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Molecular Endocrinology, doi:10.1210/me.2003-0050
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Molecular Endocrinology 18 (6): 1499-1508
Copyright © 2004 by The Endocrine Society

Insight into Mutation-Induced Activation of the Luteinizing Hormone Receptor: Molecular Simulations Predict the Functional Behavior of Engineered Mutants at M398

Francesca Fanelli, Miriam Verhoef-Post, Marianna Timmerman, Annelieke Zeilemaker, John W. M. Martens and Axel P. N. Themmen

Dulbecco Telethon Institute and Department of Chemistry (F.F.), University of Modena and Reggio Emilia, 4100 Modena, Italy; and Department of Internal Medicine (M.V.-P., M.T., A.Z., J.W.M.M., A.P.N.T.), Erasmus MC, 3000 DR Rotterdam, The Netherlands

Address all correspondence and requests for reprints to: Francesca Fanelli, Dulbecco Telethon Institute and Department of Chemistry, University of Modena and Reggio Emilia, Via Campi 183, 41100 Modena, Italy. E-mail: fanelli{at}unimo.it.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In this study, molecular simulations have been combined with site-directed mutagenesis experiments to explore M398(2.43), a LH (lutropin) receptor (LHR) site in helix 2 susceptible to spontaneous activating mutations, and to develop a computational tool for predicting the functionality (i.e. active or nonactive) of LHR mutants.

Site-directed mutagenesis experiments engineered 15 different substitutions for M389(2.43), which resulted in variable levels of constitutive activity, inversely correlated with the size of the replacing amino acid. This inverse correlation is suggested to be mediated by I460(3.46), M571(6.37), and Y623(7.53), the tyrosine of the NPxxY motif. In fact, size reduction at position 398(2.43), which is concurrent with constitutive receptor activity, releases the van der Waals interactions found in the wild-type LHR between M398(2.43) and these three amino acids, resulting in structural modifications in the proximity to the E/DRY/W motif. An increment, above a threshold value, in the solvent accessibility of the cytosolic ends of helices 3 and 6 is the main structural feature shared by the active mutants of the LHR. This feature has been successfully used for predicting the functionality of the engineered mutants at M398(2.43), proving that molecular simulations can be useful for in silico screening of LHR mutants.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
IN THE RHODOPSIN FAMILY of G protein-coupled receptors (GPCRs), the glycoprotein hormone receptors have a special place. The last exon of these receptors encodes the transmembrane domain that carries the seven-transmembrane helices, which are typical of the GPCR superfamily. Hormone binding occurs in the large glycosylated extracellular domain that consists of several leucine-rich repeats flanked by cystine-rich clusters, connected to the transmembrane domain by a less well-conserved hinge region (1, 2, 3, 4).

Two glycoprotein hormones, LH and human chorionic gonadotropin (hCG), exert their effects through binding to the LH receptor (LHR) (1, 4). In the testis, LH (hCG in the fetus) stimulates androgen production by the Leydig cells, which are in number and differentiation dependent on correct LH signaling. In the ovary, LH has two major functions. The midcycle LH peak triggers the ovulatory response of the preovulatory follicles and the subsequent release of the oocyte. Furthermore, LH stimulates androgen production by the follicular theca cells, which is subsequently converted to estrogens in the granulosa cells. After successful fertilization, the activity of the corpus luteum is supported by the placental hCG.

The essential role of correct LHR function is exemplified by the effect of mutations in the LHR gene on sex differentiation and gonadal function (4). Mutations that cause inactivation of the LHR result in complete pseudohermaphroditism in men and anovulation in women. In addition, missense LHR mutations have been identified in boys with very early familial precocious puberty [familial male-limited precocious puberty (FMPP)]. These mutations cause constitutive activity of the LHR in the absence of ligand, resulting in testicular androgen production with the corresponding pubertal changes. Most of the amino acid changes causing constitutive activity are located in the transmembrane and cytosolic portions of helix 6, although such mutations have been found also in helices 1, 2, 3, and 5 (4, 5, 6).

