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Molecular Endocrinology, doi:10.1210/me.2005-0521
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Molecular Endocrinology 20 (12): 3351-3363
Copyright © 2006 by The Endocrine Society

Structural Differences in the Hinge Region of the Glycoprotein Hormone Receptors: Evidence from the Sulfated Tyrosine Residues

Marco Bonomi, Marta Busnelli, Luca Persani, Gilbert Vassart and Sabine Costagliola

Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, Université Libre de Bruxelles (M.Bo., G.V., S.C.), and Service de Génétique Médicale (G.V.), Hôpital Erasme, B-1070 Brussels, Belgium; Department of Medical Sciences (M.Bo., M.Bu., L.P.), University of Milan, and Laboratory of Endocrinological Research, Istituto Auxologico Italiano Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), 20095 Cusano Milanino and Fondazione IRCCS Ospedale Maggiore di Milano, Mangiagalli e Regina Elena, 20100 Milan, Italy

Address all correspondence and requests for reprints to: Marco Bonomi, Laboratory of Endocrinological Research, Istituto Auxologico Italiano Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Via Zucchi 18, I-20095 Cusano Milanino-Milan, Italy. E-mail: dmbonomi{at}tin.it.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Tyrosine sulfation is a late posttranslational modification of proteins that takes place in the Golgi network. In the past few years, this process has been identified as an important modulator of protein-protein interactions. Sulfated tyrosine residues have recently been identified in the C-terminal, so-called hinge region of the ectodomain of glycoprotein hormone receptors [TSH, LH/chorionic gonadotropin (CG), and FSH receptors] and were shown to play an important role in the interaction with their natural ligands. The position of two sulfated tyrosine residues in a Y-D/E-Y motif appears perfectly conserved in the alignment of TSH and LH receptors from different species, and site-directed mutagenesis experiments demonstrated that sulfation of the first residue of this motif was responsible for the functional effect on hormone binding. In contrast, the corresponding motif is not conserved in the FSH receptor, in which the first tyrosine residue is missing: the Y-D/E-Y motif is replaced by F333DY335. We extend here our previous observation that, in this case, it is sulfation of the second sole tyrosine residue in the motif that is functionally important. An LH/CG receptor harboring an F331DY333 motif (i.e. displaying decreased sensitivity to human CG) was used as a backbone in which short portions of the FSH receptor were substituted. Segments from the FSH receptor capable of restoring sensitivity to human CG were identified by transfection of the chimeras in COS-7 cells. These experiments identified key amino acid residues in the hinge region of the FSH receptor associated with the functional role of the second sulfated tyrosine residue in a Y-D/E-Y motif, allowing for efficient hormone binding. The experiments represent strong evidence that structural differences in the hinge regions of FSH and LH/CG receptors play a significant role in hormone-receptor-specific recognition.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE GLYCOPROTEIN HORMONE receptors [TSH receptor (TSHR), LH/choriogonadotropin (CG) receptor (CGR), and FSH receptor (FSHR)] constitute a G protein-coupled receptor (GPCR) subfamily characterized by a large (359–414 residues) amino-terminal extracellular domain (ECD) and a rhodopsin-like serpentine domain (TMD). The high sequence identity of the three glycoprotein hormone receptor serpentine domains (~70%) makes them functionally interchangeable in chimeric constructs, at least for stimulation of the cAMP regulatory cascade (1, 2). In contrast, the respective ECDs are less similar (~40%), which has been related to the specificity of hormone recognition and binding (1, 2, 3, 4, 5, 6, 7). These N-terminal domains are composed of two cysteine clusters, flanking 11 leucine-rich repeats (LRRs) (7, 8, 9), which have been shown to contain the structural determinants involved in recognition specificity (1, 6, 7, 10). These are mainly responsible for the establishment of electrostatic interactions specific to each hormone-receptor couple. They involve residues of the ß-sheets constituting one of the surfaces of the LRR portion of the ectodomain (6, 7, 8, 9, 11). In addition to hormone-specific ionic interactions, we have recently demonstrated that efficient binding to and activation of glycoprotein hormone receptors require non-hormone-specific ionic interactions involving sulfated tyrosine residues located in the C-terminal region of their ectodomains (12).

Tyrosine-O-sulfation is a late posttranslational modification event taking place in the Golgi immediately before delivery of proteins to the plasma membrane or secretion (13). Protein tyrosyl-sulfation has been shown to play an important role in the interactions of a wide variety of proteins with their respective partners: examples include P-selectin and P-selectin glycoprotein 1 ligand (PSGL-1) (14, 15), platelet glycoprotein Ib{alpha} (GpIb{alpha}) on the platelet surface and von Willebrand factor (16, 17), and cholecystokinin and its receptor (18). In the GPCR field, it has been demonstrated that some of the chemokine receptors, such as chemokine receptor 5 (CCR5), undergo sulfation of specific tyrosine residues in their amino-terminal domain. The resulting negative charges contribute to the binding of its natural ligands macrophage inflammatory protein (MIP)-1{alpha} and MIP-1ß, as well as of HIV-1 glycoprotein 120 protein (19, 20). Very recently, tyrosine sulfation of some extracellular matrix proteins has been demonstrated, by mass spectrometry, to be involved in stabilization of the fibrillar/collagen network (21).

