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J. Biol. Chem., Vol. 281, Issue 42, 31638-31646, October 20, 2006
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1
12
From the
III Medical Department, University of Leipzig, Philipp-Rosenthal-Strasse 27, D-04103 Leipzig, Germany, Leibniz-Institut für Molekulare Pharmakologie, Robert-Roessle-Strasse 10, D-13125 Berlin, Germany, and ¶NIDDK, National Institutes of Health, Clinical Endocrinology Branch, Bethesda, Maryland 20892
Received for publication, May 18, 2006 , and in revised form, July 14, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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30 kDa) interact with the N-terminal extracellular domain (ECD) of their specific receptors (1, 2), TSHR, follicle-stimulating hormone receptor, and luteinizing hormone/choriogonadotropin receptor, which are members of the seven-transmembrane-spanning receptor family (3-7).
The large N-terminal extracellular domain is a common structural characteristic of all GPH receptors. The N-terminal ECD of the TSHR (Met-1-Asp-410) can be subdivided into (all numbers are hTSHR specific): (i) the extreme N-terminal tail (including the signal peptide), (ii) the cysteine box 1 (C-b1; Cys-24-Cys-41), (iii) the 11 leucine-rich repeats (LRR; Pro-37-Tyr-279) and (iv) the hinge region (Pro-280-Asp-410), which can be subdivided further into (v) the central cysteine box 2 (C-b2; Pro-280-Cys-301), (vi) the cysteine box 2/3 linker region (C-bl; Asn-302-Ile-389), and (vii) the cysteine box 3 (C-b3; Cys-390-Asp-410) located close to transmembrane helix 1.
In addition to the activation mediated by TSH, the receptor can be activated by constitutively activating mutants (CAMs) (8, 9), by mutants leading to promiscuous hormone interactions (10), tryptic action (11), and deletions in the ECD and serpentine domain (12-14). Several pathogenic gain of function mutants in the ECD of the TSHR characterized by increased constitutive activity (S281N, I, or T) have been identified (15-17). Mutational studies at Ser-281 of the TSHR (18) and corresponding positions of the luteinizing hormone/choriogonadotropin receptor (Ser-277) and follicle-stimulating hormone receptor (Ser-273) demonstrated the importance of this serine for the stabilization of the WT receptor activity state of all GPH receptors (19, 20).
The epitopes C-b2, back to back with C-b3 following the LRR domain, were not included in the crystallized x-ray structure of a follicle-stimulating hormone receptor ectodomain fragment (LRR) (2). It is established that the C-b2 and 3 potentially interact via disulfide bonds (21-23) and that mutations disrupting the disulfide bridge lead to constitutive activation (24, 25). In a previous study we identified new residues within C-b3 that are also involved in receptor stabilization and intramolecular signal transduction based on a suggested homologous molecular model for the C-b2/C-b3 unit (26). By site-directed mutagenesis we defined a region (Asp-403 Glu-404 Asn-406) of the TSHR ectodomain that can act as a switch for constitutive activity. Moreover, except Glu-404, which is TSHR specific, Asp-403 and Asn-406 are conserved within the GPH receptors. We suggested that parts of C-b2 and C-b3 are arranged as a tightly packed structural signaling interface between the N-terminal ECD and the serpentine domain that constrains the partially active, basal conformation of WT TSHR.
To gain further insight into receptor activation caused by mutations at positions Asp-403, Glu-404, and Asn-406, we now characterize the three CAMs in C-b3 in detail by side chain variation. The second aim of this study was the characterization of adjacent amino acids (Thr-399Asp-410) to identify further residues involved in the signaling process. Indeed, mutations at Pro-400 and Pro-407 also lead to constitutive activation of the TSHR. Moreover, tryptic clipping within C-b2 at an undefined site in the region of a cluster of positively charged amino acids (Lys-287-Arg-293) close to Ser-281 also leads to receptor activation (27). Considering the tightly packed structural arrangement of C-b2 and C-b3 via disulfide bridges, we assumed that mutation of additional amino acids within C-b2 might also lead to constitutive activation. We show the alanine mutation of Lys-291 in the C-b2 results in a new CAM and that Lys-291 functions as a further conformational anchor in the C-b2/C-b3 unit. Our data suggest a fundamental role of C-b2 and C-b3 in receptor functions, including stabilization of the basal and hormone-activated receptor conformations.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Cell Culture and Transfection—COS-7 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin (Invitrogen) at 37 °C in a humidified 5% CO2 incubator. Cells were transiently transfected in 24-well plates (5 x 104 cells/well) with 0.5 µg of DNA/well for cAMP accumulation and in 48-well plates (2.5 x 104 cells/well) with 0.25 µg of DNA/well for TSH binding analysis. COS-7 cells were transfected using GeneJammer ® transfection reagent (Stratagene). Controls included transfection of the wild-type human TSH receptor and transfection of the pSVL vector alone.