At the present time, no structural data exist on the glycoprotein hormone receptors. So far, three molecular models of the transmembrane domains of the LHR, with or without the extracellular and intracellular loops, have been built following ab initio or comparative modeling approaches (2, 3, 7, 8, 9).

In the present study, experimental mutagenesis has been combined with molecular dynamics (MD) simulations, to gain insight into the molecular mechanism of mutation-induced LHR activation and to challenge the ability of the LHR models to predict the functional behavior (i.e. active or nonactive) of engineered mutants. Engineered mutants concern M398(2.43) [the amino acid numbering in parenthesis, used only for the amino acids in the helix bundle, is that proposed by Ballesteros and Weinstein (10)],1 an amino acid susceptible to spontaneous activating mutations (11, 12, 13). Indeed, mutation of M398(2.43) to T has been found in several independently identified FMPP patients and causes constitutive activity of the LHR when tested in vitro (11, 12, 13). The choice of this particular amino acid as target of our study has been dictated by the fact that, in contrast to other LHR-activating mutation sites, it has not yet been well characterized. On the same line, the molecular mechanism by which this amino acid exerts a control on the basal activity of the LHR is still obscure.

Predictions of the functionality of the M398(2.43) mutants have been carried out through both the ab initio model of the LHR previously presented (8) and a new model (i.e. comparative model) achieved by comparative modeling, by using the rhodopsin structure as a template (1, 2, 3, 9, 14). Herein, an evaluation of the predictive abilities of the two LHR models is also discussed.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Experimental Mutagenesis of M398(2.43)
To investigate the role of M398(2.43) on the basal receptor activity, we have performed 15 different substitutions of the native methionine, spanning the spectrum of chemicophysical properties of the natural amino acids. A representative example of a few mutants of M398(2.43) is given in Fig. 1AGo. Transient transfection of the wild-type (WT) LHR cDNA resulted in a robust cAMP-responsive element (CRE) reporter activity to increasing concentrations of hCG (Fig. 1AGo). The low basal reporter activity is approximately 2-fold higher than the activities found in cells that were transfected with the empty expression vector pSG5 (inset in Fig. 1AGo), whereas the maximal response to 1000 ng/ml hCG is approximately 45-fold. Cell surface binding of [125I]hCG was used to determine dissociation constant (Kd) and maximal binding (Bmax) values through Scatchard analysis. The Kd values of the LHR mutants at M398 were all in the low nanomolar range (Table 1Go). To allow for a comparison, the Bmax values were normalized to the value of the WT receptor. A typical Scatchard analysis for the WT receptor is shown in Fig. 1BGo, yielding a Bmax of 340 fmol/mg protein. The M398(2.43)T LHR mutant represents the original mutation that we identified in a FMPP patient (11). This mutant receptor shows increased basal activity (3.6-fold compared with the WT LHR), an increase that is comparable to that seen for the extensively studied D578(6.44)G mutant (11, 15) (Fig. 1AGo, inset). The maximal response of the M398(2.43)T mutant is lower than that of the WT LHR (i.e. 58% of the WT LHR value; Fig. 1Go and Table 1Go). However, this is due to the much lower cell surface expression of the M398(2.43)T mutant as compared with the WT LHR (i.e. 16% of WT LHR value; Table 1Go). The basal activity of the M398(2.43) mutants has been calculated by measuring the CRE reporter activity in the absence of hormone, correcting for transfection efficiency and normalizing to the activity of the WT receptor. These normalized activities are presented as activity per cell surface binding site (Table 1Go). The mutants M398K, -R, -D, -G, and -N display poor expression (i.e. <10% of the WT LHR) and have been, therefore, excluded from further analysis. We found that amino acids bulkier than the native methionine (i.e. W, Y, and F) attenuate basal CRE activity, whereas amino acids smaller than methionine, (i.e. V, L, A, T, S, and C) induce increases in the basal receptor activity (Table 1Go). Interestingly, the M398(2.43)D, -G, and -N mutants display remarkable constitutive activity (Table 1Go) despite their barely detectable cell surface expression below 10% of the WT LHR. The inverse relation between size of the amino acid at position 2.43 and hormone-independent receptor activity is accounted for by the inverse linear correlation (r = –0.73, excluding alanine) between the specific activity of the M398(2.43) mutants and either the solvent-accessible surface area or the van der Waals volume (i.e. taken from Ref.16) of the replacing amino acid (Table 1Go).