In the mammalian TSHR and LH/CGR, the tyrosine residues undergoing O-sulfation are part of a highly conserved Y-D/E-Y motif located 27 residues upstream from the first transmembrane helix (12). We have shown by site-directed mutagenesis that sulfation of only the first tyrosine residue in the motif is required to observe high-affinity interaction of these receptors with their cognate hormones (12). In contrast, the mammalian FSHR contains an F-D/E-Y motif in the homologous position, and previous results have shown that the second and sole tyrosine residue in the motif is implicated in sulfation-dependent sensitivity to FSH (12). In the present study, we demonstrate that the need for sulfation of the second tyrosine residue of the motif, as in the wild-type human (h) FSHR, can be conferred to the hLH/CGR by keeping specific key amino acid residues from the hinge region of the hFSHR. Therefore, these results provide original evidence for a relevant functional role of structural differences in the hinge region of the hFSH and hLH/CG receptors, which is the only region of this GPCR class that has not been crystallized so far but has been modeled using only a comparative approach (7, 22).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Sulfation of Nonhomologous Tyrosine Residues Is Required for High-Affinity Binding and Activation of the hLH/CG and hFSH Receptors by their Respective Agonists
The sulfation motifs in hLH/CGR and hTSHR (Y-D/E-Y) are well conserved (see Fig. 1Go), containing two potentially sulfated tyrosine residues. Both residues have effectively been shown to be sulfated in the hTSHR (12). In the FSH receptors from all mammalian and bird species the Y-D/E-Y motif is replaced by F-D/E-Y in the homologous position (see Fig. 1Go), with an additional tyrosine residue located three amino acids upstream in mammals (Y330-T-E-F333-D-Y335, in human).


Figure 1
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  		Fig. 1. Sulfation Region in the Three Glycoprotein Hormone Receptors of Various Species

Alignment of glycoprotein hormone receptor sequences of various species (numbering refers to the human sequences) in the Y-D/E-Y motif region (underlined). The Y-D/E-Y motif is extremely well conserved in the TSHRs and LH/CGRs but not in FSHRs (see Discussion).

 
By performing concentration effect curves, we confirmed and extended our previous results (12) concerning the loss of sensitivity associated with single or double mutation of the various tyrosine residues in hLHR/CGR or hFSHR construct (see also Fig. 2Go and Table 1Go). The concentration-effect curves of hLHR and hFSHR double mutants (Y331-E-Y333 -> F-E-F; Y330-x-x-F-D-Y335 -> F-x-x-F-D-F) both presented a rightward shift, corresponding to EC50 values higher than those of the wild-type receptors (EC50 of wild-type LH/CGR = 0.02 ± 0.003 IU/ml, EC50 of F331-E-F333 = 0.155 ± 0.05 IU/ml; EC50 of wild-type FSHR = 0.04 ± 0.003 IU/ml, EC50 of F330-x-x-F-D-F335 = 0.81 IU/ml; see also Fig. 2Go, A and B, and Table 1Go). The concentration-effect curves of the single mutants confirmed that in the hLH/CGR only the first tyrosine (Y331) is functionally important for activation by hCG. The loss of sensitivity to hCG is similar in the single F331-E-Y333 and double F331-E-F333 mutants (see Fig. 2AGo and Table 1Go). On the contrary, for the hFSHR, sensitivity to human recombinant (hr) FSH depends on the presence of the second sulfated tyrosine (Y335). The loss of sensitivity to hrFSH is similar in the single Y330-x-x-F-D-F335 and double F330-x-x-F-D-F335 mutants, and the F330-x-x-F-D-Y335 single mutant behaves as the wild type (see Fig. 2BGo and Table 1Go). No differences were observed in terms of maximal response in cAMP for all the single mutants in the two receptors, whereas we observed a slight decrease in the F330-x-x-F-D-F335 double mutant (see Fig. 2Go, A and B, and Table 1Go).


Figure 2
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  		Fig. 2. Concentration-Effect Curves in hLHR/CGR and hFSHR Sulfation Mutant

Concentration-effect curves for the (A) hLH/CGR mutants and (B) hFSHR mutant, respectively, under stimulation of increasing concentrations of hCG or hFSH. COS-7 cells transiently transfected with the various constructs (see Materials and Methods) were stimulated by the concentration of hCG or hFSH indicated, and intracellular cAMP values were determined. The results are expressed as picomoles per milliliter. The Prism computer program (GraphPad Software, Inc.) was used for curve fitting and for EC50 determination. Cell surface expression of the (C) hLHR/CGR mutants and (D) hFSHR mutants, measured by flow cytofluorometry (see Materials and Methods). Results are expressed as arbitrary fluorescence units (AFU). BAS, Basal value.

 

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  		Table 1. Summary of the Constructs Data Shown

 
All the mutants were expressed at the cell surface as well as their wild-type counterpart, as demonstrated by flow immunocytometry with specific monoclonal antibodies (mAbs) (see Fig. 2Go, C and D, and Table 1Go).

Whereas the F331-E-Y333 (Y331F hLH/CGR) and Y330-x-x-F-D-F335 (Y335F hFSHR) mutants show decreased sensitivity to hCG or hrFSH, respectively, we demonstrated that stimulation by other agents or means remained unaffected (see supplemental data, published on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org).

Evidence that the effect of substitution of Y331 and Y335, respectively, in hLHR and hFSHR was secondary to the loss of sulfation at these positions is provided by the differential sensitivity of wild-type and mutated constructs to the inhibitory action of NaClO3. As expected, this treatment decreases the sensitivity to hCG or hrFSH in the wild-type and single unaffected mutants, whereas it is without effect on the two mutants in which the putatively sulfated tyrosine residues have been replaced by phenylalanine (see supplemental Figs. 2 and 3).