Fluorescence-activated Cell Sorter Analysis—Nonpermeabilized cells were detached from the dishes 48 h after transfection using 1 mM EDTA and 1 mM EGTA in phosphate-buffered saline and transferred in Falcon 2054 tubes. Cells were washed once with phosphate-buffered saline containing 0.1% bovine serum albumin and 0.1% NaN3 before incubation with the primary antibody. The first step of the permeabilized cell assay included cell fixation with 1% paraformaldehyde for 10 min on ice following an incubation with phosphate-buffered saline containing 0.1% bovine serum albumin and 0.1% NaN3 and 0.2% saponin for 30 min. Saponin was supplemented in all subsequent buffers. Afterward, cells were incubated for 1 h with a 1:200 dilution of a mouse anti-human TSHR monoclonal antibody 2C11 (MAK 1281; Linaris, Wertheim-Bettingen; 10 µg/ml) in the same buffer. Cells were washed twice and incubated at 4 °C for 1 h in the dark with a 1:200 dilution of fluorescein-conjugated F(ab')2 rabbit anti-mouse IgG (Serotec, Oxford, UK). Cells were washed twice and fixed with 1% paraformaldehyde before fluorescence-activated cell sorter analysis (FACscan; BD Biosciences). Receptor expression was determined by the mean fluorescence intensity. The WT TSHR was set at 100%, and receptor expression of the mutants was calculated according to this. The percentage of signal-positive cells corresponds to transfection efficiency, which was 70% of viable cells for each mutant in each experiment.
Radioligand Binding Assay—48 h after transfection, competitive binding studies were performed as previously described (29). Obtained data were analyzed assuming a one-site binding model using the fitting module of GraphPad Prism 4.0 for Windows.
cAMP Accumulation Assay—Measurement of cyclic AMP (cAMP) accumulation was performed 48 h after transfection. Cells were preincubated with serum-free Dulbecco's modified Eagle's medium without antibiotics containing 1 mM 3-isobutyl-1-methylxanthine (IBMX; Sigma) for 30 min at 37 °C in a humidified 5% CO2 incubator. Subsequently, cells were stimulated in the same medium supplemented with 100 milliunits/ml bTSH (Sigma) for 1 h. After termination of the reaction by aspiration of the medium, cells were washed once with ice-cold phosphate-buffered saline and then lysed by addition of 0.1 N HCl. After collecting and drying of the supernatants, cAMP content of the cell extracts was determined using the cAMP AlphaScreenTM assay (PerkinElmer Life Sciences) according to the manufacturer's instructions.
Linear Regression Analysis of Basal Activity as a Function of TSHR Expression Determined by 125I-bTSH Binding—COS-7 cells were transiently transfected in 24-well plates (5 x 104 cells/well) with various concentrations of WT and TSHR constructs (50, 100, 150, 200, 250, and 300 ng DNA/well). For radioligand binding assays, cells were incubated for 4 h at room temperature in the presence of 180,000-200,000 cpm of 125I-bTSH supplemented with 5 milliunits/ml nonlabeled bTSH. The effect of expression level on basal cAMP accumulation was analyzed according to Hjorth et al. (30). The obtained results for TSH binding and basal cAMP accumulation at various DNA concentrations were used to calculate the constitutive activity of the mutants. Linear regression analysis of basal activity as a function of TSHR expression determined by 125I-bTSH binding (slope) was determined by blotting TSH binding (x-axis) versus basal cAMP accumulation (y-axis) using the linear regression function of GraphPad Prism 4.03 for Windows. The slope of the WT TSHR was set at 1, and slopes of the mutants were calculated according to this.