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  		Fig. 1. hCG Dose-Response Curves of WT, M398(2.43)T, -L, and -K and D578(6.44)G LHR Mutants

A, HEK293 cells were co-transfected with a cAMP-responsive reporter plasmid (pCRE6Lux), a ß-galactosidase expression plasmid (pRSVlacZ), and the expression vector pSG5 carrying no insert (pSG5, open squares), WT LHR cDNA (WT, open circles), or different mutant LHR cDNAs [(M398(2.43)T, closed circles; M398(2.43)L, closed triangles; M398(2.43)K, inverted open triangles; and D578(6.44)G, closed squares (11 15 )]. hCG-responsive luciferase activity was determined and corrected for transfection efficiency by normalizing to the ß-galactosidase activity. Inset, The basal activity of the M398(2.43)T, -L, and -K as well as of the D578(6.44)G (DG) mutants in the absence of hCG is displayed compared with the WT LHR; histograms refer to a single experiment. B, HEK293 cells were transfected with the WT LHR cDNA as described in Materials and Methods. Different amounts of purified [125I]hCG were incubated for 2 h with intact HEK293 cells transfected with the WT LHR cDNA in the presence or absence of excess of unlabeled ligand. The ratio of bound over free (B/F) was expressed as function of the concentration of bound ligand. The arrow indicates the Bmax (464 pM), which corresponds to a binding capacity of 340 fmol/mg total protein.

 

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  		Table 1. Experimental Data and Computational Indices for the M398(2.43) Mutants

 
Structure-Based Definition of a Molecular Descriptor for Functionality Predictions through the Comparative Model of the LHR
Molecular descriptors able to account for the structural differences between active and nonactive LHR mutants have been previously defined on the ab initio model (8). The definition of equivalent descriptors on the comparative model of the LHR followed the same approach as that used for the ab initio model (8). This approach consisted in comparing the structural features of the WT LHR with those of the spontaneously active and inactive LHR mutants known thus far (4, 5, 6). This training set of WT and mutant LHR forms is constituted by the average minimized structures derived from MD simulations.

The most significant feature, which makes the constitutively active mutants different from the nonactive ones, is the increase in solvent accessibility, as compared with the WT LHR, of the cytosolic extensions of helices 3 and 6. This effect is due, at least in part, to rigid body motions of helices 2, 3, 4, and 6. In particular, in the active forms, outward motion of helix 3 and the consequent detachment of helix 4 from helix 2 occur, which are marked by a change in the interaction pattern of W491(4.50). In fact, in the WT LHR, the highly conserved tryptophan interacts with N400(2.45), whereas, in the majority of the active mutants, it is directed toward the putative membrane space (Fig. 2Go). Furthermore, reciprocal motions of helices 3 and 6 occur, which are marked, at least in part, by the breakage of the charge-reinforced H-bonding interaction found in the WT LHR between R464(3.50) and D564(6.30) (Fig. 2Go). The movements of helices 3, 4, and 6 are concurrent with a change in the conformation/orientation of the second and third intracellular loops and the consequent opening of a solvent-accessible cleft in the proximity to the cytosolic extensions of helices 3 and 6 (Fig. 2Go). This effect is properly described by the solvent-accessible surface area computed over the amino acids R464(3.50), T467(3.53), I468(3.54), and K563(6.29) [solvent accessible surface area (SAS), Table 2Go], which include the arginine of the E/DRY/W motif (1, 9). Indeed, the SAS index is below 50 Å2 in the WT LHR and its inactive mutants, whereas it is above that threshold in the constitutively active mutants (SAS, Table 2Go; Fig. 2Go, cyan dots). It is noteworthy that the amino acids that participate in SAS do not contribute by the same extent in the different structures. The differences in this index are small but significant and the threshold of 50 Å2 properly separates the nonconstitutively active LHR mutants from the constitutively active ones.



