Certain Activating Mutations within Helix 6 of the Human Luteinizing Hormone Receptor May Be Explained by Alterations That Allow Transmembrane Regions to Activate Gs

Amy N. Abell, Daniel J. McCormick and Deborah L. Segaloff

Department of Physiology and Biophysics (A.N.A., D.L.S.) The University of Iowa College of Medicine Iowa City, Iowa 52242
Department of Biochemistry and Molecular Biology (D.J.M.) Mayo Clinic/Mayo Foundation Rochester, Minnesota 55905


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Male-limited gonadotropin-independent precocious puberty (MPP) is frequently associated with mutations of the human LH/CG receptor (hLHR) that result in constitutively active hLHRs. Many such activating mutations have been identified in transmembrane 6 of the hLHR, with the substitution of Asp-578 being the most frequently observed mutation. Mutagenesis of a transmembrane helix of a G protein-coupled receptor can cause local alterations in the conformation near the mutated residue, allosteric changes elsewhere in the protein, and/or changes in the interhelical packing of the receptor. Therefore, while it has been hypothesized that activation of the receptor by mutations of Asp-578 may arise via alterations in the interactions of helix 6 with other transmembrane helices and/or by allosterically altering the conformation of the third intracellular loop, it has not been possible to ascertain the role of the sixth transmembrane helix per se in activating Gs in the mutated full-length receptor. Recently, however, we have shown that a peptide KMAILIFT, corresponding to the juxtacytoplasmic portion of helix 6 of the hLHR, is capable of activating Gs. These results suggest that helix 6 itself can directly interact with Gs. Importantly, the KMAILIFT peptide did not include Asp-578, which lies just C-terminal to this sequence. We show herein that a peptide extended to include Asp-578 (KMAILIFTDFT) is a poor activator of Gs. However, if the peptide is synthesized with the aspartate replaced with either a glycine or tyrosine, substitutions that are found in some patients with MPP, these peptides have Gs-stimulating activity. Additionally, a transmembrane 6 peptide with the substitution of Ile-575 with leucine, another mutation found in MPP, mimicked the activating effects of this mutation in the full-length receptor. The ability of peptides in which Asp-578 or Ile-575 is substituted to mimic the activating effects of these mutations in the full-length receptor suggests that the sixth transmembrane helix represents a site for direct interaction with Gs. In addition to the stimulatory effects of transmembrane 6 peptides, peptides corresponding to the juxtacytoplasmic portions of the fourth, fifth, and seventh helices were also able to stimulate Gs. These results are consistent with the hypothesis that the transmembrane helices may form a pocket for interaction with Gs and that constitutive activation of the hLHR may involve the opening of the pocket formed by these helices, thus exposing Gs-binding sites on these helices.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The LH/CG receptor (LHR) is a member of the superfamily of G protein-coupled receptors having seven membrane-spanning regions connected by alternating intracellular and extracellular loops, an extracellular N terminus, and an intracellular cytoplasmic tail (1). LHR occupation by either LH or hCG results primarily in the activation of Gs and increased cAMP production (1, 2). LHR signaling is absolutely required for testosterone production and masculinization of the male. Recent studies have identified several mutations of the human (h)LHR that cause constitutive activation of the hLHR, resulting in male-limited gonadotropin-independent precocious puberty (MPP) (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14). The substitution of Asp-578 in helix 6 with glycine is the most prevalent mutation (3, 4). However, substitution of Asp-578 with tyrosine, a rarer mutation, causes an earlier presentation of MPP (4). Both D578G and D578Y mutations cause constitutive activation of the hLHR (15), resulting in testosterone secretion by Leydig cells in the context of low prepubertal levels of LH and the premature onset of puberty in boys.

