Functional Role of Transmembrane Helix 7 in the Activation of the Heptahelical Lutropin Receptor

Krassimira Angelova, Prema Narayan, J. Paul Simon and David Puett

Department of Biochemistry & Molecular Biology University of Georgia Athens, Georgia 30602-7229


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A member of the G protein-coupled receptor superfamily, the LH receptor (LHR), and the two other glycoprotein hormone receptors are distinguished from the other members by the presence of a relatively large N-terminal extracellular domain that is responsible for high-affinity ligand binding. Transmembrane helix (TMH) 7 of LHR is amphipathic, with an extended face containing only hydrophobic side chains and another containing both hydrophobic and polar side chains with potential hydrogen bond donor and acceptor functions. Since several reports have shown the importance of this helix in ligand-mediated signaling, we have used Ala scanning mutagenesis to study eight amino acid residues of rat LHR that are invariant in the three glycoprotein hormone receptors, Leu586, Val587, Asn593, Ser594, Cys595, Asn597, Phe604, and Thr605. The wild type (WT) and mutant cDNAs were transiently transfected into COS-7 cells for characterization by human CG (hCG) binding and cAMP production. No differences were detected in dissociation constants (Kds) or basal cAMP production relative to WT LHR, but three categories of LHR mutants were distinguished from WT LHR based upon their expression levels and responsiveness to hCG: 1) comparable or higher expression but reduced ligand responsiveness (N593A and C595A), 2) reduced expression and ligand responsiveness (N597A and T605A), and 3) comparable expression and responsiveness (L586A, V587A, S594A, and F604A). Three other mutants, C595M, F604Y, and T605Y, were comparable to WT LHR in ligand responsiveness. To provide more information on Asn593 and Asn597, a total of 12 replacements were investigated. Of considerable interest and potential significance was the finding that many of the replacements in LHR resulted in either loss of function (N593A, Q, S; N597R) or gain of function (N593R and N597Q), this being the first evidence of a position in LHR that, depending upon the nature of the amino acid residue, can result in constitutive activation and/or diminished responsiveness to ligand. The results of molecular modeling and energy minimization of TMHs 6 and 7, based on a postulated interaction between Asp556 (TMH 6) and Asn593/Asn597 (TMH 7), indicated that, while there is not a correlation between function and predicted energies of WT LHR and the mutants, reorientation of one or both helices is responsible for the functional changes observed. Possible interactions of TMHs 3 and 4 and of 5 and 6 were suggested by molecular modeling. Ten mutants were prepared of two amino acid residues that are invariant in the glycoprotein hormone receptors and have side chain hydrogen bond donor and acceptor function, Glu429 in TMH 3 and Asn513 in TMH 5. Expression levels and hCG-mediated signaling were reduced in most of the LHR mutants, but none of these exhibited constitutive receptor activation. We conclude that Glu429 is not critical for receptor function, while Asn513 appears to be particularly important in receptor folding and/or trafficking. The results reported herein indicate an important role for TMH 7, and particularly Asn593 and Asn597, in the process of receptor activation. Moreover, these two asparagines, although in close proximity to each other in TMH 7, are quite distinct in function as evidenced by certain replacements that can lead to loss of function in one and gain of function in the other.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The LH receptor (LHR) (1), along with the receptors for FSH and TSH, form a subfamily of the superfamily of G protein-coupled receptors (2). These particular heptahelical receptors are distinguished by their large extracellular N-terminal domain that accounts for roughly half the size of the receptor and is responsible for high-affinity ligand binding (3, 4, 5, 6, 7). LHR is expressed on gonadal cells and is required for normal reproductive function in males and females and for male sexual differentiation (8). In addition to the well established signaling pathway involving Gs and activation of adenylyl cyclase (1, 2), it has been reported that phospholipase Cß is also activated, perhaps by the ß{gamma} heterodimer released from Gi and Gs (9).

