Service de Génétique Médicale (G.S., G.V.), Hôpital Erasme, B-1070 Bruxelles, Belgium; Institut de Recherche Interdisciplinaire en Biologie Humaine et Nucléaire (G.S., C.G., I.N., G.V., S.C.), Université Libre de Bruxelles, Campus Erasme, B-1070 Bruxelles, Belgium; Service de Conformation des Macromolécules Biologiques (C.G.), Université Libre de Bruxelles, 1050 Bruxelles, Belgium; Laboratori de Medicina Computacional (L.P.), Unitat de Bioestadística, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain
Address all correspondence and requests for reprints to: Gilbert Vassart, Institut de Recherche Interdisciplinaire en Biologie Humaine et Nucléaire, Université Libre de Bruxelles, Campus Erasme, 808 route de Lennik, B-1070 Bruxelles, Belgium. E-mail: gvassart{at}ulb.ac.be.
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ABSTRACT |
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INTRODUCTION |
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The glycoprotein hormone receptors exhibit a bipartite structure with a clear functional dichotomy: the amino-terminal half constitutes an ectodomain responsible for high affinity binding of the hormones (14, 15, 16, 17, 18); the carboxyl-terminal portion with a rhodopsin-like serpentine structure containing seven transmembrane -helices is responsible for transduction of the signal, mainly to Gs (see Fig. 1A
; Refs. 19, 20, 21). The ectodomain contains a central portion of nine leucine-rich repeats (LRR) bordered by two cysteine-rich domains, typical of the large family of LRR proteins (22). Structural models of the extracellular part of the glycoprotein hormone receptors based on the atomic structure of the porcine ribonuclease inhibitor (23) have been proposed (24, 25, 26). According to these models, each LRR comprises 2024 amino acids forming a ß-strand followed by an
-helix. The LRR units are arranged with their ß-strands and
-helices parallel to a common axis and organized spatially to form a horseshoe-shaped molecule with the ß-strands and
-helices making the concave and convex surfaces of the horseshoe, respectively (Fig. 1B
). By analogy with the atomic structure of the ribonuclease-ribonuclease inhibitor complex (23), it is assumed that hormones make contact, mainly but not exclusively, with the ß-sheets of the inner concave portion of the horseshoe.
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In 1998, we described a familial case of gestational hyperthyroidism caused by a mutation of one of these exposed residues in the ectodomain of the TSHr (27). The mutation substituted an arginine for the lysine in position 183, with the result that the mutant displayed widening of its specificity toward hCG. Because of the very high plasma concentrations of hCG during pregnancy, the patients suffered from severe hyperthyroidism, requiring treatment with antithyroid drugs throughout pregnancy. In the present study, we attempted to understand in molecular terms the structure-phenotype relationships of this mutant in light of the structural model described above. To this aim, we studied by site-directed mutagenesis and functional assays in transfected COS cells the effects of substituting amino acids with different physicochemical properties for lysine 183. The results indicated unexpectedly that the gain of function toward hCG displayed by the K183R mutant could be mimicked by all different amino acid substitutions tested in this position. Molecular dynamics simulations suggested that the gain of function might be secondary to the release of a nearby glutamate residue (E157) from a salt bridge with K183. This hypothesis was supported by further site-directed mutagenesis experiments showing that the presence of an acidic residue in position 157, or in its vicinity, was required to observe the increase in sensitivity to hCG.