The mechanism by which mutations produce constitutively active hLHRs is not understood. A mutation may produce constitutive activation indirectly through changes in receptor conformation that expose distant Gs contact sites or directly by altering a site that physically contacts Gs. Although the sites of the hLHR that contact Gs remain mostly undetermined, studies of other Gs-coupled receptors have led to the prediction that the carboxyl-terminal region of the third intracellular loop of the hLHR will be involved in coupling to Gs (16, 17, 18). Recent studies of the hLHR have suggested that interhelical interactions of helix 6 with helix 5 (19) and helix 7 (20) are important in the ligand-independent activation of the hLHR. Thus, based on the location of Asp-578 in the middle of helix 6 of the hLHR, it has been postulated that mutations of Asp-578 produce constitutively active hLHRs by altering interhelical interactions, thus exposing regions of the third intracellular loop. An alternative possibility, i.e. that mutations may alter the direct interaction of helix 6 with Gs, although not excluded, had not until recently been addressed (21).

Because mutagenesis of Asp-578 may cause local changes in the conformation of helix 6 and/or conformational changes elsewhere, it has not been feasible to address the direct role of helix 6 in Gs activation using this approach. Therefore, we have turned to an alternate approach in which peptides corresponding to intracellular or transmembrane regions of the hLHR have been tested for their ability to directly activate Gs (21). With synthetic peptides, the direct effect of regions of a protein can be examined without concerns for conformational changes induced by mutagenesis of the protein. Several studies in other systems using synthetic peptides have identified short stretches of sequence that mimic the function of the corresponding region of the protein (18, 22, 23, 24, 25, 26). For example, Neubig and co-workers (25, 27) identified a peptide corresponding to the C-terminal half of the third intracellular loop of the {alpha}2-adrenergic receptor that stimulates Gi/o. Using peptides corresponding to regions of G proteins, Hamm and co-workers (28, 29, 30, 31) identified regions of the visual system G protein transducin (Gt) that interact with rhodopsin, and regions of Gs that interact with the ß-adrenergic receptor. Recent studies from our laboratory have demonstrated that the peptide KMAILIFT, corresponding to the juxtacytoplasmic region of helix 6 (see Fig. 1Go), activates adenylyl cyclase by stimulating Gs (21). These data clearly point to a role for helix 6 of the hLHR in activating Gs. Interestingly, this initially described stimulatory peptide does not contain the aspartate corresponding to Asp-578, which lies just C-terminal to the KMAILIFT sequence (Fig. 1Go). This observation, coupled with reports of constitutive activation of the full-length hLHR caused by certain substitutions of Asp-578, led us to hypothesize that whereas helix 6 can activate Gs, Asp-578 may normally impede this activation. The following experiments used synthetic peptides corresponding to wild-type vs. mutated forms of helix 6 to examine the mechanism by which mutation produces constitutively active receptors. Additionally, peptides corresponding to the juxtacytoplasmic portions of the other helices were examined to identify other hLHR contact sites with Gs.



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Figure 1. Orientation and Proposed Topology of the hLHR

The deduced sequence, orientation, and proposed topology of hLHR (32 ) from which the peptides are derived are illustrated. The shaded region represents the transmembrane regions above which are the extracellular regions and below which are the intracellular regions. The boundaries for the transmembrane regions are based on the hydrophobicity plots of the rLHR (2 ). While this manuscript was in preparation, a molecular model of the hLHR based on the two-dimensional electron density map of bovine rhodopsin was published (50 ). Bolded lines indicate the proposed transmembrane boundaries suggested by this computer model (50 ). The extracellular domain and the cytoplasmic tail are not shown in this picture.