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  		Fig. 2. Average Minimized Structures of the WT LHR and of the D564(6.30)G Mutant as Achieved Following Comparative Modeling and MD Simulations

Top left, Cylinder representations of the helix bundles are shown, viewed from the intracellular side in a direction almost perpendicular to the membrane surface. The spheres, centered on the ß-carbon atoms, indicate the LHR sites found to be susceptible to spontaneous activating mutations (green spheres) as well as the LHR sites of the ERW motif and of the highly conserved polar amino acids (violet spheres). Top right, Details of selected interactions involving M398(2.43) as well as the arginine of the E/DRY/W motif. The amino acid side chains, colored according to their polarity, are seen in a direction almost parallel to the membrane surface with the intracellular side at the top. Bottom, Representation of the WT LHR (left) and of the D564(6.30)G constitutively active mutant (right) seen from the intracellular side in a direction perpendicular to the membrane surface. The seven-helices are represented by cylinders whereas the three intracellular loops are represented by thin ribbons. The extracellular loops are not shown. Helices 1, 2, 3, 4, 5, 6, and 7 are colored, respectively, in blue, orange, green, pink, yellow, light blue, and violet, whereas the intracellular loops 1, 2, and 3 are respectively colored, respectively, in light green, white, and purple. The side chains of E463(3.49), R464(3.50), W491(4.50), and D564(6.30) are represented by sticks and colored according to their polarities. The composite solvent-accessible surfaces computed over the amino acids R464(3.50), T467(3.53), I468(3.54), and K563(6.29) are also shown, represented by cyan dots.

 

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  		Table 2. Functional States (i.e. Active or Inactive) and Theoretical Descriptors for Naturally Occurring LHR Mutants

 
Computer-Based Predictions of the Functional Behavior of the M398(2.43) Engineered Mutants
MD simulations have been carried out on all the engineered M398(2.43) mutants, and the results are reported in Table 1Go.

Predictions of the functionality (i.e. active or nonactive) of the M398(2.43) mutants have been done through both the ab initio and the comparative models of the LHR. The evaluation of functionality predictions concerning the mutants M398(2.43)K, -R, -D, -N, and -G has not been done because their specific activity could not be determined (Table 1Go).

Predictions through the ab initio model have been based upon the two theoretical indices previously defined, i.e. the distance between D405(2.50) and R464(3.50) [or, in some mutants, between E463(3.49) and R464(3.50), Table 1Go] and the solvent-accessible surface area of W465(3.51) (i.e. SASW465, Table 1Go) (8). As previously reported, distances between D405(2.50) and R464(3.50) or E463(3.49) and R464(3.50) below 6.0 Å and SASW465 below 11.0 Å2 mark the nonactive forms, whereas values of the same indices, respectively, above 6.0 Å and above 30.0 Å2 mark the active forms (8). SASW465 values above or below 30.0 Å2 are associated, respectively, with the presence or absence of a solvent-accessible cleft between the second and third intracellular loops (8). The goodness of predictions is summarized in Table 1Go, where the uppercase Y means good prediction based upon both descriptors, whereas lowercase y indicates those cases, in which the SASW465 index does not account properly for the presence/absence of the cytosolic cleft. Aromatic amino acid replacements of M398(2.43) have been correctly predicted as nonconstitutively active, although these predictions are unequivocal only for the M398(2.43)W mutant (Y, Table 1Go). The presence of constitutive activity has been correctly and unequivocally predicted for the M398(2.43)I, V, T, S, and C mutants (Y, Table 1Go). The functionality of the M398(2.43)L mutant is nonpredictable (NP, Table 1Go) because the model holds characteristic features of both the inactive and active states, i.e. D405-R464 distance and SASW465 below and above the thresholds, respectively.