More direct evidence for sulfation of the Y335 residue in the FSHR is provided by the decrease in [35S]sulfate incorporation observed in a construct harboring a phenylalanine substitution at this position. For convenience, this construct was made on the background of a glycosylphosphatidylinositol-anchored ectodomain of the receptor harboring a histidine tag (see Materials and Methods; Fig. 3Go, A and B; and Ref. 5). Wild-type (F330-x-x-F-D-Y335) and Y330-x-x-F-D-F335 mutant constructs were transfected in Chinese hamster ovary (CHO) cells, and the cells were incubated with [35S](methionine+cysteine) or [35S]sulfate. Receptor constructs were affinity purified from cells extract by nickel chromatography and the resulting material was subjected to polyacrylamide gel electrophoresis and autoradiography. As can be seen in Fig. 3CGo, when similar amounts of [35S](methionine+cysteine)-labeled receptor were loaded for each construct, incorporation from [35S]sulfate was virtually absent in the Y335F mutant (Y330-x-x-F-D-F335).


Figure 3
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  		Fig. 3. FSHR ECD with a 10 Histidine Tag and a GPI Anchor

Linear sequence (A) and schematic representation (B) of the FSHR-ECD-10His construct with its signal for GPI attachment, released by PI-PLC cleavage. Incorporation of [35S]sulfate in the hFSHR mutants (C): CHO cells expressing wild-type (WT) or mutated Y-x-x-F-D-F ECD-hFSHR-10H-GPI construct (see Materials and Methods) were incubated for 24 h in DMEM without cysteine and methionine, together with 0.5 mCi each of [35S]cysteine and [35S]methionine (lanes 3 and 4), or in sulfate-free medium with 1 mCi [35S]-sulfate (lanes 1 and 2). Labeled ectodomains released with PI-PLC and purified on nickel beads were analyzed by SDS-PAGE under reducing conditions. Molecular mass markers are indicated (in kilodaltons).

 
Introduction of a Tyrosine in Position 333 of the FSHR Rescues Functionality of the Y335F Mutant
To further explore sensitivity of the hFSHR to alteration in the pattern of sulfated tyrosine residues, a tyrosine was introduced in position 333 (F333Y) of the wild-type hFSHR (Y330-x-x-Y333-D-Y335 construct) and the Y330-x-x-F333-D-F335 mutant (Y330-x-x-Y333-D-F335construct). The Y330-x-x-Y333-D-Y335 construct displayed wild-type sensitivity to hrFSH (EC50 of Y330-x-x-Y333-D-Y335 = 0.03 ± 0.007 IU/ml; EC50 of wild-type FSHR = 0.04 ± 0.003 IU/ml; see Fig. 4AGo and Table 1Go). More interesting, introduction of a tyrosine residue in position 333 of the Y330-x-x-F333-D-F335 mutant (construct Y330-x-x-Y333-D-F335) completely rescued its loss of sensitivity to hrFSH (EC50 of Y330-x-x-Y333-D-F335 = 0.05 ± 0.013 IU/ml; EC50 of Y330-x-x-F333-D-F335 = 0.44 ± 0.155 IU/ml; see Fig. 4AGo and Table 1Go). The expression level of the two mutants appears unmodified compared with the respective wild type (Y330-x-x-Y333-D-Y335, 86.71 ± 3.79% of the wild-type expression; Y330-x-x-Y333-D-F335, 85.28 ± 4.69% of the wild type expression; see Fig. 4BGo and Table 1Go). From this, we conclude that the precise position of the sulfated tyrosine is less important in the hFSHR than in either the hTSHR or the hLH/CGR. We also performed competition binding assays of the two mutants using 125I-FSH, and their behavior was similar to the wild-type receptor (data not shown).


Figure 4
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  		Fig. 4. Rescue Functionality of the Y335F Mutant in hFSHR

A, Concentration-effect curves for the hFSHR mutant under stimulation of increasing concentrations of hFSH. COS-7 cells transiently transfected with the various constructs (see Materials and Methods) were stimulated by the concentration of hFSH indicated, and intracellular cAMP values were determined. The results are expressed as picomoles per milliliter. The Prism computer program (GraphPad Software, Inc.) was used for curve fitting and for EC50 determination. B, Cell surface expression of the hFSHR mutants, measured by flow cytofluorometry (see Materials and Methods). Results are expressed as arbitrary fluorescence units (A.F.U.).

 
Sequences in the Hinge Region of the hFSHR Are Responsible for its Differential Sensitivity to Mutations of the Sulfation Motif
To attempt identification of the structures in the hFSHR implicated in its differential use of sulfated residues in the Y/F-D/E-Y motif, we generated a series of chimeras between hLH/CGR and hFSHR. Using as starting point a chimera between rat LHR and FSHR described by Braun et al. (1), we constructed first the LF2L pSVL and the LF4L pSVL chimeras, in which large portions of the FSHR were introduced in the hLHR/CGR background (see Fig. 5Go). When transfected in COS-cells, the two constructs are well expressed at the cell surface (see also Table 1Go) and they respond to hCG with a sensitivity comparable to the wild-type hLH/CGR (LF2L-pSVL EC50 = 0.02 ± 0.007 IU/ml; LF4L-pSVL EC50 = 0.02 ± 0.005 IU/ml; see also Table 1Go). These results confirm that the absence of the first tyrosine residue is without effect in the background of these chimeras and provide novel indirect information reflecting structural differences in the hinge region of the receptor class.


Figure 5
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  		Fig. 5. Chimeras between the hLHR/CGR and the hFSHR

A, Schematic representation of the different chimeras containing segments of the hFSHR (in white) inserted in a hLH/CGR background (in black) with their respective pharmacological phenotypes. The restriction sites used to make the constructs are indicated. B, Concentration-effect curves for the chimeras under stimulation of increasing concentrations of hCG. COS-7 cells transiently transfected with the various constructs (see Materials and Methods) were stimulated by concentration of hCG indicated, and intracellular cAMP values were determined. The results are expressed as picomoles per milliliter. The Prism computer program (GraphPad Software, Inc.) was used for curve fitting and for EC50 determination. C, Cell surface expression of the chimeras, measured by flow cytofluorometry (see Materials and Methods). Results are expressed as arbitrary fluorescence units (AFU).