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RESULTS |
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Modeling the Effects of Mutations at Position 183
The atomic model of the portion of TSHr ectodomain made of LRRs (from residue 54 to 254) built by Kajava et al. (24) on the template of the porcine ribonuclease inhibitor provides a structural framework to understand the possible effect of the mutations. Unrestrained molecular dynamics simulations of the TSHr ectodomain in water (see Materials and Methods) have been used to model the interactions between the side chains of the ectodomain, focusing on K183 and its surroundings. The results of these computer simulations suggest that K183 is implicated in a direct interaction with its neighbor residue, E157. As illustrated on the representative structure shown in Fig. 3 (first row, left panel), the side chain of K183 is nicely positioned to achieve a salt bridge with one of the carboxyls of glutamate 157 (observed in 80% of the structures from the production phase of the simulation). E157 is also found to interact with another basic residue, R109 (observed in 100% of the structures). Further analysis of the model shows that the numerous charged or polar residues exposed in the surroundings of K183 (the inside of the horseshoe) are predicted to form a large and complex network of polar interactions. Therefore, we computed the electrostatic potential at the accessible surface of the model, using the program GRASP, to visualize the effects of these interactions on the overall distribution of charges (see Materials and Methods). As shown in Fig. 3
(first row, right panel), the electrostatic distribution is clearly asymmetrical. The N-terminal part of the horseshoe (the left side of the picture) appears positively charged (colored in blue), due to the presence of K58, R80, R109, and K183, which completely shield the acidic residues E107 and E157. On the right side of the picture, the C-terminal part is on average negatively charged (colored in red), mainly due to the presence of D203, D232, and E251 [R255 is not included in the model of Kajava et al. (24)].
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In summary, in the wt receptor, E157 appears to be shielded by positively charged residues, namely R109 and K183. Mutations of K183 into alanine modifies strongly the electrostatic pattern, inducing an increase in negatively charged areas, in particular around position 157. Similar structural effects would be expected to result from substitutions of K183 by any non-basic amino acid, providing a satisfactory explanation for the phenotypes observed for the mutations of K183 into A, I, T, E, D, M, N, Q, or W. The increase of stimulation by hCG in the case of the conservative K183R mutation would require a different explanation, implying that the R183 mutant would achieve a different spatial organization of its electrostatic potential, when compared with the wt structure (see below and Discussion).
E157 Is Implicated in the Stimulation of TSHr K183 Mutants by hCG
To test the hypothesis that the increased responsiveness of the mutants to hCG was due to the release of E157 from a neutralizing interaction with K183, we created a series of double mutants in which, in addition to the substitutions in position 183 (K183R, A, I, T, E, W, M, N, or Q), an alanine was substituted for glutamic acid in position 157. The E157A mutation was also introduced in the wt TSHr as a control. All the double mutants and E157A were well expressed at the surface of transfected COS-7 cells (expression ranging from 47 to 83% of wt, Fig. 4A). When stimulated by high concentrations of hCG, none of the mutants displayed increase in cAMP production with the exception of the E157A+K183E mutant (Fig. 4
, B and C). This indicates an important role of E157 in the interaction with hCG, supporting the hypothesis of an indirect role for mutations at position 183. Notably, the E157A+K183E mutant responded to hCG with an EC50 (58 ± 10 IU/ml) in the same range as that of K183 single mutants (ranging from 7 to 58 IU/ml hCG, Fig. 4D
). A likely interpretation for the exceptional phenotype of this particular construct amongst the double mutants is that a glutamic acid in position 183 would somehow fulfill the role achieved by E157 in the single mutants (e.g. K183A). Molecular modeling of the E157A+K183E double mutant further supports this hypothesis. As shown in Fig. 3
(third row), mutating both E157A and K183E produces a model whose electrostatic surface potential is quite similar to that of the single K183A mutant. The left side of the model is overall positively charged, as observed for the wt and the K183A mutant, whereas the right side is negatively charged. The presence of E183 induced an extension of the negatively charged area up to the middle of the structure, making it quite similar to the K183A model. It is tempting to propose that the increased sensitivity to hCG observed in the E157A+K183E double mutant is due to the same mechanism as for most K183 single mutants (i.e. increase in negative potential around positions 157 or 183).
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Gain of Responsiveness to hCG Is Dependent on the Presence of a Negative Charge in Position 157 or 183
Aspartic acid was substituted for glutamic acid in position 157 in the wt TSHr, the K183R, K183A, and K183W mutants. All mutants were expressed at the surface of COS cells, although the E157D+K183A and the E157D+K183W mutants displayed low expression [54, 86, 13, and 10% of wt for E157D, E157D+K183R, E157D+K183A, and E157D+K183W, respectively (Fig. 5A)]. In agreement with the hypothesis, all three double mutants displayed increased response to hCG, as compared with the E157D single mutant (Fig. 5B
). Note, however, that the E157D single mutant shows also a very small response to hCG allowing determination of an EC50 value (48 ± 14 IU/ml). It is conceivable that the shorter side chain of an Asp in position 157 would be less efficiently neutralized by K183, thus allowing it to interact with hCG.