There is considerable interest in elucidating the mechanism by which binding of the heterodimeric glycoprotein hormones, with molecular masses between 30–37 kDa, to the extracellular domain leads to receptor activation. Ji and co-workers (10) have proposed a model in which high-affinity binding of ligand occurs to the extracellular domain, followed by a conformational change in which the ligand/exodomain complex interacts with the endodomain, leading to a reorientation of helices. Little, however, is known about the nature of the conformational changes of the transmembrane helices (TMHs) required for LHR activation. Lin et al. (11) have proposed a model involving reorientation of TMHs 6 and 7, and Schoneberg et al. (12) proposed a major change in the relative positions of TMHs 5 and 6. The generalized gonado-TSH receptor model proposed by Hoflack et al. (13) has an interior cleft, largely hydrophobic, that could also form a series of hydrogen bonds, contributed by all TMHs, with a portion of the bound glycoprotein hormone penetrating into the cleft. A number of studies have been reported on the functional consequences of replacing, via site-directed mutagenesis, certain amino acid residues in the TMHs (14, 15, 16, 17, 18, 19). Moreover, there are now numerous reports of naturally occurring mutations in the TMHs, some of which are loss-of-function mutations leading to hypogonadism and pseudohermaphroditism, while others lead to constitutive receptor activation, as manifested, for example, in familial and sporadic male-limited precocious puberty (see reviews in Refs. 20, 21, 22, 23). The most common mutation in the human LHR gene resulting in male-limited precocious puberty is a replacement of Asp578 (corresponding to Asp556 in rat LHR in which the 22-amino acid residue signal peptide is not included in the numbering as it is with human LHR) in TMH 6 with Gly.

We suggested earlier that TMH 7 of LHR contains a number of polar and hydrophobic side chains that can function as hydrogen bond donors and/or acceptors, e.g. Tyr590, Asn593, Ser594, Asn597, and Tyr601, and, interestingly, these amino acid residues map to a common face of the helix (17). In a rigorous molecular modeling study of helical packing in LHR, Lin et al. (11) proposed that interactions involving TMHs 6 (Thr555 and Asp556) and 7 (Asn593 and Asn597) are important in receptor activation. A number of reports have also indicated the functional importance of TMH 7 in LHR function. For example, our laboratory reported on two replacements of conserved residues in TMH 7 of rat LHR, P591L and Y601A, that diminished ligand-mediated signaling, but not ligand binding (17), as was also found for Lys583, located at the interface between exoloop 3 and TMH 7 (24, 25). Another point mutant in TMH 7 was examined, P598L, which fails to localize properly at the cell surface (17). Two reports of a naturally occurring mutation in TMH 7 of the human LHR, S616Y, corresponding to Ser594 of rat LHR, have been reported in 46,XY individuals presenting with a micropenis and Leydig cell hypoplasia (16, 26). When examined in transfected cells, human LHR with the S616Y mutation is nonfunctional, probably due to the lack of proper membrane localization (16, 26, 27). Two siblings, one 46,XX and the other 46,XY, presenting with gonadal LH resistance, were found to have a deletion of two amino acid residues in LHR, corresponding to Leu608 and Val609 (Leu586 and Val587 of rat LHR) (28). As with the Pro->Leu and Ser->Tyr replacements, the Leu-Val deletion mutant of LHR appears to be retained intracellularly. Finally, there is a preliminary report that replacement of Asn619 with Gln in human LHR (Asn597 in rat LHR) results in constitutive activation of LHR (29).

In view of the important structural and functional role of TMH 7 of LHR indicated in these studies, we have investigated several polar amino acid residues that can serve as hydrogen bond donors and acceptors, Asn593 and Asn597, and as hydrogen bond donors, Ser594 and Thr605. In addition, we have studied the role of several hydrophobic side chains, Leu586, Val587, Cys595, and Phe604. These amino acid residues were chosen because all are invariant in the three glycoprotein hormone receptors and are conserved in all G protein-coupled receptors; moreover, all but Val587 and Cys595 map to a common face of the helix. Our results demonstrate that replacements of Asn593 and Asn597 can result in loss of function, i.e. reduced responsiveness to ligand, or gain of function, i.e. constitutive activation, depending upon the chemical structure of the side chain. These experimental observations were complemented with molecular modeling and energy minimization of TMHs 6 and 7.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Molecular modeling was performed on TMH 7 of rat LHR, and schematic representations of the helix are shown in Fig. 1Go. The two prolines at positions 591 and 598 introduce kinks, and panel A presents a side view of the helix, with only the side chains displayed for those amino acid residues replaced in this study. Asn593, Ser594, and Asn597 form a patch of polar amino acid residues on one extended face of the helix near its center. Although not shown for purposes of clarity, the side chains of the two tyrosines at positions 590 and 601 fall on an extended common face of the helix, as does Thr605. Thus, all six of the side chains with hydrogen bond donor function on TMH 7, Tyr590, Asn593, Ser594, Asn597, Tyr601, and Thr605, the two asparagines also having hydrogen bond acceptor function, are interspersed with hydrophobic side chains on one side of the helix, while the opposite side contains all hydrophobic side chains. The partial amphipathic nature of TMH 7 was noted in an earlier study (17). A top view of an energy-minimized structure of TMH 7 is shown in Fig. 1BGo. This representation emphasizes the kinking introduced by the two prolines and also shows the partial amphipathic nature of TMH 7.