 

    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In a previous report, it was demonstrated that a peptide KMAILIFT corresponding to the juxtacytoplasmic region of transmembrane 6 of the rat (r)LHR-stimulated basal adenylyl cyclase activity 2-fold in membranes of untransfected 293 cells (21). This maximal stimulation elicited by the KMAILIFT peptide is approximately 75% of the maximal stimulation observed when membranes from 293 cells transfected with the rLHR are incubated with a maximally stimulatory concentration of hCG (21). The stimulation of adenylyl cyclase by the KMAILIFT peptide was further shown to be through the activation of Gs, rather than through a direct effect on adenylyl cyclase (21). For comparative purposes, the activity of the KMAILIFT peptide in membranes prepared from untransfected 293 cells is shown in Fig. 2AGo. It can be seen that concentrations of this peptide up to 100 µM stimulate cyclase activity, whereas at very high concentrations of peptide the stimulation is attenuated (Fig. 2AGo). Asp-578, the residue most frequently mutated in the hLHRs of patients with MPP, is located just C-terminal to the KMAILIFT sequence (3, 4, 8, 15).1 Based on these observations, we hypothesized that, in addition to the interactions of Asp-578 with other transmembrane helices (19, 20), Asp-578 may also directly affect the interaction of helix 6 with Gs.



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Figure 2. Effect of Asp-578 on the Ability of hLHR Helix 6 Peptides to Stimulate Adenylyl Cyclase Activity

Membranes prepared from 293 cells that do not express the hLHR were incubated for 20 min at 37 C with increasing concentrations of hLHR peptides and assayed for basal adenylyl cyclase activity as previously described (21 ). A, Data shown are expressed as a percentage of basal cyclase activity in the absence of peptide (i.e. 100% represents no change from basal activity). Basal cyclase activity for these experiments was 5.54 ± 0.73 pmol/min/mg protein. Data shown are the mean ± SEM of four experiments. B, Data shown are expressed as a percentage of basal cyclase activity in the absence of peptide (i.e. 100% represents no change from basal activity). Basal cyclase activity for these experiments was 9.86 ± 1.00 pmol/min/mg protein. Data shown are the mean ± range of two experiments.

 
To examine potential direct effects of Asp-578 on the coupling of the hLHR to Gs, a longer peptide C-terminally extended to include the next three residues of helix 6 was examined (see Fig. 1Go and Table 1Go). As shown in Fig. 2Go and in Table 2Go, the KMAILIFTDFT peptide was a poor stimulator of 293 membrane adenylyl cyclase activity as compared with the shorter KMAILIFT peptide. These data suggest that the additional residues, DFT, interfere with the ability of the sequence KMAILIFT to activate adenylyl cyclase. The KMAILIFTDFT peptide was then used as a template to prepare peptides in which the aspartate residue was substituted with either glycine or tyrosine (Table 1Go). These peptides were designed to reflect the substitutions of Asp-578 of the full-length hLHR associated with constitutive activation of the receptor as seen in MPP (3, 4, 8). As shown in Fig. 2AGo, both KMAILIFTGFT and KMAILIFTYFT peptides stimulated adenylyl cyclase activity to a greater degree than the KMAILIFTDFT peptide. At high concentrations, the KMAILIFTGFT and KMAILIFTYFT peptides were even more efficacious than the shorter KMAILIFT peptide because, unlike the KMAILIFT peptide, they did not inhibit cyclase activity at very high concentrations. As shown in Fig. 2BGo, the reduced activity of the KMAILIFTDFT peptide is not due to the negative charge of Asp-578. Thus, the KMAILIFTEFT peptide, in which the aspartate was substituted with glutamate, was also stimulatory. Interestingly, a peptide in which the aspartate was substituted with an asparagine (KMAILIFTNFT) was also active (Fig. 2BGo). Together, these data suggest that it is the addition of the aspartate, not the phenylalanine or threonine, to the KMAILIFT peptide that reduces the ability of the KMAILIFTDFT peptide to stimulate adenylyl cyclase activity. Additionally, these data suggest that substitution of the aspartate in the KMAILIFTDFT peptide alters the peptide, allowing it to better stimulate adenylyl cyclase activity. The increased activity of the helix 6 peptides corresponding to D578G, D578Y, and D578E mutations correlates well with the reported constitutive activity induced by these mutations in the full-length hLHR (Table 1Go). However, whereas the helix six peptide corresponding to the D578N mutation exhibited increased activity, the comparable substitution in the full-length receptor is without effect (Table 1Go).


