Role of the Third Intracellular Loop for the Activation of Gonadotropin Receptors

Angela Schulz, Torsten Schöneberg, Ralf Paschke, Günter Schultz and Thomas Gudermann

Institut für Pharmakologie (A.S., T.S., G.S., T.G.) Freie Universität Berlin 14195 Berlin, Germany
Medizinische Klinik und Poliklinik III (R.P.) Universität Leipzig 04103 Leipzig, Germany


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Hyperfunctional endocrine thyroid and testicular disorders can frequently be traced back to gain-of-function mutations in glycoprotein hormone receptor genes. Deletion mutations in the third intracellular (i3) loop of the TSH receptor have recently been identified as a cause of constitutive receptor activity. To examine whether the underlying mechanism of receptor activation applies to all glycoprotein hormone receptors, we created deletion mutations in the LH and FSH receptors. In analogy to the situation with the TSH receptor, a deletion of nine amino acids resulted in constitutive activity irrespective of the location of deletions within the i3 loop of the LH receptor. In contrast, only one ({Delta}563–566) of four different 4-amino acid deletion mutants displayed agonist-independent activity. Systematic examination of the structural requirements for this effect in the {Delta}563–566 mutant revealed that only deletions including D564 resulted in constitutive receptor activity. Replacement of D564 by G, K, and N led to agonist-independent cAMP formation while introduction of a negatively charged E silenced constitutive receptor activity, indicating that an anionic amino acid at this position may be required to maintain an inactive receptor conformation. Insertion of A residues up- and downstream of D564 did not perturb receptor quiescence, showing that a certain degree of spatial freedom of the negatively charged amino acid within the context of the i3 loop is well tolerated. In contrast to the results obtained with the LH receptor, deletion of the corresponding D567 from the i3 loop of the FSH receptor did not cause constitutive receptor activation, highlighting significant differences in the activation mechanism of gonadotropin receptors.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
According to the allosteric ternary complex model, G-protein-coupled receptors (GPCRs) exist in an equilibrium between inactive and active receptor conformations (1, 2). The model predicts that, even in the absence of agonist, a certain fraction of receptors will spontaneously adopt an active conformation, permitting agonist-independent G-protein activation. In keeping with this current paradigm, some receptors such as D1B dopamine and TSH receptors display significantly elevated basal activity even in the unliganded state when compared with other GPCRs (3, 4). To describe mutated receptors characterized by a shift of the isomerization equilibrium toward active conformations, the term constitutive activity has been coined. By definition, such receptors evoke clearly discernible second messenger production in the absence of agonist.

Mutational activation of GPCRs has been identified as a pathophysiological mechanism of human diseases such as retinitis pigmentosa (5), familial male-limited precocious puberty (6), and autonomous toxic thyroid adenomas (7). To date, a fair number of gain-of-function mutations in the receptors for the glycoprotein hormones TSH and LH have been identified (8, 9), providing valuable information on structural requirements of receptor quiescence. Therefore, the subfamily of glycoprotein hormone receptors may serve as a particularly suitable model system to study conformational changes accompanying receptor activation.

The structure of glycoprotein hormone receptors is predicted to consist of a large extracellular hormone-binding domain connected to a transmembrane (TM) core that shares a common molecular architecture with other GPCRs. The TM core is composed of seven {alpha}-helical TM domains (TM1–7) connected by three extracellular and three intracellular loops (10). Most missense mutations leading to constitutively active TSH or LH receptors (TSHRs or LHRs) are located in the C-terminal three TMs. It is hypothesized that interhelical interactions between TM5/TM6 and TM6/TM7 stabilize the inactive state of glycoprotein hormone receptors and that gain-of-function mutations have a destabilizing impact on these contacts (11).