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Modeling the Effects of the K183R Mutant
The above experimental results suggest that Arg, when present at position 183, interacts with a different partner than E157. In the three-dimensional (3D) model used here, three others acidic residues appear to be accessible to R183, namely D160, D203, and D232. The interatomic distances between the carbon positions of R183 and D160, D203, or D232 residues are compatible with such an interaction (9.5, 11.2, and 9.3 Å, respectively). The longer side chain of arginine compared with lysine might facilitate the interaction with these more distant residues. To identify the potential ionic partners of R183, we surveyed all possible conformations of the Arg side chain using a backbone-dependent rotamer library (Ref. 28 ; see Materials and Methods for computational details). The E157
R183 interaction is favored in 8 different conformations of the 81 conformations theoretically accessible to an Arg side chain. These 8 rotamers were observed in 31% of the crystal structures in the original protein-structure survey statistics (28). The R183
D232 interaction was favored by 6 allowed rotamers (amounting to 8% of the observed conformations in the survey), whereas the R183
D160 interaction was allowed in only 1 case (5%), and the R183
D203 interaction was never favored by any of the allowed rotamers. We modeled the three possible interactions using the most probable rotamer in each case; i.e. the g+,t,t,t conformation, the t,t,t,g+ conformation and the g+,g+,t,t conformation for the E157
R183, D160
R183 and R183
D232 interactions, respectively. The molecular modeling procedure revealed that direct interaction with D160 required the Arg side chain to extend between the ß-strands of the LRR5 and LRR6. The steric clash of R183 with the ß-branched side chain of T159 and the side chain of Y185 contributing to LRR5 and LRR6 prevents this contact (not shown here). Thus, in addition to E157, D232 appears the most likely partner for R183.
Figure 6A shows a representative structure of the unrestrained molecular dynamics simulation of the K183R mutant forming an ionic bridge with E157. Importantly, in contrast to the ammonium group of lysine, the guanidium group of arginine can form multiple interactions via its N
, NH1, and NH2 moieties. Thus, the single free O
atom of E157 (the other O
atom interacting with R109; see Fig. 3
) interacts with the N
moiety of R183 (observed in 70% of the structures from the production phase of the simulation), whereas the other N groups are exposed to the solvent and interact only with water molecules (not shown). As shown in Fig. 6B
, the ionic interaction of R183 with E157 yields a model whose electrostatic surface potential is similar to that of the wt model (Fig. 3
, first row, right panel), with the positively charged region (blue area) extending beyond the center of the horseshoe. In summary, in the case of a E157
R183 interaction, the K183R mutant would behave electrostatically in a similar way as the wt model and R183 would be strongly solvated.
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Figure 6D shows the electrostatic potential of the model displayed in Fig. 6C
, in which R183 interacts with D232 and neighboring residues. The conformation of R183 significantly influences the electrostatic map at the molecular surface. Despite the fact that the net charge of the system remains the same, the positively charged area around position 157 becomes much smaller if, instead of interacting with E157, R183 interacts with D232 (compare Fig. 6
, B and D). E157 becomes accessible, inducing a negatively charged region in the center of the model. Hence, this conformation leads to an electrostatic potential map resembling that of the K183A model (Fig. 3
, second row, right panel), providing a structural rationale to the similar of phenotype of the two mutants.
In summary, although both negative partners (E157 and D232) are geometrically accessible to R183, the R183D232 conformation would be favored by the additional stabilizing interactions (i.e. N-H··
). This conformation displays little solvation of R183 and is electrostatically close to the other K183 mutants. In the case of the wt receptor, a K183
D232 is unlikely because 1) this interaction can happen in only one allowed rotamer, representing 1% of all observed conformations (using similar criteria as for K183R) due to the shorter side chain of lysine, and 2) stabilizing interaction with surrounding side chains would not take place as a lysine has only one active group (the ammonium moiety involved in the salt bridge). K183 would therefore preferentially interact with the closer E157.