 
To identify the amino acids implicated in the functional recovery observed in the LF4L-pSVL chimera, and to better define the indirect structural differences in the hinge region emerged, we generated additional chimeric constructs exploring the role of segments located immediately upstream or downstream from the sulfation motif.

Comparison of the functional characteristics of all the chimeras demonstrated that the gain of sensitivity to hCG of an LH/CGR construct with an FDY motif depended on the presence of FSHR sequences located immediately downstream of the motif. Indeed, inclusion of only the upstream portion of FSHR sequences (construct LF7L pSVL) did not increase responsiveness to hCG (compare LF7L pSVL and F331D332Y333; see also Fig. 5Go, B and C, and Table 1Go). On the contrary, inclusion of residues located downstream of the motif resulted in a gain of sensitivity (compare LF7L pSVL and F331EY (333) mutant; Fig. 5Go, B and C, and Table 1Go). Interestingly, this gain of function could be obtained with two nonoverlapping downstream segments, each of six residues in length (LF5L pSVL and LF6L pSVL; see Fig. 5AGo and Table 1Go). Besides, it is noteworthy that an E332D substitution in the F331-D332-Y333 hLHR mutant results in a partial gain of sensitivity to hCG when compared with the F331-E-Y333 mutant (see also Fig. 5BGo and Table 1Go). Moreover, the 125I-hCG binding tested in this construct presents an intermediate IC50 in comparison with the wild-type hLHR/CGR pSVL and the F331-E-Y333 hLHR/CGR pSVL mutant (hLHR/hCGR-pSVL IC50 = 0.130 ± 0.024 IU/ml; F331-E-Y333 hLHR-pSVL IC50 = 0.025 ± 0.005 IU/ml; F331-D332-Y333 hLHR/CGR IC50 = 0.056 ± 0.01 IU/ml; see also Fig. 6Go, B and C, and TableGo 1).


Figure 6
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  		Fig. 6. Concentration-Effect Curve and hCG Binding in the hLHR Mutants

A, Concentration-effect curves for the hLH/CGR mutants under stimulation of increasing concentrations of hCG. COS-7 cells transiently transfected with the various constructs (see Materials and Methods) were stimulated by the concentration of hCG indicated, and intracellular cAMP values were determined. The results are expressed as picomoles per milliliter. The Prism computer program (GraphPad Software, Inc.) was used for curve fitting and for EC50 determination. B, Displacement curves of 125I-hCG by increasing concentration of hCG. COS-7 transfected with various constructs were incubated overnight at room temperature in the presence of 100,000 cpm of 125I-hCG and graded concentration of cold hCG (see Materials and Methods). The results are expressed as 125I-hCG-bound cpm. The Prism computer program (GraphPad Software, Inc.) was used for curve fitting and for IC50 calculation. C, 125I-hCG binding of the different constructs transfected as mentioned above (see also Materials and Methods). The graph shows the ratio of the single mutants’ total binding reported to the wild type value. D, Cell surface expression of the hLHR mutants, measured by flow cytofluorometry (see Materials and Methods). Results are expressed as arbitrary fluorescence units (AFU).

 
The D334, D336, and T345 Amino Acids in the hFSHR Are Responsible for its Differential Sensitivity to Mutations of the Sulfation Motif
A detailed analysis of the amino acidic sequence that follows the sulfated motif in the hFSHR in comparison with the aligned sequence in the chicken LHR (where, similar to the situation in mammalian FSHR, only the second tyrosine of the motif is present; see Fig. 1Go) focused our attention on two amino acids, D336 and T345, in the hFSHR (respectively G334 and R342 in the hLHR). Because their presence seems to correlate with that of an FD/EY motif, it was tempting to hypothesize that they might play a role in the choice of the functional sulfated tyrosine residue. To verify this point, we generated double and triple mutants in the hLHR, exchanging each of the above-mentioned amino acids in single or combined manner with their hFSHR counterparts. The introduction of the single substitution G334D or R342T in the F331-E-Y333 mutant did not change either sensitivity or affinity to hCG of the original construct (F331-E-Y333-D334 hLHR-pSVL EC50 = 0.08 ± 0.004 IU/ml, IC50 = 0.026 ± 0.012 IU/ml; F331-E-Y333-T342 hLHR-pSVL EC50 = 0.076 ± 0.006 IU/ml, IC50 = 0.026 ± 0.002 IU/ml; see also Fig. 6Go and Table 1Go). On the contrary, the combined substitution of the two residues (G334D and R342T) in the F331-E-Y333 mutant showed a partial recovery of the EC50 but not of the IC50 (F331-E-Y333-D334-T342 hLHR-pSVL EC50 = 0.054± 0.009 IU/ml, IC50 = 0.031 ± 0.008 IU/ml; see also Fig. 6Go and Table 1Go).