We have recently shown that the deletion of amino acid residues from the third intracellular (i3) loop of the TSHR results in agonist-independent receptor activation (12). This constitutive activity is independent of the exact position of the deleted amino acids, yet is influenced by the length of the deleted region. Since previously identified gain-of-function mutations in intracellular receptor portions were point mutations, our finding highlights a new mechanism of TSHR activation. Primary sequence comparison of glycoprotein hormone receptors reveals an overall amino acid identity of approximately 50% and of more than 70% among putative TM helices (13). The remaining structural differences most likely impinge upon receptor activation and signal transduction properties that display characteristic differences between members of the glycoprotein hormone receptor family (4, 13, 14). Our previous observation, that deletions within the i3 loop constitutively activate the TSHR, prompted us to study whether such an activation mechanism would be a general phenomenon and apply to all glycoprotein hormone receptors. Thus, we systematically studied the importance of the i3 loop for the activation of the LHR and FSH receptor (FSHR). We demonstrate in the present study that similar to the TSHR, large deletion mutations in the i3 loop of the LHR result in constitutive activation. In contrast to findings obtained with the TSHR, however, deletions of five ({Delta}558–562) or four amino acids ({Delta}563–566) resulted in a similar agonist-independent receptor activity as noted for the 9-amino acid ({Delta}558–566) deletion mutant. To analyze the structural requirements for constitutive receptor activation, various LHR deletion mutants were tested. Interestingly, an active conformation was only achieved if a conserved D residue at position 564 was included in the deleted region. Further analysis of the functional role of D564 revealed that the presence of a negatively charged amino acid at this position is required to stabilize an inactive conformation. Similar mutational studies with the FSHR did not allow the design of a receptor with comparable constitutive activity, thus suggesting a more constrained inactive conformation and an activation mechanism clearly set apart from the other two glycoprotein hormone receptors.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Functional Characterization of LHR Deletion Mutants
Using a site-directed mutagenesis approach we systematically introduced deletion mutations varying in length and location into the i3 loop of the LHR. Functional analysis of a 9-amino acid deletion ({Delta}558–566) equivalent to a deletion mutation found in the i3 loop of the TSHR in a toxic thyroid nodule (15) resulted in a 2.6-fold elevation of basal cAMP levels when compared with the wild-type LHR [LHR(wt)]. Concomitantly with constitutive receptor activity, we observed a reduced maximal cAMP response [~40% of the response seen with the LHR(wt)] to stimulation with 100 nM human CG (hCG) (Table 1Go).


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Table 1. Functional Characterization of LHR Mutants

 
This observation may relate to the significantly reduced membranous expression of LHR deletion mutants (see Table 1Go). Constitutive receptor activation was also observed after shifting the 9-amino acid deletion toward the N-terminal loop portion ({Delta}554–562), demonstrating that a 9-amino acid deletion does not have to be exactly positioned at a certain location within the i3 loop to cause constitutive receptor activation (see Table 1Go). Since both LHR deletion mutants displayed functional properties similar to the corresponding TSHR mutants (12), we set out to study in depth the importance of distinct regions of the i3 loop for maintaining an inactive state of the LHR.

The {Delta}558–566 deletion spans a stretch of amino acids that can be subdivided into a C-terminal portion highly conserved among glycoprotein hormone receptors and a less conserved N-terminal part (see Fig. 1Go). In the absence of the less conserved 5 ({Delta}558–562) or of the 4 conserved amino acids ({Delta}563–566), the mutant receptors displayed comparable constitutive activity. Maximal hCG-induced cAMP levels amounted to 55% of the LHR(wt) control (see Table 1Go). In contrast to previous findings with the TSHR, the extent of constitutive receptor activation was equivalent to the one observed with the 9-amino acid deletion mutant ({Delta}558–562). Next we asked whether a 4-amino acid deletion per se or the exact location of this deletion within the i3 loop would account for constitutive activity. Thus, we generated three additional 4-amino acid deletion mutants located in the very N-terminal ({Delta}550–553, {Delta}554–557) and C-terminal ({Delta}567–570) portions of the i3 loop. None of these mutant LHRs displayed elevated basal cAMP levels. Maximal hCG-induced cAMP levels, however, were markedly reduced. In accord with the latter findings, all four-amino acid deletion mutants showed decreased membranous expression levels in binding studies (see Table 1Go). Whereas membrane expression of most 4-amino acid deletion mutants amounted to approximately 16% of wt receptor levels, expression of the deletion mutant {Delta}567–570 located at the i3/TM6 transition was hardly detectable (see Table 1Go).