Statistics—Statistical analysis was carried out by t test using GraphPad Prism 4.03 for Windows (***, p < 0.001 extremely significant; **, p < 0.01, very significant; *, p < 0.05, significant; p > 0.05 not significant) (see Tables).
Molecular Modeling—The generation of the homology model for C-b2/C-b3, modeling procedures, and methods used have been previously described by us (26).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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70% of viable cells could be achieved as indicated by fluorescence-activated cell sorter analysis (data not shown). To identify further residues involved in the signaling process, adjacent amino acids of the CAM region Asp-403 Glu-404 Asp-406 in C-b3 were investigated by alanine substitution (Fig. 1).
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80% of the WT exhibited a cAMP signal comparable with the WT TSHR. Basal activity calculated by linear regression analysis of constitutive activity as a function of TSHR expression was analyzed to evaluate the effect of receptor density on basal cAMP accumulation independently from their cell surface expression.
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Glu-404—All substitutions of Glu-404 (Asp, Lys, Asn, and Ser) revealed a cell surface expression of over 55% of the WT TSHR (Table 1). All substitutions at Glu-404 showed an increased basal cAMP signal (1.7- to 4.5-fold relative to WT TSHR), whereas E404N revealed the highest constitutive activity (Table 1 and Fig. 2B). The only mutation that exhibited WT TSHR properties after functional characterization was E404D. All Glu-404 substitutions resulted in TSH-induced cAMP production comparable with the WT TSHR.
Asn-406—N406Q and N406S showed a cell surface expression of 70%, whereas substitutions to asparagine, lysine, and leucine revealed only a cell surface expression of 30% of the WT TSHR (Table 1). By means of the permeabilized cell assay, a receptor expression of 50% was measured for these mutants (data not shown). Except for N406K, all mutants showed an increased basal cAMP accumulation (3.0- to 6.8-fold relative to the WT). The mutant N406D revealed the highest basal cAMP value (6.8-fold over WT) of all Asn-406 substitutions and also the highest constitutive activity ascertained by linear regression analysis. (Table 1 and Fig. 2C).
Mutations of Residues in Cysteine Box 3
Data of the alanine scan in C-b3 are summarized in Table 2. Most of the alanine substitutions (T399A-D410A) showed a cell surface expression equivalent to the WT TSHR in the range of 65-100%, except for mutants P400A and F405A with 11-16% compared with WT TSHR (Table 2). In contrast to P400A at the N terminus of the region, mutant P407A at the C-terminal end revealed a cell surface expression of 60%. After permeabilization, expression levels of more than 60% were determined for P400A and F405A, suggesting an increased receptor accumulation within the cells.
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Alanine Mutations of Residues in Cysteine Box 2
Based on the model of a tightly packed structural arrangement of C-b2 and C-b3 via disulfide bridges (26) and considering activation by tryptic clipping (27), we hypothesized that apart from Ser-281 additional amino acids in C-b2 could also be sensitive for constitutive activation by mutations. Therefore, we tested by single alanine substitution whether further hydrophilic residues of C-b2, Lys-287, Asn-288, Gln-289, Lys-290, Lys-291, and Arg-293, are involved in constraining the WT TSHR conformation. All mutants revealed a cell surface expression >75% of the WT TSHR (Table 2). All alanine mutants showed a basal cAMP accumulation comparable with the WT, except K291A, which is characterized by a 3.3-fold increase of basal cAMP production (Table 2). K291A also revealed increased constitutive activity compared with the WT TSHR as determined by linear regression analysis of constitutive activity as a function of TSHR expression determined by 125I-bTSH binding (Table 2 and Fig. 2E). Alanine substitutions at positions Lys-287, Asn-288, Gln-289, Lys-290, and Arg-293 revealed no changes in basal activity and were comparable with the WT. In contrast to N288A and Q289A and R293A, which were characterized by a slightly decreased or WT-like TSH-induced cAMP response, substitutions of Lys-287, Lys-290, and Lys-291 by alanine resulted in an increased TSH-stimulated cAMP accumulation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Novel CAMs at Cysteine Boxes 2 and 3—The second aim of this study was to characterize adjacent amino acids of the CAM region Asp-403 Glu-404 Asn-406 to define the extension of this region and to identify further residues involved in the signaling process. Indeed, mutations P400A and P407D constitutively activate the TSHR. We suggest that the constitutive activation caused by the P400A mutant is based on a structural shift of adjacent residues that are involved in the stabilization of the basal TSHR conformation. Furthermore, we assume that the constitutive activation observed for mutant P407D is based on repulsion of an H-acceptor moiety (dotted arrow in Fig. 3), which we already characterized as an interaction partner for Asn-406. It is most likely localized in close spatial neighborhood to Pro-407 (Fig. 3).