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Figure 1. Schematic Model of TMH 7 (Residues 584–605) of LHR

A side view of TMH 7 (ILLVLFYPVNSCANPFLYAIFT) showing only the amino acid residues replaced in this study (A). A perfect {alpha}-helix was constructed with SYBYL (Tripos Associates Inc.) and then minimized using the Kolman all-atom force field (electrostatics included) until default convergence criteria were met. The {alpha}-carbons from each residue connected to generate the helical wheel diagram viewed from the extracellular side of the cell membrane looking toward the cytoplasmic side, and the amino acid residues investigated in this study are shown in boldface (B).

 
Ala scanning mutagenesis was initially conducted on eight amino acid residues in TMH 7 of rat LHR. After transient transfection into COS-7 cells, each of the mutant LHRs was characterized by competitive ligand binding with 125I-human (h) CG and hCG to determine the IC50, from which the Kd could be obtained. Since amino acid residue replacements in TMH 7 were found to have no effect on Kd values, the specific binding at a fixed concentration of 125I-hCG, Bo, is proportional to receptor density. We also measured basal cAMP production and changes thereof in response to hCG to obtain the ED50 and maximal cAMP produced at a saturating concentration of hCG after subtraction of the basal level (Rmax). In each case, wild-type (WT) LHR was included as control, and the results are reported for Bo and Rmax as a percentage of WT LHR. The results for each of the Ala replacements are summarized in Table 1Go, and representative data are shown in Fig. 2Go for competitive binding and ligand-mediated signaling of the N593A, C595A, and N597A LHR mutants. In all cases, the Kds and basal cAMP levels are equivalent to those of WT LHR. The results can be considered in three categories based upon expression levels and ligand responsiveness. 1) For the N593A and C595A mutants, receptor expression was comparable to or exceeded that of WT LHR, but responsiveness to hCG was diminished; 2) for the N597A and T605A mutants, both expression levels and hCG responsiveness were reduced relative to WT LHR; and 3) for the mutants L586A, V587A, S594A, and F604A, receptor expression and responsiveness were like those of WT LHR.


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Table 1. Binding and Signaling Parameters of Ala Replacements in TMH 7 of LHR1

 


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Figure 2. Competition Binding Curves between 125I-hCG (50 pM) and hCG and hCG-Mediated Increases in cAMP for WT LHR and Three Mutants in TMH 7, N593A, C595A, and N597A

Binding results are given in the upper three panels for WT LHR (•) and mutant LHR ({circ}). The cAMP changes in response to hCG of WT LHR ({blacktriangledown}) and mutant LHR ({triangledown}) are shown in the bottom three panels. The data represent the mean ± M of three independent transfections.

 
To address the question of basal cAMP levels and Rmax dependence on receptor density over the range investigated, we determined maximal ligand-mediated signaling at various levels of expression for the Ala-containing receptor mutants. From the data presented in Fig. 3Go, there is only a very slight dependence of Rmax on receptor density for WT LHR. The receptor mutants L586A, V587A, and S594A, but not N593A, C595A, N597A, F604A, or T605A (data not shown for the latter), also showed a slight dependence of Rmax on receptor expression levels, and there was no significant change in basal cAMP values with receptor density for WT and the mutant LHRs (data not shown).



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Figure 3. Relationship between Rmax and Receptor Density (Proportional to Bo) for Transfected COS-7 Cells Expressing WT and Mutant LHRs

Plots (log10-log10) of Rmax vs. Bo were constructed for WT and mutant LHRs. Each point represents a separate transfection, and the means ± range of duplicate measurements are shown. The data were fit by linear regression, and the solid line shows the best fit with 95% confidence limits (P < 0.05) given by the dashed lines. There is a slight dependence of maximal cAMP response to hCG and apparent receptor density for WT LHR.

 
Other point mutants of LHR were then characterized, C595M, F604Y, and T605Y, with the replacements chosen for the following reasons. In view of the finding that the C595A mutant exhibited loss of responsiveness, we chose a Met replacement since it is more hydrophobic than Ala and has a sulfur-containing side chain, but is devoid of thiol function. The F604Y replacement was made to ask if an aromatic side chain with hydroxyl function would alter expression and signaling since the Ala replacement did not. Finally, since the T605A mutant exhibited reductions in both receptor expression and signaling, we replaced the side chain with an aromatic side chain, while retaining the hydroxyl function of Thr. As shown in Table 2Go, the receptor expression levels, basal cAMP values, and hCG-mediated cAMP increases were comparable to WT LHR, with the exception of the C595M mutant that expressed at a lower level. Thus, it appears that the more hydrophobic Met, compared with Ala, restores signaling at position 595 (Cys); position 604 (Phe) can tolerate Ala or Tyr replacements equally well; and the more hydrophobic side chain, Tyr, is tolerated better at position 605 (Thr) than Ala.