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Figure 1. Localization of LHR Mutations within the i3 Loop

Putative arrangement of the LHR within the lipid bilayer highlighting amino acid sequences of the third intracellular loop of the wt human LHR and the mutants constructed. Deleted amino acids are indicated by dashes; amino acid substitutions are highlighted in italics, and amino acid residues that are conserved within the group of glycoprotein hormone receptors are shown in boldface. Acronyms for each mutation are indicated on the left.

 
The results obtained so far indicated that the amino acid cluster 563–566 contains determinants responsible for maintaining the inactive receptor conformation. Subsequently, we attempted to identify relevant structural elements within this portion by further dissecting the 563–566 portion of the i3 loop, deleting only 2 and then single amino acids. Membrane insertion of the 2-amino acid deletion mutants {Delta}563–564 and {Delta}565–566 was 45.9 and 27.6% of wt levels, respectively, and hCG stimulation yielded similar maximal cAMP levels (see Table 1Go). However, only the mutant LHR-{Delta}563–564 displayed constitutive activity (see Table 1Go). Functional analysis of the single-amino acid deletion mutants LHR-{Delta}563 and -{Delta}564 proved that the absence of a D at position 564 is responsible for the constitutive activity (see Table 1Go).

To test whether the agonist hCG elicited cAMP formation in cells expressing the LHR(wt) or various constitutively active mutants with different potencies, concentration-response studies were performed. As shown in Fig. 2Go, EC50 values for the LHRs that were investigated differed at most by a factor of 2.6 [LHR(wt): 0.31 nM; {Delta}558–566: 0.44 nM; {Delta}563–566: 0.41 nM; {Delta}563–564: 0.87 nM; {Delta}564: 0.79 nM].



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Figure 2. Concentration-Dependent hCG-Stimulated cAMP Accumulation in COS-7 Cells Expressing the wt and Constitutively Active Mutant LHRs

cAMP accumulation assays were performed in COS-7 cells transiently transfected with wt and mutant LHR plasmids. Cells were incubated with increasing concentrations of hCG, and increases in cAMP levels were determined as described in Materials and Methods. All data are presented as means (-fold of basal cAMP levels of the wt LHR) of two independent experiments each performed in duplicate.

 
Mutational Characterization of Amino Acid Position 564
To further characterize the importance of D564 for receptor quiescence, we replaced D564 by an E residue. Expression of LHR-D564E did not reveal signs of constitutive receptor activity, but rather led to a reduced basal cAMP formation when compared with the LHR(wt) (see Table 1Go). Next, we replaced the free carboxyl moiety of D564 by the corresponding carboxyl amide by changing D to N (D564N). In contrast to the results obtained with the D564E mutant, LHR-D564N showed ligand-independent cAMP accumulation. Introduction of a noncharged G and a positively charged K at position 564 conferred constitutive activity upon the mutated receptor (see Table 1Go). Maximal hCG-stimulatable cAMP formation, as well as membranous receptor expression of the latter three missense mutants, closely resembled the situation encountered in the case of wt receptor expression (see Table 1Go).

Next, we speculated that the immediate conformational environment within the i3 loop of position D564 may have a bearing on the activity state of the receptor. To address this issue, we inserted three additional A residues upstream (insA562) or downstream (insA564) of D564. None of these mutations entailed significant changes in basal or in agonist-induced intracellular cAMP levels upon functional expression of the respective receptors (see Table 1Go).

Screening for a Basic Amino Acid in the i2 Loop That Interacts with D564
In a first attempt to identify a positively charged amino acid potentially involved in a salt bridge with D564, all conserved basic amino acids in the second intracellular (i2) loop were replaced by A residues, and the resulting mutants were tested for constitutive activity (Fig. 3AGo). None of the tested LHR mutants imparted constitutively elevated basal cAMP levels on the transfected cells. All i2 mutants were functionally expressed at the cell surface as illustrated by maximal agonist-dependent cAMP formation that resembled that observed with the LHR(wt) (Fig. 3BGo).