Functional classification of the M398 mutants through the comparative model has been done by means of the SAS index described above (Tables 1Go and 2Go). The goodness of predictions through the SAS index is reported in Table 1Go, where uppercase Y and N mean, respectively, good and incorrect prediction.

Molecular simulations on the comparative model, consistently with the experimental findings, suggest that substitutions of M398(2.43) with hydrophobic or polar amino acids, which have smaller dimensions than a methionine (i.e. replacements with D, V, L, A, T, S, and C), induce an increase of SAS over the threshold, similarly to the spontaneously active LHR mutants found in FMPP patients (Figs. 2Go and 3Go; Tables 1Go and 2Go). In contrast, augmentation in size (with respect to the native methionine) of the replacing amino acid at position 398(2.43) (i.e. replacements with W, Y, and F) confers to the cytosolic domains the features of the nonactive forms, i.e. SAS below the threshold (Table 1Go and Fig. 3Go). The M398(2.43)I mutant, which is characterized by a very low basal activity, displays the features of the nonactive LHR forms.



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  		Fig. 3. Average Minimized Structures of the WT LHR as well as of the Mutants of M398(2.43) to W, Y, I, V, S, and C, as Achieved Following Comparative Modeling and MD Simulations

The structures are viewed from the intracellular side in a direction almost perpendicular to the membrane surface. The extracellular domains are not shown in this figure. The side chains in the mutated position are colored in red and represented by van der Waals spheres. The composite solvent-accessible surfaces computed over the amino acids R464(3.50), T467(3.53), I468(3.54), and K563(6.29) are also shown, represented by green dots.

 
In summary, excluding from the evaluation those mutants that are nonexpressed [i.e. M398(2.43)K, -R, -D, -N, and -G), the ab initio model predicts correctly the functionality of 10 of 11 M398 mutants by using two indices; however, predictions are unequivocal only in six cases (i.e. Y in Table 1Go). In contrast, the comparative model predicts correctly the functionality of all the mutants but one, by using only one descriptor.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this study, we have used computer-simulated and experimental mutagenesis to explore M398(2.43), an LHR site in helix 2 known to be susceptible to spontaneous activating mutations (5).

The majority of the 15 different substitutions for M398(2.43) that have been engineered in this study resulted in increased basal activity levels, as compared with the WT LHR (Table 1Go). Interestingly, the basal activity levels of the M398(2.43) mutants are inversely correlated with size/shape descriptors such as either the solvent-accessible surface area or the van der Waals volume of the replacing amino acid, i.e. the lower the size of the amino acid at position 2.43, the higher the basal activity of the LHR.

For both the ab initio and the comparative model of the LHR, the analysis of the average minimized structures of the WT and of the majority of the spontaneously active and inactive mutants known so far has been instrumental in the development of theoretical descriptors, which discriminate the inactive from the active LHR forms (i.e. this work and work described in Ref.8). These descriptors have been challenged in their ability to predict the functional behavior (i.e. active or nonactive) of the M398(2.43) mutants engineered in this work. The theoretical index developed on the comparative model performs better in functionality predictions than those developed on the ab initio model (Table 1Go). These results are in line with the knowledge that comparative modeling produces more reliable results than ab initio modeling, at least in the homologous domains between the target and the template (i.e. the transmembrane helices for the LHR and rhodopsin, respectively) (17). The comparative model can, hence, be considered an advancement over the ab initio model, which has been extremely useful, however, for developing the computational approach (8). The index employed for functionality predictions through the comparative model is the solvent-accessible surface area computed over R464(3.50), T467(3.53), I468(3.54), and K563(6.29) (i.e. SAS, Tables 1Go and 2Go). Indeed, only the M398(2.43)I mutant, which barely displayed constitutive activity, has been misclassified by the SAS index, indicating that this descriptor, by using a 50Å2 cutoff, is able to predict the functionality of the M398(2.43) mutants. The effectiveness of SAS as hallmark of presence or absence of constitutive activity has been also recently validated on a series of single and double engineered LHR mutants involving D578(6.44) (9).