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Table 2. Expression and Signaling Parameters of Cys595, Phe604, and Thr605 Replacements in TMH 7 of LHR1

 
Additional studies focused on Asn593 and Asn597 for several reasons. The N593A mutant exhibited interesting properties in that expression was greater than that of WT LHR, yet signaling was diminished. Also, we showed earlier that the two Asns in TMH 7 mapped to a face of the helix that contained the other side chains with hydrogen bond donor or acceptor function, e.g. Tyr590, Ser594, Tyr601, and Thr605, and suggested that this region may be involved in ligand-mediated signaling (17). Finally, using molecular modeling Lin et al. (11) suggested that Asp556 in TMH 6 may interact with the two Asns in TMH 7 as part of receptor activation.

In these studies, as with the C595M, F604Y, and T605Y LHR mutants, signaling was based on measurements of basal cAMP and maximally hCG-stimulated cAMP. As shown in Table 3Go, the Asn593->Ala, Gln, and Ser replacements yielded LHR mutants that expressed at levels comparable to WT LHR, yet responsiveness to hCG was blunted; the Asn593->Lys replacement exhibited properties like those of WT LHR. The Asn593->Asp and Glu replacements resulted in reduced expression and responsiveness, the latter perhaps attributable to the low receptor number and/or inherent loss of function. Of considerable interest is the Asn593->Arg substitution. This LHR mutant, which expresses at a level comparable to WT LHR, is constitutively active and responsive to hCG. Figure 4Go shows the basal and stimulated cAMP levels for two mutant forms of LHR relative to WT LHR and represents a loss-of-function mutant, N593A, and a gain-of-function mutant, N593R. Comparable studies were performed with Ala, Gln, Asp, Lys, and Arg replacements of Asn597 (Table 3Go). Compared with WT LHR, the N597A, N597D, and N597K mutants expressed at lower levels and exhibited reduced responsiveness to hCG; basal cAMP values were similar to that of WT LHR. The N597R mutant exhibited a basal cAMP level like that of WT LHR, but responded poorly to hCG. Thus, the Asn597->Arg replacement represents a loss-of-function mutant. The N597Q mutant expressed at a level comparable to that of WT LHR and, like the N593R mutant, exhibited constitutive receptor activation as judged by the increase in basal cAMP; moreover, responsiveness to hCG was like that of WT LHR. The basal and cAMP levels of the N597R (loss-of-function mutant) and the constitutively active mutant, N597Q, are shown in Fig. 5Go.


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Table 3. Expression and Signaling Parameters of Asn593 and Asn597 Replacements in TMH 7 of LHR1 and Relative Energies from Energy Minimization of TMHs 6 and 7 Interactions

 


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Figure 4. Basal and Maximal cAMP Levels for LHR Mutants in TMH 7 with Replacements at Asn593

The two-mutant forms of LHR have comparable receptor densities (cf. Table 3Go): one is a loss-of-function mutant (N593A) (A), and the other is a gain-of-function mutant (N593R) (B). The cAMP levels measured in the presence of hCG have not been corrected for basal levels.

 


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Figure 5. Basal and Maximal cAMP Levels for LHR Mutants in TMH 7 with Replacements at Asn597

The two mutant forms of LHR have similar receptor levels (cf. Table 3Go). The N597R mutant represents a loss-of-function mutant (A), and N597Q is a gain-of-function mutant (B).