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Figure 3. Functional Analysis of Conserved Basic Amino Acids within the LHR i2 Loop by Alanine Scanning Mutagenesis

To identify a cationic contact site for D564, all conserved basic amino acids within the i2 loop were replaced by A using a site-directed mutagenesis approach (A). The various LHR mutants were expressed in COS-7 cells, and cAMP accumulation assays were performed as described in Materials and Methods (B). Basal (open bars) and agonist-induced cAMP levels (100 nM hCG; filled bars) are presented as means ± SEM of two independent experiments, each carried out in triplicate.

 
hCG-Induced Phospholipase C Activation
To study the influence of constitutively activating mutations on the phospholipase C effector system (13, 16), we examined all LHR mutants for their ability to generate inositol phosphates (IPs). Expression of the LHR(wt) consistently led to a 3- to 4-fold increase in IP levels after stimulation with 100 nM hCG (see Table 1Go). Most of the LHR mutants ({Delta}558–566, {Delta}563–566, {Delta}550–553, {Delta}554–557, and {Delta}567–570) that were expressed at considerably lower receptor densities than the wt receptor did not respond to agonist challenge with significant IP formation. It should be noted, however, that none of the mutant LHRs showed constitutive activity toward the IP-signaling pathway. Surprisingly, expression of several receptor mutants that constitutively activated the Gs/adenylyl cyclase pathway ({Delta}564, D564G, D564K, and D564N) was accompanied by a profound enhancement of maximal hCG-induced IP accumulation. However, we had to abandon our initial suspicion of a causal relationship between ligand-independent cAMP production and a more efficient coupling to the phospholipase C system, because expression of additional mutant receptors (insA562 and insA564) not constitutively activating Gs also entailed a marked increase of maximal agonist-induced IP levels when compared with the wt receptor (Fig. 4Go and Table 1Go). These observations reflect an increased efficacy of the agonist hCG acting at the mutant receptors. On the contrary, the EC50 values of the wt and mutant LHRs [LHR(wt): 7.0 nM; {Delta}564: 11.2 nM; D564E: 7.2 nM; insA562: 7.4 nM; insA564: 7.2 nM] showed that there was no significant change in the potency of hCG to stimulate phospholipase C via various LHR mutants (see Fig. 4Go).



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Figure 4. hCG-Stimulated IP Accumulation in COS-7 Cells Expressing the wt and Selected Mutant LHRs

Determination of IP levels was performed in COS-7 cells, transiently transfected with wt and mutant LHR plasmids. Cells were incubated with increasing hCG concentrations, and increases in IP levels were determined as described in Materials and Methods. All data are presented as means (-fold of basal) of two independent experiments each performed in duplicate.

 
Expression and Characterization of FSHR i3 Loop Deletion Mutants
Since deletions within the i3 loop of two glycoprotein hormone receptors, i.e. TSHR and LHR, can result in constitutive activity, we sought to investigate whether such a receptor activation mechanism also applies to the third member of this receptor subfamily, the FSHR (Fig. 5AGo). Expression of FSHR-{Delta}561–569 did not confer elevated basal cAMP levels to transfected COS-7 cells (Table 2Go and Fig. 5BGo). The mutant receptor showed a poor membranous expression of [125I]FSH binding sites and responded only weakly to porcine (p) FSH (800 nM). Subsequently, we omitted only the C-terminal 3 amino acids (to preserve the four-S stretch in the FSHR i3 loop) from the conserved portion of the original 9-amino acid deletion to generate FSHR-{Delta}567–569 (see Fig. 5AGo). In addition, we deleted D567 corresponding to D564 in the LHR (see Fig. 5AGo). Whereas FSHR-{Delta}567–569 showed a drastically reduced membranous expression of binding-competent receptors and poorly responded to FSH challenge (see Table 2Go and Fig. 5BGo), deletion of D567 resulted in a mutant receptor that did not constitutively couple the mutant FSHR to the Gs/adenylyl cyclase system. Subsequently, we replaced D567 in the i3 loop by a G residue to generate FSHR-D567G, which has been reported to epitomize the only constitutively active FSHR known so far (17). If anything, expression of FSHR-D567G showed the tendency to slightly increase basal cAMP production, although this effect was not significant (see Table 2Go). Taking into account, however, that membrane expression of binding-competent receptors after transfection of COS-7 cells with FSHR-D567G cDNA is reduced by 60% when compared with FSHR(wt) (see Table 2Go), we cannot exclude with certainty that functional characteristics of the latter mutant may indeed mirror some degree of constitutive activity. Upon FSHR(wt) expression in COS-7 cells, we did not observe FSH-stimulatable phosphoinositide breakdown. Therefore, this signaling pathway was not analyzed further for the various FSHR mutants.