The SAS index properly describes the solvent accessibility of the cytosolic extensions of helices 3 and 6, which, in the active states, become more exposed to the solvent, as compared with the nonactive ones. Values of the SAS index above the threshold are generally concurrent with the opening of a cleft between the second and third intracellular loops. Our results are consistent with the results of cross-linking, spin labeling, and scanning accessibility experiments on rhodopsin [reviewed by Meng and Bourne (18)]. In fact, mapping these experimental data onto the rhodopsin structure suggests that activation by light opens a cleft at the cytoplasmic end of the helix bundle with separation of transmembrane helices 3 and 6 and increased exposure of the inner faces of helices 2, 3, 6, and 7 (18). Finally, our hypotheses are consistent with the results of experiments that implicate the involvement of the cytosolic end of helix 6 in G protein activation by the LHR (19, 20, 21).

Interestingly, molecular simulations provide an explanation for the observed inverse correlation between size of the replacing amino acid at position 398(2.43) and basal activity level of the mutant receptor. In fact, reducing the size of the amino acid at position 398(2.43) reduces its ability to perform local van der Waals interactions and confers the characteristics of the active forms to the cytosolic extensions of helices 3 and 6, which hold the E/DRY/W motif (Figs. 2Go and 3Go). The communication between the mutation site in helix 2 and the receptor portions close to the E/DRY/W motif is suggested to involve, at least in part, I460(3.46), M571(6.37), and Y623(7.53) of the NPxxY motif. In fact, size reduction at position 398(2.43), which is concurrent with constitutive receptor activity, releases the van der Waals interactions found in the WT LHR between M398(2.43) and these three amino acids (Fig. 2Go). Decrease in size, together with the ability to donate H bond to the backbone carbonyl oxygen atom of P394(2.39) as well as to accept H bonds from K570(6.36) or from N619(7.49), is suggested to be the trigger of the constitutive activity of the M398T and M398S mutants, which, together with M398C, display significantly higher specific activity than the other constitutively active M398 mutants (Table 1Go). The consequent change in the packing interactions at the cytosolic halves of 1) helices 2 and 7, 2) helices 2 and 3, 3) helices 3 and 6, and 4) helices 6 and 7, as shared by the constitutively active mutants at M398, is concurrent with increased solvent accessibility of the cytosolic extensions of helices 3 and 6 (i.e. SAS above the threshold) and weakening of the interactions that involve the arginine of the E/DRY motif in the WT LHR. In contrast, and consistent with the experimental findings, replacing M398(2.43) with aromatic amino acids that strongly interact with I460(3.46), M571(6.37), and Y623(7.53) retains the features of the nonactive forms, i.e. SAS below the threshold and presence of strong interactions between R464(3.50) of the E/DRY/W motif and both E463(3.49) and D564(6.30) (Fig. 2Go). Equivalent interactions concerning the E/DRY arginine characterize the inactive state of rhodopsin (14). Thus, M398(2.43) favors tight packing of the cytosolic halves of helices 2, 3, 6, and 7. Interestingly, the amino acids, which are suggested to mediate the transfer of the structural modification from the mutation site in helix 2 [i.e. position 398(2.43)] to the cytosolic ends of helices 3 and 6, include M571(6.37), an amino acid susceptible to spontaneous activating mutations, and the functionally important Y623(7.53) of the NPxxY motif. The results of this study suggest that there is a structural link between the highly conserved tyrosine in helix 7 and the E/DRY motif, in line with the inferences of a recent study on rhodopsin suggesting that the NPxxY(x)5,6F and D/ERY motifs provide, in concert, a dual control of the activating structural changes in the photoreceptor (22).