When we operated the same amino acids changes in the F331D332Y333 mutant background, we observed an intermediate sensitivity and affinity for single G334D and the R342T mutations (F331D332Y333-D334 hLHR-pSVL EC50 = 0.034 ± 0.011 IU/ml, IC50 = 0.132 ± 0.028 IU/ml; F331-D332-Y333-T342 hLHR-pSVL EC50 = 0.036 ± 0.013 IU/ml, IC50 = 0.131± 0.029 IU/ml; see also Fig. 6Go and Table 1Go), whereas a complete functional recovery was seen for the combined mutants (F331D332Y333-D334- T342 hLHR-pSVL EC50 = 0.022 ± 0.004 IU/ml, IC50 = 0.170 ± 0.042 IU/ml; see also Fig. 6Go and Table 1Go).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Tyrosine O-sulfation is a late posttranslational modification conferring strong anionic properties to specific domains of extracellular proteins (13). The importance of this modification is illustrated in knockout mice in which the two tyrosylprotein sulfotransferases enzymes [TPST-1 and TPST-2 (23, 24)] have been invalidated (13, 25). The phenotype of the mice indicates that the two isoenzymes have distinct biological roles in vivo, with some relevance to reproductive physiology, which may have some bearing on the present observation in the gonadotropin receptors. In particular, TSPT1–/– mice appear healthy but have 5% lower average body weight than wild-type controls. Fertility is normal, but TSPT1–/– females have significantly smaller litters than wild type due to fetal death (13). On the other hand, TSPT2–/– mice are severely delayed in growth. The reproductive performance of the male, but not the female, is severely decreased, probably due to abnormalities in sperm transport, or capacitation, and/or fertilization. However, other gonad-related parameters are reported to be normal (testicular weight and serum FSH, LH, and testosterone levels) (13). Clear evidence that lack of sulfation of specific tyrosine residues may have functional consequences in vivo in humans is provided by patients with mild/moderate hemophilia caused by a Y1680F mutation in coagulation factor VIII (26). Sulfation of Y1680 in factor VIII is required for its optimal binding to von Willebrand factor (26). von Willebrand factor acts as carrier protein increasing the circulating half-life of factor VIII in the plasma (27).

Our previous studies, centered mainly on the hTSHR, demonstrated unambiguously that both tyrosine residues of a Y-D-Y motif, in the C-terminal portion of the ectodomain of the receptor, were sulfated (12, 28). Inhibition of sulfation by incubation of cells in 10 mM sodium chlorate, or mutation of the first (but not of the second) tyrosine in the motif to Phe, resulted in a 1-log decrease in sensitivity of the receptor to TSH (12). Preliminary results suggested that a similar situation involves also the gonadotropin receptors (12).

Alignment of the TSHRs and LH/CGRs from a series of species indicates strong conservation of a Y-D/E-Y motif, whereas the FSHRs displayed a different sequence in the homologous position, Y330-x-x-F-D/E-Y335, with the first conserved tyrosine replaced by Phe and a Tyr residue located two amino acids upstream (see Fig. 1Go). Our present results demonstrate that, similar to the situation in hTSHR, wild-type receptor sensitivity to hCG is strictly dependent on the presence of the first tyrosine residue of the Y-D/E-Y in the hLH/CGR (see Fig. 2AGo). Treatment with an inhibitor of sulfation, NaClO3, affected only the wild-type and the second tyrosine mutant, whereas it was without effect on the hormone sensitivity of a mutant in which the first tyrosine was eliminated (see supplemental Fig. 2). On the contrary, in the hFSHR, the first (upstream) tyrosine residue is dispensable, and wild-type receptor sensitivity to hrFSH is observed in both Y330-x-x-F-D-Y335 (wild-type sequence) and F330-x-x-F-D-Y335 mutants (Fig. 2BGo). The conclusion that sulfation of Y335 is functionally important in the wild-type FSHR is supported by two types of experiments: 1) the loss of incorporation of [35S]sulfate in the second tyrosine mutant (Y330-x-x-F-D-F335) demonstrates unambiguously that this residue is effectively sulfated (see Fig. 3CGo); 2) the differential effect of NaClO3 on sensitivity to FSH observed for wild-type FSHR and the F330-x-x-F-D-Y335 mutant (strong decrease in sensitivity), when compared with the Y330-x-x-F-D-F335 mutant (unaffected) (see supplemental Fig. 3), indicates the functional role of this sulfation event. Interestingly, and contrary to the situation in the TSHR and LH/CGR, some latitude is allowed for the position of the sulfated tyrosine in the FSHR, because wild-type receptor sensitivity to FSH is observed in a Y330-x-x-Y333-D-F335 construct (Fig. 4AGo).

From this we conclude that wild-type receptor sensitivity to their respective agonists require sulfation of tyrosine residues in all three glycoprotein hormone receptors, but with less stringent structural constrains for the hFSHR.

It has been known for a long time that binding of glycoprotein hormones to their receptors are strongly dependent on electrostatic interactions (3, 4, 7, 8, 29, 30, 31, 32). Putting together the present observations and previous data in which we have identified structural determinants implicated in recognition specificity (6), we suggest that binding of glycoprotein hormones to their receptors involves both hormone-specific ionic interactions with specific residues of the LRR motifs and less (or non-) specific ionic interactions of hormones with sulfated tyrosine residues. This is in line with the results by Karges et al. (33) concerning an N-terminal truncated hLHR/CGR (LHR-Fur316), which is still stimulated by hCG even though with decreased maximal response and increased EC50; and with the data by Angelova et al. (34), where human {alpha}-subunit analogs act as partial agonist of the hTSHR. A series of basic residues present in the {alpha}-subunit common to all three glycoprotein hormones (3, 32) or the glycosylated oligosaccharide chains whose composition may be crucial for glycoprotein hormone intrinsic bioactivity (35, 36) provide strong candidates for non-hormone-specific interaction with the sulfated motifs. Nevertheless, interactions of sulfated tyrosine residues with basic amino acids conserved in the different ß-subunits cannot be excluded. Considering the TSHR model proposed by Kleinau et al. (7), in which they identified a novel epitope acting as intramolecular signaling interface, the Y-E/D-Y sulfated motif is located in the middle between the Cys283/ Phe286 and the epitope D403EFNPC408 and, in the absence of direct structural data, we can hypothesized that it could be implicated, after interaction with the hormone, in inducing the conformational changes in the receptor that allow the signaling to the G protein. The different sensitivity of the hFSHR to position change of tyrosine residues in the sulfation motif, when compared with the hTSHR or hLH/CGR, raised the possibility, however, that some specificity dictated by the hormone might exist. Our experiments with a hFSHR mutant capable of responding to hCG (F26 mutant; see also Ref. 6) directly addresses this point. Contrary to what is observed with wild-type hLH/CGR, on the hFSHR-F26 background no difference in sensitivity to hrCG (data not shown) was observed for constructs bearing F333-D-Y335 (as in the wild-type hFSHR) or Y333-D-Y335 (as in the hLH/CGR). However, these two mutants behave slightly differently when stimulated by hrFSH (see supplemental Fig. 4 and Table 1). The difference in sensitivity of the two [(F26) Y330-x-x-Y333-D-F335 and (F26)Y330-x-x-Y333-D-Y335] mutants to hrFSH, but not to hCG, suggests that recognition of the sulfated motifs by hrFSH and hCG may involve different structural determinants.