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Figure 5. Localization of FSHR Mutations and Functional Characterization of Mutant Receptors

To characterize mutant FSHRs (A), COS-7 cells were transfected with the different constructs, and cAMP accumulation assays were performed as outlined in Materials and Methods. (B) Data obtained from three independent experiments, each performed in triplicate, are presented as -fold of basal cAMP levels of the FSHR(wt) (means ± SEM).

 

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Table 2. Functional Characterization of FSHR Mutants

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We recently identified deletion mutations within the i3 loop of the TSHR resulting in marked constitutive receptor activation (12). As the number of deleted amino acids, rather than their exact position within the i3 loop, is the major determinant of constitutive TSHR activation and because 75% of the i3 loop could be deleted without rendering the receptor unresponsive to agonist, we speculated that the deletion per se led to constitutive receptor activity by allowing critical amino acid residues within TM6 to productively interact with Gs (12, 18). To investigate whether this activation mechanism applied to all glycoprotein hormone receptors, we functionally characterized i3 loop deletion mutants of the human LHR and FSHR.

Functional analysis of an LHR 9-amino acid deletion ({Delta}558–566) corresponding to the one originally described in the TSHR (12), as well as of an N-terminally shifted deletion ({Delta}554–562), revealed pronounced agonist-independent cAMP formation, thereby supporting a mechanism of receptor activation initially worked out for the TSHR (12). Sequence comparison within the family of glycoprotein hormone receptors reveals that the 9-amino acid deletion ({Delta}558–566) can be subdivided into a highly conserved C-terminal and a less conserved N-terminal portion. In contrast to previous findings with the TSHR, deletion of the nonconserved ({Delta}558–562) or conserved ({Delta}563–566) stretch of amino acids from the LHR i3 loop did not result in a reduction of constitutive activity when compared with the original {Delta}558–566 mutant. These initial findings prompted us to hypothesize that, in the LHR, deletion of 4 amino acid residues may already be sufficient for maximal constitutive receptor activity. However, expression of only one 4-amino acid deletion mutant (LHR-{Delta}563–566) of 4 ({Delta}550–553, {Delta}554–557, {Delta}567–570) was accompanied by constitutive activation of the Gs/adenylyl cyclase system. This indicates that a loss of distinct amino acids is responsible for constitutive activity. Subdividing the {Delta}563–566 mutation into 2- and single-amino acid deletions finally revealed that the loss of D564 resulted in agonist-independent cAMP formation comparable to that observed with the original {Delta}558–566 mutant. A point mutation at amino acid position 564 (D564G) was first identified in a patient with familial male-limited precocious puberty (19). The corresponding mutation (D619G) in the TSHR causing a toxic thyroid nodule (20) in conjunction with the marked constitutive activity elicited by deleting D619 (12) underline the importance of this conserved D within the i3 loop for maintaining an inactive receptor conformation. Replacement of D564 by E (D564E) extended the carboxylate side chain of D present in the LHR(wt) by one methylene group while preserving the negative charge. This conservative substitution did not lead to constitutive receptor activity. However, introduction of a positively charged amino acid (D564K) and conversion of D564 to N, a similarly sized but uncharged residue that retains the ability to serve as a hydrogen bond acceptor, resulted in constitutive activity. These data indicate that a negative charge at position 564 in the LHR’s i3 loop may be necessary to keep the receptor in the inactive conformation. Thus, one may hypothesize that the negatively charged D564 stabilizes a positively charged amino acid residue within the receptor via a salt bridge constraint. While this manuscript was being reviewed, Kosugi and colleagues (21) also stressed the importance of D564 for the stabilization of an inactive state of the LHR.

