Laboratory of Molecular Endocrinology, Institute of Human Virology, Medical Biotechnology Center, 725 W. Lombard St., N457, Baltimore, MD 21201. Tel.: 410-706-0993; Fax: 410-706-4574; E-mail: grossman{at}umbi.umd.edu.
1   The abbreviations used are: TSH, thyroid-stimulating hormone; hTSH, human TSH; hTSHbeta , human TSH beta -subunit; CG, choriogonadotropin; FSH, follitropin; LH, lutropin; rh, recombinant human; CHO, Chinese hamster ovary; PCR, polymerase chain reaction; wt, wild-type.

REFERENCES

  1. Pierce, J. G., and Parsons, T. F. (1981) Annu. Rev. Biochem. 50, 465-495 [CrossRef][Medline] [Order article via Infotrieve]
  2. Lapthorn, A. J., Harris, D. C., Littlejohn, A., Lustbader, J. W., Canfield, R. E., Machin, K. J., Morgan, F. J., and Isaacs, N. W. (1994) Nature 369, 455-461 [CrossRef][Medline] [Order article via Infotrieve]
  3. Wu, H., Lustbader, J. W., Liu, Y., Canfield, R. E., and Hendrickson, W. A. (1994) Structure (Lond.) 2, 545-558 [Medline] [Order article via Infotrieve]
  4. Segaloff, D. L., and Ascoli, M. (1993) Endocr. Rev. 14, 324-347 [Abstract]
  5. Szkudlinski, M. W., Grossmann, M., and Weintraub, B. D. (1996) Trends Endocrinol. Metab. 7, 11-20
  6. Moyle, W. R., Campbell, R. K., Rao, S. N. V., Ayad, N. G., Bernard, M. P., Han, Y., and Wang, Y. (1995) J. Biol. Chem. 270, 20020-20031 [Abstract/Free Full Text]
  7. Baenziger, J. U. (1994) in Glycoprotein Hormones (Lustbader, J. W., Puett, D., and Ruddon, R. W., eds), pp. 167-174, Springer-Verlag New York Inc., NY
  8. Bielinska, M., and Boime, I. (1995) in Glycoproteins (Montreuil, J., Vliegenhart, J. F. G., and Schachter, H., eds), pp. 565-587, Elsevier Science Publishers B.V., Amsterdam
  9. Combarnous, Y. (1992) Endocr. Rev. 13, 670-691 [Medline] [Order article via Infotrieve]
  10. Grossmann, M., Szkudlinski, M. W., Zeng, H., Kraiem, Z., Ji, I., Tropea, J. E., Ji, T. H., and Weintraub, B. D. (1995) Mol. Endocrinol. 9, 948-958 [Abstract]
  11. Grossmann, M., Szkudlinski, M. W., Tropea, J. E., Bishop, L. A., Thotakura, N. R., Schofield, P. R., and Weintraub, B. D. (1995) J. Biol. Chem. 270, 29378-29385 [Abstract/Free Full Text]
  12. Grossmann, M., Szkudlinski, M. W., Dias, J. A., Xia, H., Wong, R., Puett, D., and Weintraub, B. D. (1996) Mol. Endocrinol. 10, 769-779 [Abstract]
  13. Szkudlinski, M. W., Teh, N. G., Grossmann, M., Tropea, J. E., and Weintraub, B. D. (1996) Nat. Biotechnol. 14, 1257-1263 [Medline] [Order article via Infotrieve]
  14. Keutmann, H. T., Mason, K. A., Kitzmann, K., and Ryan, R. J. (1989) Mol. Endocrinol. 3, 526-531 [Abstract]
  15. Santa Coloma, T. A., and Reichert, L. E., Jr. (1990) J. Biol. Chem. 265, 5037-5042 [Abstract/Free Full Text]
  16. Chen, F., and Puett, D. (1991) J. Biol. Chem. 266, 6904-6908 [Abstract/Free Full Text]
  17. Moore, W. T., Burleigh, B. D., and Ward, D. N. (1980) in Chorionic Gonadotropin (Segal, S. A., ed), pp. 89-126, Plenum Press, New York
  18. Campbell, R. K., Dean-Emig, D. M., and Moyle, W. R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 760-764 [Abstract]
  19. Moyle, W. R., Campbell, R. K., Myers, R. V., Bernard, M. P., Han, Y., and Wang, X. (1994) Nature 368, 251-255 [CrossRef][Medline] [Order article via Infotrieve]
  20. Dias, J. A., Zhang, Y., and Liu, X. (1994) J. Biol. Chem. 269, 25289-25294 [Abstract/Free Full Text]
  21. Morris, J. C., McCormick, D. J., and Ryan, R. J. (1990) J. Biol. Chem. 265, 1881-1884 [Abstract/Free Full Text]
  22. Medeiros-Neto, G., Herodotou, D. T., Rajan, S., Kommareddi, S., de Lacerda, L., Sandrini, R., Boguszewski, C. S., Hollenberg, A. N., Radovick, S., and Wondisford, F. E. (1996) J. Clin. Invest. 97, 1250-1256 [Abstract/Free Full Text]
  23. Costagliola, S., Swillens, S., Niccoli, P., Dumont, J. E., Vassart, G., and Ludgate, M. (1992) Endocrinology 75, 1540-1544
  24. Ascoli, M. (1981) Endocrinology 108, 88-95 [Abstract]
  25. Yoo, J., Zeng, H., Ji, I., Murdoch, W. J., and Ji, T. H. (1993) J. Biol. Chem. 268, 13034-13042 [Abstract/Free Full Text]
  26. Sarkar, G., and Sommer, S. S. (1990) BioTechniques 8, 404-407 [Medline] [Order article via Infotrieve]
  27. Chen, F., Wang, Y., and Puett, D. (1991) J. Biol. Chem. 266, 19357-19361 [Abstract/Free Full Text]
  28. Jeevanram, R., Blithe, D., Liu, L., Wehmann, R., and Nisula, B. (1990) in Glycoprotein Hormones (Chin, W. W., and Boime, I., eds), pp. 395-402, Serono Symposia U. S. A., Norwell, MA
  29. Matzuk, M. M., Keene, J. L., and Boime, I. (1989) J. Biol. Chem. 264, 2409-2414 [Abstract/Free Full Text]
  30. Keutmann, H. T., Charlesworth, M. C., Mason, K. A., Ostrea, A., Johnson, L., and Ryan, R. J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 2038-2042 [Abstract]
  31. Yoshimura, M., and Hershman, J. M. (1995) Thyroid 5, 425-434 [Medline] [Order article via Infotrieve]
  32. Ibanez, C. F. (1994) J. Neurobiol. 25, 1349-1361 [Medline] [Order article via Infotrieve]
  33. Sairam, M. R., Linggen, J., Sairam, J., and Bhargavi, G. N. (1990) Biochem. Cell Biol. 68, 889-893 [Medline] [Order article via Infotrieve]
  34. Liu, C., Roth, K. E., Lindau Shepard, B. A., Shaffer, J. B., and Dias, J. A. (1993) J. Biol. Chem. 268, 21613-21617 [Abstract/Free Full Text]
  35. Ji, I., Zeng, H., and Ji, T. H. (1993) J. Biol. Chem. 268, 22971-22974 [Abstract/Free Full Text]
  36. Fiddes, J. C., and Talmadge, K. (1984) Recent Prog. Horm. Res. 40, 43-78 [Medline] [Order article via Infotrieve]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.