 
Energy minimization of the interaction between TMHs 6 and 7 gave the results presented in Table 3Go. Since the calculations are based on charged side chains of the amino acid residues, Asp, Glu, Lys, and Arg, it is not surprising that Asp-Lys and Asp-Arg interactions appear to be more favorable, e.g. -18 to -30 kcal/mol relative to the putative Asp-Asn interactions in WT LHR (11). Figure 6Go shows a side-view schematic of the energy-minimized structures of native TMHs 6 and 7. This structure was used as a starting point to evaluate the effect of replacement of Asn593 and Asn597 with Gln and Arg to give the four point mutants, N593Q, N593R, N597Q, and N597R. These were chosen since N593R and N597Q are constitutively activating mutants and N593Q and N597R are loss-of-function mutants. The appropriate amino acid residues were substituted in TMH 7, and the new helix 6-helix 7 interaction was minimized. The results of these calculations, along with WT LHR as control, are presented in Fig. 7Go as viewed from the cell exterior toward the interior. This vantage point was chosen since there is little shift in the vertical arrangement of the helices in the energy-minimized structures of these four mutants, but noticeable reorientation, e.g. twisting or turning, does occur. The Gln replacements, not surprisingly, are accommodated more readily than the Arg replacements. The two activating mutations, N593R and N597Q, lead to dramatic shifts in TMHs 6 and 7, respectively. The two loss-of-function mutants, N593Q and N597R, also lead to helix reorientation, but of a somewhat different nature. The Arg597 side chain, in particular, cannot be fully extended as can the Arg593 side chain, and thus projects into the interior of the helical array presenting a hydrogen bond donor to this microenvironment. In all cases, reorientation of TMHs 6 and 7 can alter the conformation of the third intracellular and extracellular loops, as well as the cytoplastic tail of LHR.



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Figure 6. Schematic Model of TMHs 6 and 7 in WT LHR Emphasizing the Predicted Interhelical Interactions

A side view of the energy-minimized structure of TMHs 6 and 7 with the putative interaction (11 ) between Asp556 (TMH 6) and Asn593/Asn597 (TMH 7) highlighted.

 


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Figure 7. Schematic Models of TMHs 6 and 7 in WT and Four Mutant LHRs

A top view, i.e. from the cell exterior toward the cytoplasmic side, between positions 556 and 593/597 for WT LHR (A) and the four point mutants N593Q(B), N593R(C), N597Q(D), and N597R(E). All putative hydrogen bonds between D556 and N593/N597, Q593/N597, R593/N597, N593/Q597, and N593/R597 were between 2.6–2.7 Å; moreover, the N593R mutant is predicted to have an intramolecular hydrogen bond, between R593 and N597 of 2.8 Å.

 
In summary, the Asn593->Ala, Gln, and Ser and Asn597->Arg replacements represent loss-of-function mutants; the Asn593->Asp, Glu and the Asn597->Ala, Asp, and Lys replacements may as well, but the reduced level of receptor expression compromises such a conclusion. The Asn593->Arg and Asn597->Gln replacements yield constitutively activating LHR mutants that can also respond to hCG.

As indicated by molecular modeling (11), there are large interhelical contact surfaces between TMHs 3 and 4 and potential interactions between Glu429 in TMH 3 and Ser472 in TMH 4; moreover, Asn513, one of the few polar amino acid residues in TMH 5, is unique in the three glycoprotein hormone receptors and may interact with TMH 6. Thus, we investigated the functional roles of Glu429 and Asn513 in TMHs 3 and 5, respectively, by site-directed mutagenesis. These two amino acid residues are invariant in the three glyco-protein hormone receptors (2). As shown in Table 4Go, the E429D LHR mutant expressed poorly and, not surprisingly, the responsiveness to hCG was minimal. Other replacements with Ala and Gln yielded mutants that gave expression levels and Rmax values comparable to those of WT LHR. The E429S mutant exhibited a reduction in both expression and signaling. The Asn513->Gly, Ala, Gln, Leu, Asp, and His replacements resulted in receptor mutants that expressed poorly and failed to respond well to hCG as monitored by Rmax values. There was no evidence of constitutive action in any of the Glu429 or Asn513 LHR mutants, and the reduced expression levels for many of the replacements preclude any conclusion regarding loss of responsiveness.


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Table 4. Expression and Signaling Parameters of Glu429 (TMH 3) and Asn513 (TMH 5) Replacements of LHR1

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
This study has identified three amino acid residues of TMH 7 of LHR that appear to be involved in ligand-mediated signaling, Asn593, Cys595, and Asn597. In earlier work we identified similar roles for Pro591 and Tyr601, but not Tyr590 (16). Thus, of the six side chains in TMH 7 that map to a common face of the helix and exhibit potential hydrogen bond donor and/or acceptor function, Tyr590, Asn593, Ser594, Asn597, Tyr601, and Thr605, three seem to have a functional role in signaling. Interestingly, the three functional side chains, Asn593, Asn597, and Tyr601, are in close proximity to each other. Pro591 and Cys595 map to a common segment on the extended hydrophobic face of TMH 7 opposite that of the Asn593/Asn597/Tyr601 patch. The role(s) for Pro591 and Cys595 may be structural only; alternatively, the peptide carbonyl group of proline and the side chain of cysteine may serve as hydrogen bond acceptor and donor, respectively. It is quite possible that these two opposite faces of TMH 7 may be required for helix reorientation in ligand-mediated signaling. The naturally occurring loss-of-function human LHR mutant, S616Y (Ser594 in rat LHR), has been reported to result in poor cell surface localization (16, 26, 27). Our results on the S594A mutant in rat LHR show that WT-like expression and function occur; thus, the bulky side chain of tyrosine probably interferes with proper helix packing and perhaps trafficking to the cell surface. There is no evidence that the hydroxyl group of this serine is essential for signaling.