To assess the importance of the conformational environment of D564 within the i3 loop for maintaining the inactive receptor conformation, we inserted three additional A residues up- and downstream of position 564, generating LHR-insA562 and -insA564. The relative shift of the conserved D within the i3 loop did not result in constitutive receptor activity. Our results show that while a negatively charged amino acid residue in the C-terminal part of the i3 loop is required for receptor quiescence, there is some degree of spatial freedom.

As the deletion of D619 from the TSHR i3 loop, as well as a D619G replacement, is sufficient to render the receptor constitutively active (12), a common activation mechanism appears to apply to both glycoprotein hormone receptors. Therefore, we initiated a search for a positively charged amino acid residue in the cytoplasmic portions of the LHR that could potentially provide for a counter ion for D564 to form a salt bridge. Because the i2 loop of the TSHR and LHR has been suggested to participate in Gs activation (22, 23), we initially focused on basic amino acids within this loop that are conserved among glycoprotein hormone receptors and replaced these residues by A. However, none of the five A mutants showed signs of ligand-independent activation and, therefore, most probably does not represent the site of a positively charged contact partner for D564. Systematic mutagenesis studies are underway to probe other locations at the cytoplasmic receptor aspect to identify a basic amino acid residue involved in maintaining a salt bridge constraint. Identification of such an amino acid would greatly enhance our knowledge on the spatial arrangement of intracellular receptor loops.

Experimental evidence combined with molecular modeling suggested that the central R residue in the conserved DRY motif, a structural hallmark of all GPCRs belonging to the rhodopsin family, is stabilized in a ‘polar pocket’ formed by several highly conserved polar amino acids located at the cytoplasmic aspect of different TMs (24). Thus, we considered R464 in the i2 loop of the LHR representing the center piece of the DRY motif (permutated to ERW in the glycoprotein hormone receptors) to be a potential contact partner for D564. Our A scanning approach revealed the noteworthy fact that R464 at the TM3/i2 transition can be replaced by A without major disturbance of LHR signaling upon transient expression of the mutant in COS-7 cells. An exchange of the conserved R for H, however, led to a drastic decrease of maximal hCG-induced cAMP formation in stably transfected HEK 293 cells (25). Functional studies with mutated m1 muscarinic receptors have shown that a charge-conserving R-for-K exchange results only in modest impairment of receptor function (26), suggesting that the nature of the replacing amino acid residue, and not only the loss of the conserved R, significantly contributes to functional properties of the resulting mutant receptors. Mutational studies with rhodopsin (27), the V2 vasopressin (28), and {alpha}1B-adrenergic receptors (24) indicated that replacement of the conserved R in the DRY motif by various different amino acids virtually abolishes G-protein coupling, and the conserved R has been implicated as a central trigger of GDP release from the G protein {alpha}-subunit (29). Our results clearly show that although the conserved R enhances LHR coupling to Gs, it cannot be regarded as the general and indispensable molecular switch for G-protein activation via all GPCRs.

In addition to the Gs/adenylyl cyclase system, the activated LHR is also known to stimulate phospholipase C (13, 16). We noticed that several LHR mutants (D564G, D564N, D564K, {Delta}564) modified at position 564 caused constitutive coupling to Gs and additionally profoundly enhanced agonist-induced IP accumulation, suggesting a connection between constitutive cAMP formation and agonist-dependent phospholipase C activation. Interestingly, two A insertion mutants, LHR-insA562 and -insA564, which do not cause constitutive cAMP formation, also responded to hCG challenge with a markedly increased phosphoinositide breakdown. These results show that ligand-independent adenylyl cyclase stimulation does not represent a prerequisite for an efficient coupling to the phospholipase C-signaling pathway, yet both signaling events appear to be independent.