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Table 1. Peptides Corresponding to Helix 6 of the wild-type hLHR or Constitutively Active Mutants Thereof

 

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Table 2. Comparison of hLHR Peptide-Stimulated Cyclase Activity in 293 and S49-wt Membranes

 
To confirm that the hLHR helix 6 peptides that stimulate basal adenylyl cyclase activity in 293 cell membranes are doing so via an activation of Gs, the ability of the LHR peptides to stimulate adenylyl cyclase activity was examined in S49 wild-type (S49-wt) cell membranes as compared with S49 cyc- cell membranes that lack Gs{alpha} (33). Previous studies from our laboratory (21) confirmed that the S49-wt and S49 cyc- cell membranes behaved as predicted from initial characterizations of these cells (34, 35). Thus, S49-wt membranes exhibited basal, NaF-, and forskolin-stimulated cyclase activity. However, cyc- membranes exhibited low levels of basal cyclase activity and did not respond to NaF, but displayed forskolin-stimulated cyclase activity at comparable levels to that observed in wild-type membranes (21, 33, 35). In each experiment presented herein utilizing S49-wt and cyc- cell membranes, basal, NaF-, and forskolin-stimulated cyclase activities were routinely included as internal controls. As shown in Fig. 3Go, in S49-wt cell membranes the KMAILIFTDFT peptide was much less potent than the KMAILIFTGFT and KMAILIFTYFT peptides. Importantly, even at very high (300 µM) peptide concentrations, KMAILIFTDFT, KMAILIFTGFT, and KMAILIFTYFT peptides caused no detectable increases in adenylyl cyclase activity in S49 cyc- cell membranes (Fig. 3Go), although comparable levels of forskolin-stimulated adenylyl cyclase activity were observed in the S49 cyc- cell membranes as compared with the S49-wt cell membranes, confirming that the levels of adenylyl cyclase in the two membrane preparations were equivalent (Fig. 3Go).



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Figure 3. Effect of hLHR Helix 6 Peptides on S49-wt and S49 cyc- Adenylyl Cyclase Activity

Crude membranes were prepared from S49-wt or S49 cyc- mouse lymphoma cells as previously described (21 ). S49-wt membranes (open symbols) were incubated for 20 min at 30 C with increasing concentrations of hLHR peptides and assayed for basal adenylyl cyclase activity as previously described (21 ). S49 cyc- membranes (closed symbols) were incubated for 20 min at 30 C with 300 µM of hLHR peptides and assayed for basal adenylyl cyclase activity as previously described (21 ). Data shown are a representative experiment of three such experiments. All points represent the mean ± range of duplicate determinations. The control data for this experiment, expressed as the mean ± range for duplicate determinations of adenylyl cyclase activity in picomoles per min/mg of protein, were the following: for S49-wt membranes basal, 2.06 ± 0.13; NaF, 36.27 ± 1.21; forskolin, 55.80 ± 1.14; for S49 cyc- membranes basal, 0; NaF, 0; forskolin, 46.87 ± 1.23.

 
Although the general effects of LHR peptides on membranes from 293 cells and from S49 cells were similar, there were differences in the absolute stimulatory activities of the peptides in these two cell types.2 Table 2Go compares the effects of incubation of 293 and S49-wt membranes with 300 µM LHR peptides on basal cyclase activity. In all cases, LHR peptides produced a greater percent increase in basal cyclase activity in S49-wt cell membranes as compared with 293 membranes. For example, defining basal cyclase activity as 100%, incubation of 293 membranes with 300 µM KMAILIFTYFT peptide resulted in a cyclase activity of 284.5 ± 10.1%, whereas incubation of S49-wt membranes with this peptide resulted in a cyclase activity of 428.8 ± 30.3% (Table 2Go). However, the fold stimulation of cyclase activity by either the KMAILIFTYFT or KMAILIFTGFT peptides as compared with the KMAILIFTDFT peptide was the same for both cell types (Table 2Go), indicating that the relative effects of each peptide were similar in the two cell types.