The most surprising and significant observations from the current study are that replacement of either Asn593 or Asn597 can lead to loss-of-function mutants (Ala593, Gln593, Ser593, and Arg597) or to constitutively activating mutants (Arg593 and Gln597). The observation that replacement of a given side chain can result in either loss of function or gain of function, depending upon the nature of the side chain, is indeed intriguing. The importance of Asp556 (Asp578 in human LHR) in TMH 6 has been well documented in LHR function (18); our results argue for pivotal roles, albeit less dramatic, of Asn593 and Asn597 as well. The most common naturally occurring mutation leading to familial or sporadic male-limited precocious puberty is replacement of Asp in TMH 6 with Gly, which results in a 4.7-fold increase in basal cAMP over that of WT LHR (18). However, not all naturally occurring gain-of-function mutations increase basal cAMP to this extent. The A572V mutation in TMH 6 of human LHR also leads to male-limited precocious puberty, but gives only a 3-fold increase in basal cAMP levels (30). The fold-increase in basal cAMP levels noted with the N593R mutant is comparable to this value.

A naturally occurring mutation in TMH 7 of the TSH receptor, Asn670->Ser (corresponding to position 593 in rat LHR), was found to lead to constitutive activation (31). Interestingly, in rat LHR we found that the same replacement led to a loss-of-function mutant. Replacement of Asn593 with Arg yields a gain-of-function LHR mutant and responsiveness to hCG; hence, the Arg side chain, in addition to its role in constitutive activation, is able to mimic Asn in LHR responsiveness to hCG. Likewise, Gln behaves similarly when replacing Asn597.

It has been suggested that the inactive conformation of the LHR is stabilized by interactions involving Thr555 and Asp556 in TMH 6 with Asn593 and Asn597 in TMH 7 and that weakening the hydrogen bonds between them can cause constitutive receptor activation (11). Our results on Ala replacements of Asn593 and of Asn597 are not totally consistent with this model; otherwise, one of the individual Ala replacements should lead to constitutive receptor activation, and this was not observed. It is of interest that the formation of an intrahelical hydrogen bond between Asn593 and Asn597 in TMH 7 may be required for complete ligand-mediated activation of LHR, and, if so, it is not surprising that Ala replacements of either of the two asparagines results in a loss-of-function mutant. (We prepared a double mutant of LHR, N593A/N597A, but it failed to express at sufficiently high levels for evaluation.) The close proximity of Asn593 and Asn597 is demonstrated in Fig. 1AGo. Movement of TMH 6 has been proposed to be involved in the activation process (11), and our results strongly argue that TMH 7 is reoriented as well.

The results of molecular modeling and energy minimization are based on ionizable side chains of Asp, Glu, Lys, and Arg residues. In bacteriorhodopsin, water is present in the interhelical channel, and many of the ionizable side chains are charged (32, 33, 34). Unfortunately, no comparable data exist for LHR. The energy minimizations were also performed considering only interhelical interactions between 6 and 7. The important contributions of other TMHs and bound phospholipids, as well as the constraints imposed by the third intracellular and extracellular loops, were not included in the calculations. Thus, the energies reflect a localized interaction of TMHs 6 and 7. Not surprisingly, Lys and Arg replacements of Asn at positions 593 and 597 in TMH 7 yielded the lowest energies resulting from the formation of ion-ion interactions with Asp556 in TMH 6, while Asp and Glu replacements gave higher energies due to charge repulsion. These results may of course be misleading if the side chains are not ionized. Gln substitutions of Asn at positions 593 and 597 are only slightly less favored than Asn, e.g. 2–4 kcal/mol higher energy. The other replacements such as Ala at positions 593 and 597 and Ser at 593 result in unfavorable energies of 18–30 kcal/mol.