To examine whether i3 loop deletions lead to constitutive activity of all glycoprotein hormone receptors, we also created mutant FSHRs. The human receptor, however, did not tolerate the deletion of 9 or 3 amino acid residues from its third intracellular loop. These results were not unexpected, as extensive mutational studies with the rat FSHR had already shown that this glycoprotein hormone receptor is particularly prone to improper folding and intracellular retention when only slightly modified (30, 31). Deletion from the FSHR i3 loop of D567, which corresponds to D564 in the LHR and D619 in the TSHR, did not interfere with membrane expression. However, in contrast to our results with the TSHR and LHR, the corresponding FSHR mutant was functionally equivalent to FSHR(wt) and did not present any evidence for constitutive activity. Therefore, one cannot escape the conclusion that, in the case of the FSHR, the molecular events underlying receptor activation differ from those operative in the other two glycoprotein hormone receptors. Our results are in accord with a recent study on hybrid LHRs/FSHRs emphasizing the importance of TM5/TM6 interactions for keeping gonadotropin receptors in an inactive state (14). In the FSHR, interhelical contacts between TM5 and TM6 appear to be more constrained to secure an inactive receptor state that cannot be destabilized by activating mutations or deletions in the i3 loop.

We previously suggested a model of TSHR activation in which deletion mutations within the i3 loop are assumed to lose the contact of TM5 to TM6 (12). Considering the present results obtained with LHR, agonist- or mutation-induced disruption of a salt bridge involving the i3 loop may also contribute to the activation of LHRs and TSHRs. In the FSHR the impact of the i3 loop on receptor activation clearly differs from the scenario that prevails in the TSHR and LHR. In the future, it will be enlightening to correlate these in vitro findings with distinct biological functions of the members of the glycoprotein hormone receptor family.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation of Mutant LHR and FSHR Genes
The cDNA of the human LHR in the pSG5 vector (Stratagene, La Jolla, CA) was inserted into the eukaryotic expression vector pcD-PS (32) via an EcoRI site in the polylinker and is further referred to as LHR-pcD-PS. A 1-kb fragment of 3'-untranslated region was removed by site-directed mutagenesis (33). The cDNA of the human FSHR was amplified by PCR from poly(A+) RNA isolated from human granulosa lutein cells. Briefly, two overlapping PCR fragments were amplified using the primer pairs PR1 5'-GCGGAATTCATGGCCCTGCTCCTGGTCTCTTTG-3'/PR2 5'-GCTGGCTTCCATGAGG-GCGACAAG-3' and PR3 5'-CTGCCTACTCTGGAAAAGCTTGTC-3'/PR4 5'-CGCGGATCCTGAGTTTTGGGCTAAATGA-CTTAG-3'. The PCR reactions were performed with the Expand High Fidelity system (Boehringer, Mannheim, Germany) under the following conditions (35 cycles): 1 min at 94 C, 1 min at 62 C, and 2 min at 68 C. To obtain full-length cDNA, the two overlapping PCR fragments were used as template, and the FSHR was amplified by applying the primer pair PR1/PR4. After a restriction enzyme digest with EcoRI/BamHI, the PCR product was initially cloned into the pSG5 vector (Stratagene). The cDNA sequence was confirmed by direct sequencing. Since all constructs used in this study were inserted into the pcD-PS vector, the FSHR cDNA was subsequently subcloned into this expression vector using the BglII sites within the coding sequence of the FSHR and the polylinker. The remaining cDNA coding for the N-terminal portion of the human (h)FSHR was introduced by employing standard PCR mutagenesis techniques using the SmaI site of the polylinker and the Bsu36I site in the coding sequence.