Gain of Function of K183 Mutants Is Specific for hCG
Finally, we explored whether the gain of function displayed by K183 mutants was restricted to stimulation by hCG or could also be observed for TSH or FSH. Some of the mutants (K183R, A, I, T, E, W, or D; E157A; E157A+K183R, A, I, T, E, W, or D) were challenged with TSH and the EC50 values were determined. As illustrated in Fig. 7 and Table 1
, all mutants were readily stimulated by TSH; for the single mutants, their EC50 values were in the same range (0.0370.104 mIU/ml) as for the wt receptor (0.060 ± 0.013 mIU/ml); for the double mutants, EC50 values were generally higher (0.1790.581 mIU/ml) except for E157A+K183E and E157A+K183D, which displayed a wt value (0.047 ± 0.010 and 0.088 ± 0.022 mIU/ml, respectively). Similarly, the wt TSHr and K183R, A, or W mutants were subjected to stimulation by FSH. No stimulation of cAMP production could be observed at the maximal concentration assayed (30 IU/ml, i.e. 103 times the EC50 for the FSHr; data not shown). From these experiments, we conclude that the overall 3D structure of the mutants is well preserved (as they are all readily stimulated by TSH) and that the gain of function, when present, is specific of hCG.
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DISCUSSION |
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Molecular dynamics simulations carried out with the model of Kajava et al. (24) as a starting point indicated that K183 would be involved in a salt bridge with the nearby E157, suggesting that the gain of function toward hCG of mutants in position 183 would be secondary to the generation of a negatively charged area in this portion of the ectodomain. The observation that in the double mutant series, with E157 mutated to alanine and K183 replaced by various amino acids, only the E157A+K183E and E157A+K183D constructs showed that responsiveness to hCG is a strong argument in favor of this hypothesis. Altogether, our results suggest that hCG mainly needs an accessible acidic residue in position 157 of the ectodomain (or in the nearby 183 position, like in the E157A+K183E and E157A+K183D double mutants) to stimulate the mutant TSH receptors.
Ironically, the mutant described originally, K183R, is the most difficult to fit in the above model. Indeed, one would intuitively assume that both an arginine and a lysine, when present in position 183, would be able to establish a salt bridge with E157. Our experimental results imply that this is not the case, suggesting that, when an arginine is present in 183, it would interact with a different partner than E157. Molecular modeling indicates that the free side chain of D232 is the likely alternative partner for R183. However, available methods do not allow the determination of whether the conformation of R183 pointing toward D232 is more stable than the conformation of R183 pointing toward E157. In both conformations, R183 forms an ionic interaction with a single oxygen atom (of E157 or D232). Notably, the Arg conformation achieving interaction with E157 is stabilized by hydrogen bonds between the other N groups of R183 and surrounding water molecules, whereas the interaction with D232 is additionally stabilized by hydrogen bonds with the side chains of other residues: Y185, Y206, and N208.
The formation of the TSHr-hCG complex is a function, among others parameters, of the interaction energy between the ligand and the receptor, and the energy required to displace, totally or partially, the water molecules from the interacting surface. The observation that the phenotype of the K183R mutant is highly similar to that of the K183A mutant (see Fig. 2C) suggests that R183 does not directly participate in the interaction between the receptor and hCG and that no desolvation of R183 is required for hCG binding. The side chain of R183 interacting with E157 is exposed to the interacting surface (see Fig. 6A
) and is heavily solvated. This is at variance with the comparable K183A and K183R phenotypes. In contrast, the side chain of R183 interacting with D232 is hidden to the ligand and is weakly solvated, as every polar moiety is involved in at least one hydrogen bond with other side chains of the receptor (namely Y185, Y20, and N208; see Fig. 6C
). Thus, we strongly favor the hypothesis that R183 would interact mostly with D232 and rarely with E157.