Furthermore, the exchange to bulkier side chains at Pro-400 and Pro-407 (Asp, Lys, Leu) led to a receptor conformation that is completely or partially inactive for ligand-mediated cAMP signaling (Table 3). The inhibited signaling by mutants at Pro-400 is most likely caused by strongly decreased cell surface expression and implies a disturbed receptor folding. Mutant E409A is characterized by a strongly decreased capability for cAMP-mediated signaling and a cell surface expression of 83% (Table 2). At the luteinizing hormone/choriogonadotropin receptor the mutation of the corresponding Glu-354 to Lys is a loss of function mutant, characterized by complete loss of cAMP-mediated signaling (33). Mutant D410N is also known as a pathogenic loss of function mutant of the TSHR (34) with a binding capability similar to the WT TSHR. Furthermore, E409K and D410K are characterized by a cell surface expression of 63 and 90%, respectively, of the WT TSHR but a complete loss of cAMP-mediated signaling (35). Therefore, we assume that Glu-409 and Asp-410 are necessary to maintain the hormone-induced active conformation of GPH receptors and they are constituents of an intramolecular activator. We therefore named these amino acids "activator component" (red boxed in Fig. 3B). Mutants at Pro-407 with decreased activity but reasonable cell surface expression indicate the ability of Pro-407 to stabilize the hormone-induced active state, very likely via a backbone orientation that is suitable for the activator component comprising Glu-409 and Asp-410.
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Amino Acid Variation on CAM Positions Asp-403, Glu-404, and Asn-406—We have recently shown that alanine and lysine mutations of the amino acids Asp-403, Glu-404, Asn-406 at C-b3 near transmembrane helix 1 lead to constitutive activation of the TSHR (26). Here, we studied further side chain variations at these positions. Mutations of Asp-403 to the bulky hydrophobic amino acid leucine, and even to the charge keeping glutamate but extending the side chain length, showed high constitutive activation, whereas substitutions with reduced side chain length such as asparagine and serine do not exhibit constitutive activity. Therefore, we hypothesize a tight spatial environment by van der Waals interactions surrounding Asp-403, which would explain that smaller amino acids are tolerated and maintain the basal WT TSHR conformation at this position (Table 1 and Fig. 3). In contrast, at position Glu-404 substitutions to amino acids of variable characteristics, except to aspartate, are CAMs. Thus for Glu-404 we assume an H-donator as interaction partner (Fig. 3). All substitutions at Asn-406 except the mutant N406K are CAMs, implying that the interaction partner of Asn-406 should provide a negative charge or at least a strong capacity of accepting H-bond(s) (Fig. 3). Together with our previous data (26) the results of this study show that constitutive activation at Asp-403, Glu-404, and Asn-406 seems to be initiated by disruption of a constraining hydrogen bond network or a destabilization of a tightly packed environment surrounding C-b2 and C-b3 that is necessary for the maintenance of the basal state of the WT TSHR (Fig. 3).
Modulation of Signaling Activity by Amino Acids of Cysteine Boxes 2 and 3—Our data provide (a) properties of potential interaction partners for the amino acids Asp-403, Glu-404, Asn-406 recently identified as CAM positions; and (b) evidence that adjacent prolines (Pro-400, Pro-407) are important for stabilizing the basal WT conformation of the TSHR. These two prolines are also molecular key players for the receptor activation process at C-b2 and C-b3 induced by TSH and provide a backbone orientation that is required for an activator component, which very likely comprises Glu-409 and Asp-410.