In conclusion, the results of this study provide insight into the LHR structure and function, suggesting also that molecular simulations can be used for in silico screening of LHR mutants as well as for designing new mutants able to antagonize or strengthen the basal activity of activating mutations.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Construction of the Mutant LHR Expression Plasmids
The coding region of the LHR, extended with an immunotag (HA1) at the C terminus (23) was placed downstream of the simian virus 40 large T antigen promoter in the expression plasmid pSG5 (11, 24), resulting in pSG5-hLHR-HA1. The HA1-tag does not affect expression and signal transduction of the WT LHR (results not shown). The amino acid changes at position 398(2.43) were introduced in the expression vector pSG5-hLHR-HA1 using a previously described approach (11). The forward mutant primers are given in Table 3Go; the complementary sequences were used as reverse mutant primers. For these mutations, a PCR amplification with the flanking primers 1030for (5'-CCGATGTGCTCCTGAACC-3') and 1430rev (5'-GTCCAGGTGAATAGCATAG-3') was performed. The resulting fragments were exchanged with the corresponding fragments in pSG5-hLHR-HA1 after digestion with Bsu36I (position 1084) and XbaI (position 1383). The mutant expression vector obtained is designated pSG5-hLHRM398(2.43)X, where X indicates the relevant amino acid residue at position 398(2.43). The double mutants were constructed by exchanging the Bsu36I-XbaI fragment containing the M398(2.43)T mutation with the corresponding fragment in LHR expression vectors carrying the following mutations: S616(7.46)Y, I625(7.55)K, M571(6.37)I, and D578(6.44)G (7, 45). Primers were purchased from Eurogentec (Seraing, Belgium), and mutagenesis was verified by DNA sequence analysis of the exchanged fragments including restriction sites and flanking DNA.


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  		Table 3. Oligonucleotides Used to Create Amino Acid Changes in the Human WT LHR cDNA (see Materials and Methods)

 
In Vitro Expression and Signaling of Mutant LHRs
Human embryonic kidney (HEK)293 cells were maintained as described previously (11). For the estimation of hormone-dependent induction of cAMP and total cell surface binding, subconfluent HEK293 cells were transiently transfected (25) with 2 µg of the cAMP-reporter plasmid pCRE6Lux (26), 1 µg pRSVlacZ (27) as a control for transfection efficiency, and 10 µg pSG5, pSG5-hLHR-HA1, or pSG5-hLHRmutant and 7 µg carrier DNA per 75-cm2 culture flask. Two days after transfection the cells were trypsinized and plated in 24-well tissue culture plates (Nunc, Roskilde, Denmark) for luciferase and ß-galactosidase measurements, and in a 75-cm2 tissue culture flask (Nunc) for total cell surface hCG binding. To determine the cAMP-reporter response, cells were incubated the next day in culture medium containing 0.1% BSA and the indicated concentration of hCG (Organon International, Oss, The Netherlands). After 6 h, the cells were lysed and luciferase activity was determined (28). For each mutant LHR, a complete hormone dose-response curve was constructed. hCG binding to intact cells was performed as described previously (29, 30). Briefly, single-point binding assays or multiple-point Scatchard analysis on intact cells were performed in isotonic Tris-buffer (10 nM Tris-HCl, pH 7.5; 5 mM MgCl2; 5 mM NaN3; 200 mM sucrose; 0.1% BSA) with [125I]hCG for 1 h in the presence or absence of 2000-fold excess of unlabeled ligand. After binding, bound and free ligand were separated by centrifugation and counted in a scintillation counter. Because the hormone binding affinity of the WT LHR receptor is solely dependent on the extracellular domain (31, 32), we have used single-point binding assays to determine the expression level of the mutants. To better compare the functional behavior of the LHR mutants, basal and hormone-stimulated activities as well as surface binding of radiolabeled hCG have been normalized to the values of the WT.