From an evolutionary point of view, the presence of a Y-D/E-Y motif in mammalian [where both tyrosine residues are sulfated (12)] and fish TSHR, in mammalian LH/CGR, and in Cynops FSHR suggests that such a motif likely represents the ancestral sequence (see Fig. 1Go). This would imply that in fish and bird LH/CGRs, and in almost all available FSHR (except Cynops pyrrhogaster, lizard and snake), the first Tyr residue would have been lost. Because this is the functionally important residue in both mammalian TSHR and LH/CGR, it is likely that sulfation of the two residues was functionally redundant in the ancestral receptors and that subsequent divergence introduced structural differences, locally, restricting functional relevance to the first tyrosine in hTSHR and hLH/CGR. In the hFSHR, receptor structure would have retained the capability to use the second tyrosine, as in the wild-type receptor, or even to tolerate substitution of the motif from F333-D-Y335 to Y333-D-Y335 (Fig. 4AGo). If this hypothesis is correct, one would expect the hFSHR to harbor (a) domain(s) in its structure capable of conferring to the hLH/CGR the ability to use the second tyrosine. This fits exactly with what is observed in LF2L and LF4L chimeras, in which small segments of the hFSHR have been introduced in the ectodomain of the hLH/CGR (Fig. 5Go and Table 1Go). By generating additional chimeras (from LF5L to the F-D-Y hLHR pSVL mutant; see also Fig. 5Go and Table 1Go) we first demonstrated that the important domains in the hFSHR are localized downstream of the sulfated motif (see Fig. 5Go and Table 1Go). More precise mutants were engineered to finally point out the specific hFSHR residues (see Fig. 6Go and Table 1Go) that confer to this receptor a differential sensitivity to substitution of tyrosine residues in the sulfation motif. Whether these amino acids (D334, D336, and T345 in hFSHR) are able to influence directly the sulfation process or play a relevant role in the balance of the charges in the interaction between the hormone and its receptor remains an open question. The observation of the rescued phenotype of the F331-D332-Y333-D334-T342 mutant in hLHR (see Fig. 6Go and Table 1Go) with the introduction of the three hFSHR determinants (D334, D336, and T345) agrees with the evolutionary scenario depicted above. It also confirms that the differential sensitivity to substitution of tyrosine residues in the sulfation motif displayed by the hTSHR-hLH/CGR couple and the hFSHR is mainly associated with structural differences in the receptors, rather than in the hormones. Of interest, these results provide novel indirect information reflecting a structural characteristic in the hinge region of the hFSHR that is not shared by the hLH/CGR and hTSHR, and this is even more important considering that this domain of the glycoprotein hormone receptors is the joint between the ECD and the TMD, and, so far, it is the sole domain of this subfamily that is still not crystallized but only modeled using a comparative approach (7, 8, 22). The future will tell whether this peculiarity of an important domain of the hFSHR has any relation with the functional differences it displays in terms of constitutive activity (4, 37), its ability to be activated by mutations (38), or its susceptibility to promiscuous activation by hCG when mutated in the serpentine domain (39, 40, 41, 42).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Reagents
Plasmid pBluescript SK+ was from Stratagene (La Jolla, CA); plasmid pSVL was from Amersham Pharmacia Biotech (Roosendaal, The Netherlands); restriction enzymes were from Life Technologies (Merelbeke, Belgium) and New England Biolabs, Inc. (Beverly, MA). Pfu Turbo polymerase was from Stratagene. Platinum Pfx polymerase was from Life Technologies. mAbs 16B5 (6) and 1E3 (our unpublished results) were obtained by genetic immunization with the cDNA coding for the hLH/CGR as already described (43) and are directed against a linear epitope between amino acids 1 and 161 (our unpublished results). mAb 5B2 was obtained following the same protocol using a cDNA coding for the hFSHR (6). Purified hCG was obtained from Sigma (St Louis, MO). hrCG was obtained from Sigma. hrFSH was obtained from Organon (Brussels, Belgium). hrTSH was obtained from Genzyme Corp. (Cambridge, MA). [125I]hCG was obtained from PerkinElemer (Brussels, Belgium).