Based on our molecular model-driven characterization of mutants we can now outline a refined and extended intramolecular signaling region at the junction of the N-terminal ECD with the serpentine domain. The epitope Pro-280 and Ser-281 on the N-terminal side of C-b2, for which a mechanism of receptor activation by constitutively active mutants via disturbance of structural constraints is suggested (18, 19), was named the anchor I fragment (green boxed in Fig. 3B). On the other hand five amino acids Pro-400, Asp-403, Glu-404, Asn-406, Pro-407 at C-b3 can activate the TSHR by mutations, causing release of constraints responsible for stabilization of the basal WT TSHR conformation, which corresponds to a region named anchor II fragment (green boxed, Fig. 3B). Mutants of amino acids Pro-407, Glu-409, and Asp-410 (C-b3) have a strong effect on hormone-induced signaling by partial inactivation of the TSHR for hormone-induced receptor activation, and we hypothesize that Pro-407 stabilizes the hormone-induced active state very likely via a backbone orientation that is suitable for the activator component comprising Glu-409 and Asp-410. Furthermore, we identified the alanine mutant of Lys-291 as a new CAM in C-b2. The shared effect of receptor activation by tryptic cleavage at a cluster between Lys-287 and Arg-293 or single mutations of the positively charged amino acid Lys-291 at C-b2 leads in both cases to a release of the constrained portion at C-b2. Here, we identified the conformational anchor for C-b2 (Lys-291), which we named anchor III fragment (green boxed in Fig. 3B).
The described CAM positions in C-b2/C-b3 give an image of conformationally anchored fragments (Fig. 3, A and B). Moreover, the side chain mutagenesis studies narrow down the characteristics of the potential interaction partners for this region by characterizing their properties. Based on our findings, we suggest the following scenario for a molecular activation mechanism mediated by C-b2 and C-b3. The three anchor fragments and one activator component are tightly packed and flank the disulfide bridge that holds C-b2 and C-b3 tightly together in the center. Obviously, only the epitope Lys-287-Lys-290 and Arg-293 is accessible, because mutations have no effects on signaling, which is consistent with a reported tryptic cleavage region (27) (Fig. 3, A and B). All three anchor fragments stabilize the basal TSHR conformation. It is very likely that the three highly conserved prolines (blue at Fig. 3, A and B) assist in this process by their defined backbone conformation. Anchor fragments II and III are firmly locked by H-bond interactions. Anchor fragment I is held by tight "knob and hole" van der Waals interactions with its environment (18, 25). Because anchor fragment I follows back-to-back after the LRR domain harboring the hormone binding site, it is conceivable that a conformational change of the LRR upon hormone binding could be transferred to anchor fragment I. Such a conformational change could subsequently displace the identified sensitive C-b2/C-b3 portions relative to each other (Fig. 3B, violet double arrows). Regardless of the type of conformational interferences on the tightly constrained C-b2/C-b3 WT conformation, such as side chain modifications, tryptic cleavage, or hormone stimulation (Fig. 3B, red open arrows), they always release one of the anchor fragments. In all cases this would be followed by a conformational displacement of the sensitive C-b2/C-b3 portions against each other, which in turn could result in receptor activation. In this process, the cysteine bridge plays a pivotal role as a fulcrum. Very likely such displacements also influence the framework between the N-terminal ECD and the serpentine domain (31, 32).
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1.
1 Both authors contributed equally to this work.
2 To whom correspondence should be addressed. Tel.: 49-341-9713200; Fax: 49-341-9713209; E-mail: pasr{at}medizin.uni-leipzig.de.
3 The abbreviations used are: GPH, glycoprotein hormone; TSH, thyroid-stimulating hormone; bTSH, bovine TSH; TSHR, TSH receptor; hTSHR, human TSHR; CAM, constitutively active mutant; C-bl2/3, cysteine box2/3 linker; C-b1, cysteine box 1; C-b2, cysteine box 2; C-b3, cysteine box 3; ECD, extracellular domain; LRR, leucine-rich repeat; WT, wild type.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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