Overall, the predicted energies do not correlate with the functional data. For example, N593R, but not N593K, results in constitutive activation of the LHR mutant, and N597R, but not N597K, leads to a loss-of-function mutant; yet, the energies are comparable. Likewise, N593Q produces a loss-of-function mutant, while N597Q gives constitutive activation. The results of energy minimization, however, argue strongly for helix reorientation and perhaps a minimal displacement concomitant with certain amino acid residue replacements at positions 593 and 597 in TMH 7. Such changes in relative conformation of one or both helices presumably account for the different functional data found such as loss of function or gain of function; moreover, structural changes may adversely affect expression levels of the receptor.

The simplest scheme that has been proposed to account for G protein-coupled receptor function involves two conformations, one inactive, Ro, and one active, R*, that can undergo interconversion, Ro<- R*. In this model, ligand is assumed to have a low affinity for the Ro conformation and a high affinity for that of R*. Thus, in the presence of ligand, the equilibrium is shifted toward the right, resulting in increased signaling. In the absence of ligand, most of the receptors are in the Ro conformation; the occasional conformational conversion to R* results in a non-zero level of basal cAMP. (In the case of LHR and COS-7 cells, however, we do not find a strong correlation between basal cAMP levels and receptor density at the levels at which we normally express.) A somewhat more complex scenario would involve, rather than a cooperative transition between Ro and R*, a series of intermediate steps (35) as depicted in the following equation, where each intermediate conformation is somewhat more active than the preceding one and j denotes a general intermediate form of which there may be many: R0 {leftrightarrow} R1 {leftrightarrow} R2 {leftrightarrow} Rj {leftrightarrow} R*.

In this model, one could argue that a given constitutively active mutation would convert Ro to a more active form, e.g. R1, R2, or Rj, and that addition of ligand would yield the L·R* state characterized by maximal cAMP production, as obtained, for example, with WT receptor.

We have identified LHR mutants that exhibit WT-like basal cAMP production but cannot respond fully to ligand, i.e. loss-of-function mutants. This may represent a case of the mutant receptor in state Ro that can adopt either state R1, R2, or Rj, but not R*, in the presence of ligand. We have characterized other mutants that are constitutively active and highly responsive to ligand. These mutants may adopt state R1 or R2 in the absence of ligand and thus exhibit an elevated level of cAMP, and furthermore they may be able to complete all of the intermediate steps to form a WT-like L·R* complex.

Realizing that replacement of transmembrane amino acid residues with quite different structures, e.g. Asn593->Arg in TMH 7, may lead to a conformation that cannot be adopted by WT LHR, a more general representation involving separate schema for loss-of-function and constitutively activating mutations may be more appropriate. Also, it has been proposed, based upon strong supporting experimental evidence, that the native conformation of hCG is not required for activity (36); it seems reasonable that a number of conformations of LHR involving the region encoded by exon 11, i.e. the transmembrane helices and intra/extracellular loops, are possible. Another consideration is that the ligand may induce one or more receptor conformations. Finally, the different rates of internalization reported for gain-of-function LHR mutants (37, 38), compared with WT LHR, may also be a factor.

In conclusion, considering our results on Pro591 and Tyr601 from earlier work (17), we have identified three amino acid residues in close proximity on one face of TMH 7 of LHR, Asn593, Asn597, and Tyr601, and two residues in close proximity on the opposite face of the helix, Pro591 and Cys595, that are involved, either directly or indirectly, in ligand-mediated signaling. In addition, a novel and unusual finding was that replacement of Asn593 or Asn597 can result in either a loss-of-function or a gain-of-function mutant, depending upon the nature of the side chain. Molecular modeling and energy minimization indicated a reorientation and/or displacement of one or both TMHs 6 and 7 concomitant with certain functional replacements of D556, N593, or N597. It seems likely that a reorientation of TMHs 6 and 7 of LHR is required for proper signaling in WT LHR. Finally, we found no evidence for a role of Glu429 in TMH 3, consistent with the earlier finding based on a Glu429->Gln replacement (39). Asn513 appears to be important in receptor folding and/or membrane trafficking, but there is no evidence that it is involved in ligand-mediated signaling.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture and Transient Transfection
COS-7 cells, obtained from American Type Culture Collection (Manassas, VA), were grown in a monolayer culture in DMEM supplemented with 10% (vol/vol) FBS, 50 U/ml penicillin, 50 µg/ml streptomycin, 50 µg/ml gentamycin, and 0.125 µg/ml Amphotericin (Life Technologies, Inc., Gaithersburg, MD). Cells were maintained at 37 C in humidified air containing 5% CO2 and transiently transfected with 10 µg of the WT or mutant cDNA using Lipofectamine as recommended by Life Technologies (Gaithersburg, MD).