It must be stressed that there may be other structural interpretations to our observations. We must be open to the possibility that the horseshoe model of the ectodomain, which is certainly incorrect in its details, might be grossly incorrect altogether. Another way to account for the gain of function of mutants in position 183 is to consider that K183 would be free in wt TSHr and repulsive for hCG. Removal of the lysine would then result in an increase in sensitivity for hCG. This hypothesis would fit the negative determinant theory (12, 30). However, it would face the same difficulty to account for the phenotype of the original K183R mutant, which would be expected to exhibit a similar repulsive behavior. Also, this scenario would not provide an explanation to the phenotypes of the E157A+K183X double mutants. Whatever the final structural explanation for our observations, which will have to wait for crystallographic data, our results establish a definite relationship between the gain of function of K183 mutants and the presence of an acidic amino acid in position 157.
The Homologue Residue to K183 of TSHr Is also a Lysine in LHr
In the LH/CGr, the residue corresponding to K183 is also a lysine (K180, see Fig. 8 for an alignment of the ectodomains of TSHr, LH/CGr, and FSHr). Bhowmick et al. (31) substituted R, Q, G, and E for K180 in LH/CGr (K158 in their numbering). Considering the loss of binding of hCG to the K180E mutant, they suggested that K180 in LH/CGr is important for binding of hCG. Our results suggest that, in the wt TSHr, this lysine being involved in a salt bridge with E157 would be neither attractive nor repulsive for hCG, whereas in K183 mutants, it could obviously not play a role in the gain of function toward hCG. We can interpret this apparent discrepancy in two ways. First, the role of lysine 183 in TSHr and lysine 180 in LH/CGr must be critically dependent on their surrounding amino acids, which are significantly different in TSHr and LH/CGr (see Fig. 8
), in particular when considering charged and polar residues. In the same spirit, the geometry of the interaction of hCG with the TSHr and LH/CGr may be different, implying that hCG could make contacts with different homologous residues in the ectodomains of the two receptors. It must also be noted that the conclusions of Bhowmick et al. about an important role of K180 for the binding of hCG in LH/CGr are drawn from loss-of-function mutants, while ours about the neutral role of K183 in the TSHr are drawn from gain-of-function mutants, which avoids the pitfalls related to misfolding and/or absence from cell surface.
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Intriguingly, all the glycoprotein hormone receptors [even a distant Drosophila homologue (34)] have an acidic residue in the position homologous to E157 in the TSHr. The functional or structural meaning of this observation is expected to be different whether the residue would be involved, or not, in a neutralizing interaction with a nearby basic residue. Although not amenable to direct experimental investigation, one hypothesis would be that an acidic residue was present in this position in the ancestral glycoprotein hormone receptor, and that it was implicated in recognition of the ancestral hormone. Although it was conserved in all, in some receptors, such as the LH/CGr, it would still play that role [in keeping with this view, an E132K mutant of LH/CGr displays decreased binding of hCG (26)], whereas in others, such as the TSHr, it would have been neutralized as part of the evolutionary mechanism leading to the specificity of recognition in the various hormone/receptor couples. In addition, and compatible with the above hypothesis, it is possible that an acidic residue in position homologous to 157 would be important for the precise structural integrity of the glycoprotein hormone receptors. The small increase in EC50 for TSH in E157A and most double mutants would be compatible with this view.
Additional Natural Mutations in Position 183 of TSHr Are Expected to be Found in Gestational Hyperthyroidism
It must be kept in mind that the gain of function of the K183 mutants toward hCG is modest. As noted in the original description for the K183R mutant, and confirmed in the present study for the other K183 substitutions, the affinity for hCG is too low to permit detection of specific I125hCG binding to transfected COS cells. The EC50 of the best mutants for hCG is in the range of 10 mIU/ml, which is about 3 orders of magnitude higher than with wt LH/CGr (27). In view of the very high concentrations of hCG during the first trimester of pregnancy, this modest increase in responsiveness of K183R to hCG was, however, sufficient to cause severe hyperthyroidism (27). The present results indicate that other spontaneous mutations of K183 would be expected to produce a similar phenotype. Indeed, six of the mutations tested (K183R, N, Q, M, E, T) could result from a single nucleotide substitution in the K183 codon. This poses the question of why familial gestational hyperthyroidism remains presently limited to the description of a single family. Amongst plausible explanations one may cite: 1) a strong negative selection due to the reduction in reproductive fitness of the women carrying the mutations; 2) the possibility that some of the mutants tested here in COS cells (e.g. K183E and K183T) would be expressed at too low a level in thyrocytes to cause significant stimulation of cAMP production. Note, however, that in COS cells, K183M, N, and Q mutants have the same capacity to stimulate cAMP production in response to hCG as K183R (see Fig. 2B); 3) the possibility that the situation would simply be underdiagnosed. Considering the steep increase in hCG during the first trimester of pregnancy, biological hyperthyroidism might develop too quickly to be diagnosed before miscarriage would occur. As fetal loss, with rapidly falling hCG, constitutes the "natural cure" of the condition, spontaneously resolving hyperthyroidism may remain undiagnosed in these patients. If this holds true, it might be valuable to search for this type of mutation amongst patients with recurrent spontaneous abortions.