Plasmid Constructions
Mutants Y331F (F-E-Y), Y333F (Y-E-F), and the double mutant Y331F-Y333F (F-E-F) in hLHR were obtained by QuikChange site-directed mutagenesis with specific oligonucleotides (sequences available upon request), and then subcloned in the hLH/CGR in pSVL between XhoI and EcoRV restriction sites. Mutants Y330F (F-x-x-F-D-Y), Y335F (Y-x-x-F-D-F), and the double mutant Y330F-Y335F (F-x-x-F-D-F) in hFSHR were obtained by QuikChange site-directed mutagenesis with specific oligonucleotides (sequences available upon request), and then subcloned in the hFSHR in pSVL between BsmBI and XbaI restriction sites. The F333Y (Y-x-x-Y-D-Y) and S273I mutations in the wild-type hFSHR and in the Y-x-x-F-D-F mutant hFSHR background were introduced by QuikChange site-directed mutagenesis with specific oligonucleotides (sequences available upon request), and then subcloned in the final plasmid. The F26 hFSHR mutant has been described previously (6). It harbors two mutations in the extracellular domain (K104N, K179G), exchanging two amino acids for the hLH/CGR counterparts, which makes it respond to hCG (6). Two new double mutants, [(F26)Y-x-x-Y-D-Y] and [(F26)Y-x-x-Y-D-F], were obtained by subcloning the corresponding single mutants (Y-x-x-Y-D-Y and Y-x-x-Y-D-F) in the F26 constructs between BsmBI and XbaI. The double mutants Y331F-G334D (F-E-Y-D), Y331F-R342T (F-E-Y-T), and Y331F-E332D (F-D-Y) and the triple mutant Y331F-G334D-R342T (F-E-Y-D-T) were obtained by QuikChange site-directed mutagenesis with specific oligonucleotides (sequences available upon request), using the single mutant F-E-Y as template and then subcloned in the hLH/CGR in pSVL between BmgBI and BamHI restriction sites. The triple mutants Y331F-E332D-G334D (F-D-Y-D) and Y331F-E332D-R342T (F-D-Y-T) and the mutant Y331F-E332D-G334D-R342T (F-D-Y-D-T) were also obtained by QuikChange site-directed mutagenesis with specific oligonucleotides (sequences available upon request), using the single mutant F-D-Y as template and then subcloned in the hLH/CGR in pSVL between BmgBI and BamHI restriction sites.

Construction of FSHR ECD with a 10 Histidine Tag and a Glycosylphosphatidylinositol (GPI) Anchor
The cDNA segment encoding the GPI anchor downstream a 10 histidine tag was excised with EcoRV/Not1 from the TSHR-ECD-10H-GPI in SK+ previously described (5) and fused downstream the FSHR-ECD or FDF-ECD. The FSHR-ECD-10His construct with its signal for GPI attachment is illustrated in supplemental Fig 3. This construct was then subcloned in pEFIN3, a bicistronic vector developed at EUROSCREEN (Brussels, Belgium) for stable transfection in CHO cells.

Construction of Chimera between hLH/CGR and hFSHR
A series of chimeras were made in which segments of the ectodomain of the hFSHR were substituted for their counterpart in the hLH/CGR. To this aim, AflII sites were engineered at positions 256–257 and 252–253 of the hLH/CGR and hFSHR, respectively. Following destruction of the natural AflII site in the hFSHR allowed subcloning a 117-residue segment of the hFSHR in hLH/CGR between the AflII and EcoRV sites to obtain a first chimera named LFL-pSVL. The LF2L-pSVL chimera (see Fig. 5AGo) was obtained by subcloning, between the AflII and BsmBI in the LFL-pSVL chimera, a fragment of hLH/CGR obtained by PCR amplification with specific primers harboring the two restriction sites. The LF4L-pSVL chimera (see Fig. 5AGo) was constructed by a strategy involving PCR amplification of two complementary fragments: the first encoded amino acids 257–310 of the hLH/CGR, with the 3' primer containing a sequence corresponding to positions 307–311 of the hFSHR. It used the wild-type hLH/CGR as template. The second encoded amino acids 307–358 of the hFSHR, with the 5' primer containing a sequence corresponding to positions 305–310 of the hLH/CGR. It used chimera LF2L as template. After annealing of the two fragments, the final PCR product was amplified with external primers presenting an AflII site at the 5' end an EcoRV site at the 3' end. It was subcloned in the LFL-pSVL construct. LF5L-pSVL, LF6L-pSVL, and LF7L-pSVL (see Fig. 5AGo) were created sequentially by QuikChange site-directed mutagenesis with specific oligonucleotides (sequences available upon request), starting from LF4L-pSVL and then using the previous chimera as template. Finally, they were subcloned in the hLH/CGR in pSVL between the XhoI and BstBI restriction sites. All constructs were introduced in DH5{alpha}F’ competent cells, and recombinant DNA from selected clones was purified and sequenced for confirmation of the nucleotide sequence of the PCR-generated areas.

Transfection Experiments
COS-7 cells were used for all transient expression experiments, which were performed according to two different protocols (6). Briefly, 300,000 or 2 million cells were seeded in 3.5-cm or 10-cm Petri dishes, respectively, and transfected the following day by the diethylaminoethyl-dextran method (44). For 3.5-cm dishes, after 48 h cells were used for flow immunocytofluorometry (fluorescence-activated cell sorting, FACS), cAMP determinations, and ligand binding. For 10-cm dishes, cells were detached by trypsinization after 72 h, centrifuged, resuspended in 16 ml culture medium, and seeded in 24-well plates (1 ml/well). At d 5, cells were used for flow immunocytofluorometry and cAMP determinations. Duplicate dishes were used for each assay. Cells transfected with pSVL alone were always run as negative controls.