Ab Initio Modeling of the LHR WT and Its Mutants
Ab initio modeling of the LHR was achieved following the procedure previously described (8). The LHR input structure previously built was used to produce the input structures for the receptor mutants considered in this work. Minimization and MD simulations of the receptor models were performed using CHARMM (33) according to the computational protocol previously described (8). For the M398 mutants considered in this study, MD runs of 150 psec were performed following the same heating and equilibration setup as that employed for the longer MD simulations. The results reported were collected every 0.5 psec during the last 100 psec of the equilibrated MD trajectory. Finally, for each mutant, the structures averaged over the 200 structures stored during the production phase were used for the comparative analysis.

The comparison between ab initio model of the WT LHR and rhodopsin structure has been reported elsewhere (2, 3).

Three-Dimensional Model Building and Molecular Simulations of the Comparative Model
In this work, we built a new model of the LHR by means of the comparative modeling program MODELER (34), employing rhodopsin structure as a template (14). Different modified rhodopsin templates were probed, which lacked the third intracellular loop as well as the first and/or the third extracellular loops. For each of the different templates, MODELLER generated 25 models. Among the 200 models finally obtained, six models were selected for their low restraint violation, and the low number of faulty main-chain and side-chain conformations and contacts. Polar hydrogens were added to these models, and they were subjected to automatic and manual rotation of the side-chain torsion angles when in nonallowed conformation, by using rotamer libraries. Following these computations, 62 final models were selected, differing in the conformation of a few amino acid side chains, and subjected to energy minimization and MD simulation by means of CHARMM (33), following the same computational protocol as that previously employed for simulating the ab initio LHR model (8). The selected input structure of the WT LHR was built according to the alignment shown in Fig. 4Go.



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  		Fig. 4. Sequence Alignment between Rhodopsin and LHR

The sequence alignment, which has been used for achieving the selected input structure of the LHR by comparative modeling, employing the rhodopsin structure as a template, is shown. A modified rhodopsin template lacking the segments 101–106, 229–235, and 240–242 has been used. The asterisk on helix 7 indicates the breakpoint in the helix that precedes the tilted segment corresponding to the rhodopsin’s helix 8.

 
The minimized coordinates of the WT and of the M398(2.43)T and D564(6.30)G constitutively active mutants were used as starting points for 1050 psec of MD runs. For all the other mutants considered in this study MD, runs of 150 psec were carried out using the same heating and equilibration setup as that employed for the longer MD simulation. Finally, for the WT and all the simulated mutants, the structure averaged over the 200 structures collected (every 500 steps) during the 50,500–150,000 step interval and minimized were used for the comparative analysis.


   ACKNOWLEDGMENTS
 
We thank Dr. Susanna Cotecchia for helpful suggestions. F.F. acknowledges technical support received from CICAIA (Centro Interdipartimentale di Calcolo Automatico ed Informatica Applicata), University of Modena.


   FOOTNOTES
 
This work was supported by Telethon-Italy Grant TCP00068 (to F.F.) and by a grant from The Netherlands Organization for Scientific Research (to A.P.N.T.). F.F. is an Assistant Telethon Scientist.

Present address for J.W.M.M.: Department of Internal Oncology, Erasmus MC, Box 1738, 3000 DR Rotterdam, The Netherlands.

Abbreviations: CRE, cAMP-responsive; GPCRs, G protein-coupled receptors; FMPP, familial male-limited precocious puberty; hCG, human chorionic gonadotropin; HEK, human embryonic kidney; LHR, LH receptor; MD, molecular dynamics; SAS, solvent accessible surface area; WT, wild-type.

1 According to the numbering system by Ballesteros and Weinstein (10 ), every amino acid identifier starts with the helix number, followed by the position relative to a reference residue among the most conserved amino acid in that helix. That reference residue is arbitrarily assigned the number 50. Back

Received for publication February 10, 2003. Accepted for publication March 5, 2004.


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TOP
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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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