In conclusion, our results point to lysine 183 and glutamic acid 157 in the ectodomain of TSHr as two important residues implicated in determining the specificity of recognition by TSH and hCG. Investigation of their role by a combination of site-directed mutagenesis experiments and molecular dynamic simulations, and the coherent emerging picture in the context of the available structural model of TSHr ectodomain, illustrates the promises of the approach. In the absence of crystallographic data of hormone-receptor complexes, a similar strategy should now be used to tackle in a more global way the problem of the evolution of specificity in all three glycoprotein hormone receptors.
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MATERIALS AND METHODS |
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Construction of the TSHr Mutants
Mutations (K183R, A, I, T, E, W, D, M, N, or Q; E157A; E157D) were introduced in the hTSHr by QuikChange (Stratagene) site-directed mutagenesis method, starting with hTSHr in pBluescript SK+, as previously described (35, 36). The primers are available upon request. The appropriate mutated portions of hTSHr mutants were subcloned in pSVL-hTSHr cDNA with XhoI and AflIII to obtain the 12 mutants expected.
Mutants E157A+K183R, A, I, T, E, W, D, M, N, or Q and E157D+K183R, A, or W were constructed by subcloning: mutations in position 157 were excised from pBluescript SK+-E157A or D-hTSHr by XhoIAatII; mutations in position 183 were excised from pBluescript SK+-K183R, A, I, T, E, W, D, M, N, or Q-hTSHr by AatIIAflIII. Fragments bearing one E157 mutation and one K183 mutation were ligated in pSVL-hTSHr which had been cleaved by XhoIAflIII to obtain the 13 mutants expected.
All the constructs were amplified in DH5F'-competent cells, and recombinant DNA from selected clones was purified and sequenced for confirmation of the nucleotide sequences of the PCR-generated areas.
Transfection Experiments
COS-7 cells were used for transient expression allowing functional assays. They were transfected by the diethylaminoethyl-dextran method followed by a dimethylsulfoxide shock as described previously (37, 38). Two days after transfection, cells were used for cAMP determinations and flow immunocytofluorometry. Duplicate or triplicate dishes were used for each assay. Each experiment was repeated at least twice. Cells transfected with pSVL alone were always run as controls.
Quantification of Cell Surface Expression of TSHr Constructs by Fluorescence Activated Cell Sorter
Cells were prepared as previously described (18). After detachment, they were centrifuged at 500 x g at 4 C for 3 min and the supernatant was removed by inversion. They were incubated for 30 min at room temperature with 100 µl PBS-BSA 0.1% containing the 3G4 monoclonal antibody obtained from genetic immunization with the wt hTSHr cDNA (18). Cells were then washed with 4 ml PBS-BSA 0.1% and centrifuged as above. They were incubated on ice in the dark with fluorescein-conjugated -chain-specific goat antimouse IgG (Sigma, St. Louis, MO) 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 and resuspended in 300 µl PBS-BSA 0.1%. The fluorescence of 10,000 cells/tube was assayed by a FACScan flow cytofluorometer (Becton Dickinson and Co., Eerenbodegem, Belgium). Cells transfected by pSVL alone and by pSVL-hTSHr wt cDNA were always run as negative and positive controls, respectively.