Quantification of Cell Surface Expression of LH/CGR, FSHR, and Chimeric Receptor Constructs by FACS
Cells were prepared as previously described (44). After PBS-EDTA-EGTA detachment, they were centrifuged at 500 x g for 3 min at 4 C and the supernatant was decanted by inversion. They were then incubated for 30 min at room temperature with 100 µl PBS-BSA 0.1% containing the receptor-specific monoclonal antibody (see Reagents section for details). Cells were then washed with 2 ml PBS-BSA 0.1% and centrifuged as described above. They were incubated on ice in the dark for 30 min with fluoresceine-conjugated {gamma}-chain-specific goat antimouse IgG (Sigma) in the same buffer. Propidium iodide (10 µg/ml) was used for detection of damaged cells that were excluded from the analysis. Cells were washed as described above and resuspended in 250 µl PBS-BSA 0.1%. The fluorescence of 10,000 cells per tube was assayed by a FACScan Flow cytofluorometer (Becton Dickinson, Eerenbodegem, Belgium).

Quantification of cAMP Production Generated by LH/CGR, FSHR, and Chimeras
For cAMP determinations, culture medium was removed 48 h after transfection and replaced by Krebs-Ringer-HEPES buffer for 30 min. Thereafter, cells were incubated for 60 min in fresh Krebs-Ringer-HEPES buffer supplemented with 25 µM phosphodiesterase inhibitor Rolipram (Laboratory Logeais, Paris, France) and various concentrations of highly purified hCG (Sigma), hrCG (Sigma), hrFSH (Organon), or hrTSH (Genzyme). At the end of the 1-h incubation, the medium was discarded and replaced with 0.1 M HCl. The cell extracts were dried in a vacuum concentrator, resuspended in water, and diluted appropriately for cAMP evaluations by RIA according to the method of Brooker et al. (45). Duplicate samples were assayed in all experiments. Results are expressed in picomoles of cAMP/ml. Concentration-effect curves were fitted with the Prism computer program (GraphPad Software, Inc., San Diego, CA), and EC50 values were determined.

For cAMP determination under conditions in which sulfation is inhibited, COS cells transfected with WT and mutant receptors were washed three times with PBS 24 h after transfection and were incubated for 16 h in sulfate-free medium (ICN Biomedicals, Asse-Relegem, Belgium) in which either magnesium chlorate or sodium chlorate was added to a final concentration of 10 mM.

Binding
Ligand binding was measured, as previously described (46), on COS-7 cells transfected according to above-described protocol (3.5-cm Petri dishes), by wild-type or several mutant constructs. Cells were incubated overnight at room temperature in 1 ml of modified Hanks’ buffer without NaCl (isotonicity maintained with 280 mM sucrose), supplemented with 2.5% low-fat milk and [125I]hCG. Thereafter the cells were rapidly rinsed with the same ice-cold buffer, solubilized with 1 ml 1 N NaOH, and radioactivity was measured in a gamma-counter. All experiments were carried out in duplicate, and results are expressed as percentage of the specific binding.

Labeling of Wild-Type and Tyrosine-Mutated Ectodomain of the hFSHR
Stable CHO cell lines expressing wild-type or tyrosine-mutated (Y330-x-x-F-D-F335) ECD-10H-GPI construct were generated as previously described (5). The level of expression of wild-type and mutated constructs expressed at the cell surface as GPI-anchored proteins was measured by flow immunocytometry with the 5B2 mAb. Twenty-four hours after seeding (5 x 106 cells per 10-cm dish), cells were washed three times with PBS and incubated for 24 h in DMEM without cysteine and methionine (Sigma Aldrich, Bornem, Belgium) with 500 µCi each of [35S]cysteine and [35S]methionine (Amersham Pharmacia Biotech Benelux, The Netherlands) or in sulfate-free medium (ICN Biomedicals) with 1 mCi [35S]sulfate (Amersham Pharmacia Biotech). After being washed three times with PBS, the labeled ectodomains were released from the cells by phosphatidylinositol-specific phospholipase C (PI-PLC) and purified on nickel beads as previously described (5).

Dried pellets of purified ectodomain constructs were resuspended in 20 µl Laemmli buffer containing ß-mercaptoethanol and denaturized at 45 C for 1 h. Samples (the volumes loaded were normalized to the level of expression measured by flow immunocytometry) were analyzed by SDS-PAGE, and quantification of [35S]-labeled material was performed by PhosphorImager scanning and image analysis (Molecular Dynamics ImageQuant, Amersham, Pharmacia Biotech).


   ACKNOWLEDGMENTS
 
We thank V. Janssens for expert technical assistance.


   FOOTNOTES
 
This study was supported by the Belgian State, Prime Minister’s office, Service for Sciences, Technology and Culture; and by grants from BRAHMS, Fonds de la Recherche Scientifique Médicale, Fonds National de la Recherche Scientifique (FNRS), ARBD, Comision Interministerial de Ciencia y Tecnologia (SAF2002-01509), the Improving Human Potential of the European Community (HPRI-CT-1999-00071), and Italian Ministry of Education, University and Research (PRIN: 2004052155_005). S.C. is Chercheur Qualifié at the FNRS. M.B. is presently supported by a grant from Istituto Auxologico Italiano IRCSS.

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 10, 2006

Abbreviations: CG, Choriogonadotropin; CGR, CG receptor; CHO, Chinese hamster ovary; ECD, extracellular domain; FACS, fluorescence-activated cell sorting; FSHR, FSH receptor; GPCR, G protein-coupled receptor; GPI, glycosylphosphatidylinositol; h, human; hr, human recombinant; LRR, leucine-rich repeat; mAb, monoclonal antibody; PI-PLC, phosphatidylinositol-specific phospholipase C; TMD, rhodopsin-like serpentine domain; TPST, tyrosylprotein sulfotransferases; TSHR, TSH receptor.

Received for publication December 19, 2005. Accepted for publication July 26, 2006.


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