Mutagenesis of LHR
Mutagenesis of rat LHR, cloned in the expression vector pSVL, was performed by the in vitro site-directed mutagenesis and Quick Change site-directed mutagenesis kits as recommended by CLONTECH Laboratories, Inc. (Palo Alto, CA) and Stratagene (La Jolla, CA), respectively. To overcome the low expression levels associated with the Asn597->Gln replacement, we replaced the LHR signal sequence with that corresponding to hCGß followed by the carboxy-terminal peptide of hCGß. Mutant clones were identified by sequencing using the Sequenase Version 2.0 DNA sequencing kit (Amersham Pharmacia Biotech, Arlington Heights, IL). Mutant cDNAs were amplified, and the QIAGEN (Chatsworth, CA) plasmid maxi kit was used to obtain purified DNA.

Hormone Binding to Transfected Cells
About 16–18 h after transfection, the COS-7 cells were replated (5 x 105 cells per well) into six-well tissue culture plates and assayed for binding 24 h later. 125I-hCG (50 pM, DuPont NEN, Boston, MA) and increasing concentrations of hCG were added to each well for competitive binding assays; nonspecific binding was determined by addition of 1000-fold excess of unlabeled hormone. All determinations were performed in duplicate, and, unless stated otherwise, the data are given as mean ± SEM of two to eight independent transfections.

cAMP Assay
About 16–18 h after transfection, cells were replated (1 x 105 cells per well) into 12-well tissue culture plates. After 24 h, the cells were incubated with increasing or maximal (100 ng/ml) concentrations of hCG for 30 min at 37 C in the presence of 0.8 mM isobutylmethylxanthine (Sigma, St. Louis, MO). Incubation medium was then removed and the cells lysed in 100% ethanol at -20 C overnight. The extract was collected, dried under vacuum, and resuspended in the buffer of the 125I-cAMP assay kit. cAMP concentrations were determined by RIA as recommended by DuPont NEN. Duplicate determinations were made for each experiment, and the results are presented as mean ± SEM of two to eight independent transfections unless stated otherwise.

Data Analysis
Both binding and cAMP data were analyzed by the Prism software (Graph Pad Software, San Diego, CA). To compare the expression levels of WT and mutant LHRs, the specific binding of the WT receptor was normalized to 100%, and the specific binding of each of the mutants was given relative to that value for each transfection. For purposes of comparison of the signal transduction potency of the WT and mutant receptors, the maximal hCG-mediated cAMP production over the basal levels was normalized to 100% for the WT receptor in a given transfection, and the value obtained with the different receptor mutants, corrected for basal level, was expressed as a percentage of that of WT receptor. Basal cAMP levels are given as picomoles/ml for WT LHR and all mutants. Significance was determined by an unpaired two-tailed Students t test, with 95% confidence limits (P < 0.05).

Molecular Modeling
All molecular modeling was performed with SYBYL 6.5 Release (Tripos Associates, Inc., St. Louis, MO). TMHs 6 and 7 were constructed as individual helical segments with the following backbone torsion angles for all residues except proline: ø = -58o, {psi} = -47o, and {Omega} = 180o. Proline residues were incorporated with a ø angle of -75o with {psi} and {Omega} identical to the other amino acid residues in the helical segments. Each TMH was then minimized using the Kollman All-Atom force field using Kollman point charges. The nonbonded cutoff was set a 8.0 Å, and the default distance dielectric function was used with a dielectric constant of 1.0. Minimization was automatically terminated when the gradient fell below 0.05 kcal/mol.

The energy-minimized helices were positioned adjacent to each other such that Asn593 and Asn597 were located within hydrogen bonding distance (<3 Å) to Asp556, and the helical axes were parallel. The interacting helices were then minimized as above with the assumption that the ionizable side chains are charged. This construct is similar to that proposed by Lin et al. (11) and was used as the native, one-state model for comparison with residue replacements at positions 593 and 597.


    ACKNOWLEDGMENTS
 
We thank Drs. Neil Bhowmick and Adrian Lapthorn for their interest and assistance in the initial modeling studies of TMH 7.


    FOOTNOTES
 
Address requests for reprints to: Dr. David Puett, Department of Biochemistry & Molecular Biology, Life Sciences Building, Green Street, University of Georgia, Athens, Georgia 30602-7229.

This work was supported by NIH Research Grant DK-33973.

Received for publication September 27, 1999. Revision received December 19, 1999. Accepted for publication January 5, 2000.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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