Determination of cAMP Production
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 of the phosphodiesterase inhibitor Rolipram (Laboratoire Logeais, Paris, France) and various concentrations of highly purified hCG (Sigma), bovine TSH (Sigma), or recombinant FSH (Organon Belge SA, Brussels, Belgium). At the end of a 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 measurements by RIA according to the method of Brooker et al. (39). Duplicate samples were assayed in all experiments; results are expressed in picomoles cAMP per milliliter. Concentration-action curves were fitted with the Prism computer program (GraphPad Software, Inc., San Diego, CA).
Molecular Modeling and Molecular Dynamics Simulations of the Extracellular Domain of the TSHr
The atomic coordinates of the extracellular domain of the TSHr (24), constructed from the structure of the porcine ribonuclease inhibitor (23), were employed in the simulations. Molecular models for the mutant receptors containing the single K183A substitution and the double E157A+K183E substitutions were built from these coordinates by amino acid replacement, whereas the K183R mutant receptor was built from an exhaustive surveyed of all permitted Arg side-chain conformations. These permitted conformations of the 1,
2,
3, and
4 torsional angles were taken from a backbone-dependent rotamer library (28), from which we selected the rotamers corresponding to the backbone conformation of residue 183 of the model (
= -90° and
= 130°). Only conformations that are described to occur at least 1% of the time in the known crystal structures were considered. This leads to 27 rotamers of the 81 theoretically possible rotamers. To identify potential ionic partners of R183, we calculated the distances between the N
, NH1, and NH2 atoms of R183 and the O
1 and O
2 or O
1 and O
2 atoms of Asp and Glu residues, for all the considered conformations of the Arg side chain. To assess which rotamers were favorable for ionic interaction, we retained conformers leading to minimal N
O distance in the 2.04.0 Å range. The resulting structures were placed in a rectangular box containing Monte Carlo-equilibrated TIP3P water molecules. The periodic boxes were approximately 75 Å x 50 Å x 50 Å, and contained between 4059 and 4348 water molecules in addition to the extracellular domain. Initially, the atoms of the receptor were kept fixed, whereas the water molecules were energy minimized (500 steps). Subsequently, the entire systems were energy minimized (500 steps), heated [from 0 to 300 K in 15 picoseconds (ps)], equilibrated (from 15 to 200 ps), and the production run (from 200 to 500 ps) was carried out at constant pressure using the particle mesh Ewald method to evaluate electrostatic interactions (40). Structures were collected every 5 ps during the last 300 ps of simulation (60 structures per simulation). The molecular dynamics simulations were run with the Sander module of AMBER 5 (41 41A ; and http://www.amber.ucsf.edu/amber), the all-atom force field (42), SHAKE bond constraints in all bonds (42A ), a 2-femtosecond integration time step, and constant temperature of 300 K coupled to a heat bath. The molecular electrostatic potentials on the accessible surface of representative structures were calculated and displayed with GRASP (43 ; and http://trantor.bioc.columbia.edu/grasp) using the Cornell et al. (42) atomic charges. The interactions of R183 in the K183R mutant receptor with D232 and its neighbor residues were further characterized by ab initio quantum mechanical calculations. We performed full geometry optimization at the MP2/631G* level of theory, which is capable of describing the proposed N-H··
interactions (44) in a model system containing the following molecular fragments: the side chains of R183 (the -Cß-C
-C
-chain was replaced by a hydrogen), Y185 and Y206 (the -Cß- and OH moieties were replaced by hydrogens), and N208 and D232 (the -Cß-moieties were replaced by a hydrogen). All the quantum mechanical calculations were performed with the Gaussian 98 (Revision A.7) system of programs (45 ; and http://www.gaussian.com).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: 3D, Three-dimensional; EC50, 50% effective concentration; FSHr, FSH receptor; hCG, human CG; LH/CGr, LH and CG receptor; LRR, leucine-rich repeat; ps, picosecond; TSHr, TSH receptor; wt, wild type.
Received for publication August 10, 2001. Accepted for publication December 21, 2001.
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REFERENCES |
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