To characterize functional properties of different mutations within the i3 loop of the LHR and FSHR (Figs. 1Go and 5AGo), mutations were created by PCR mutagenesis techniques using the LHR-pcD-PS and FSHR-pcD-PS expression plasmids as a template. PCR fragments containing the mutations were digested and used to replace the corresponding BstBI/SpeI and PflMI fragments in the LHR-pcD-PS and FSHR-pcD-PS vectors, respectively. Point mutations in the i2 loop were introduced into LHR-pcD-PS using BstBI/XbaI sites. The identity of the various constructs and the correctness of all PCR-derived sequences were confirmed by restriction analysis and dideoxy sequencing with thermosequenase and dye-labeled terminator chemistry (Amersham, Arlington Heights, IL).

Transient Expression of Mutant LHRs and FSHRs and Functional Assays
COS-7 cells were grown in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37 C in a humidified 7% CO2 incubator. For transient transfections of COS-7 cells, a calcium phosphate coprecipitation method (34) was applied. In general, 20 µg of plasmid DNA per 10-cm dish or 5 µg/well (12-well dish) were transfected. For cAMP measurements, cells were split into 12-well plates (2 x 105 cells per well), transfected, prelabeled with [3H]adenine (25–50 Ci/mmol, Dupont-NEN, Brussels, Belgium), and assays were performed 3 days after transfection. For cAMP assays, cells were washed once in serum-free DMEM, followed by a preincubation with the same medium containing 1 mM 3-isobutyl-1-methylxanthine (Sigma Chemical Co., St. Louis, MO) for 20 min at 37 C in a humidified 7% CO2 incubator. Subsequently, cells were stimulated with appropriate concentrations of hCG (from pregnancy urine, 3,000 U/mg, Sigma) and porcine FSH (pFSH, isolated from porcine pituitary, 50 U/vial, Sigma) for 1 h. Reactions were terminated by aspiration of the medium and addition of 1 ml 5% trichloric acid. cAMP content of the cell extracts was determined as described previously (35).

To measure IP formation, transfected COS-7 cells were incubated with 2 µCi/ml of [myo-3H]inositol (18.6 Ci/mmol, Amersham) for 18 h. Thereafter, cells were washed once with serum-free DMEM without antibiotics containing 10 mM LiCl. hCG-induced increases in intracellular IP levels were determined by anion exchange chromatography as described (36).

To account for experimental variability inherent to transient transfection procedures, wt receptor cDNAs were included as internal controls in each transfection assay, and functional data were expressed as -fold increase of wt basal unless stated otherwise in the respective figure legends.

[125I]hCG and [125I]hFSH Binding
For radioligand binding studies, cells were harvested 72 h after transfection, and saturation binding assays were performed using membrane homogenates. Incubations were carried out for 1 h at 22 C in a 0.25 ml volume in the presence of 1.2 x 106 cpm of [125I]hCG (1800 Ci/mmol; Dupont-NEN, Brussels, Belgium) or 4 x 105 cpm of [125I]hFSH (1287 Ci/mmol; Dupont-NEN). Nonspecific binding was defined as binding in the presence of 1 µM hCG and 1.6 µM pFSH, respectively. Purchased [125I]hCG was characterized by RRA using the murine LHR stably expressed in L cells (13). Saturation binding experiments yielded a Kd value of 0.25 nM for [125I]hCG. [125I]hFSH was initially tested in binding experiments using hFSHRs expressed in COS-7 cells. Kd values of 0.6 nM were obtained for [125I]hFSH. Protein concentrations were determined by the bicinchoninc acid protein assay system (Pierce, Rockford, IL). Binding data were analyzed by a nonlinear least squares curve-fitting procedure using the program Ligand (37).


    ACKNOWLEDGMENTS
 
We would like to thank Robert Grosse for his help to clone the cDNA encoding the hFSHR. The cDNA of the human LHR was a generous gift of Dr. A. Shenker, Northwestern University Medical School, Chicago.


    FOOTNOTES
 
Address requests for reprints to: Torsten Schöneberg, Institut für Pharmakologie, Freie Universität Berlin, Thielallee 69–73, D-14195 Berlin, Germany. E-mail: schoberg{at}zedat.fu-berlin.de

This work was supported by the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie.

Received for publication May 6, 1998. Revision received October 8, 1998. Accepted for publication October 23, 1998.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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