As described above, the KMAILIFTDFT peptide exhibited a reduced ability to activate Gs as compared with the KMAILIFT, KMAILIFTGFT, and KMAILIFTYFT peptides. Clearly, the KMAILIFTDFT peptide binds and activates Gs, as measurable activity is present in cells that have Gs, but absent in S49 cyc- cells which lack Gs{alpha}. The reduced activity of the KMAILIFTDFT peptide may be due to the decreased affinity of this peptide for Gs or to a decreased ability of the peptide to activate Gs. As shown above (see Fig. 2Go), the KMAILIFTDFT peptide stimulated cyclase activity only at high (300 µM) concentrations of peptide, whereas low (10 µM) concentrations of the KMAILIFTYFT peptide were stimulatory. To determine whether the KMAILIFTDFT peptide binds to Gs at lower concentrations than are necessary for it to activate Gs and to further study the association of the KMAILIFTDFT peptide with Gs, we examined whether the KMAILIFTDFT peptide could prevent the ability of the KMAILIFTYFT peptide to activate adenylyl cyclase. Membranes from 293 cells were incubated with a minimal stimulatory concentration of the active KMAILIFTYFT peptide (10 µM) and increasing concentrations of the KMAILIFTDFT peptide. As shown in Fig. 4Go, stimulation of adenylyl cyclase activity by the KMAILIFTYFT peptide was attenuated by coincubation with increasing concentrations of the KMAILIFTDFT peptide. Although the inhibition by the KMAILIFTDFT peptide was not complete at 100 µM KMAILIFTDFT peptide (Fig. 4Go), it was difficult to examine higher concentrations of the KMAILIFTDFT peptide in this competition assay. Importantly, inhibition occurred at concentrations of the KMAILIFTDFT peptide as low as 30 µM. These data suggest that the KMAILIFTFT peptide may bind to Gs at lower peptide concentrations, but the KMAILIFTDFT peptide is a poor activator of Gs, and thus, requires higher concentrations to activate Gs.



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Figure 4. The hLHR Helix 6 KMAILIFTDFT Peptide Attenuates the Stimulation of Gs by the Active KMAILIFTYFT Peptide

Membranes prepared from 293 cells that do not express the LHR were incubated for 20 min at 37 C with 10 µM KMAILIFTYFT peptide and increasing concentrations of KMAILIFTDFT peptide and assayed for basal adenylyl cyclase activity as previously described (21 ). Data shown are expressed as a percentage of basal cyclase activity in the absence of peptide (i.e. 100% represents no change from basal activity). Data shown are the mean ± range of two experiments. Basal adenylyl cyclase activity was 8.25 ± 0.03 pmol/min/mg protein. In these experiments the KMAILIFTYFT peptide alone stimulated adenylyl cyclase activity 149 ± 20%.

 
Since substitution of Asp-578 in the KMAILIFTDFT peptide to glycine or tyrosine, substitutions that produce constitutively active full-length LHRs, resulted in peptides with enhanced abilities to activate Gs, it was important to determine whether other constitutively activating mutations found in the lower portion of helix 6 could similarly alter the activity of the KMAILIFTDFT peptide. To address this question, three additional peptides were synthesized where a residue in the KMAILIFTDFT peptide was substituted with one corresponding to that found in patients with constitutively activated LHRs (Table 1Go) (6, 7, 36). As shown in Fig. 5AGo, peptides with either the M571I or T577I substitutions had almost no effect on 293 membrane cyclase activity. However, the peptide with the I575L substitution stimulated 293 membrane cyclase activity to a greater extent than the wild-type peptide (Fig. 5AGo). Similar activities were observed in S49-wt membranes (Fig. 5BGo), and no activity was seen in S49 cyc- membranes. Therefore, of the three other activating helix 6 mutations examined, peptides corresponding to the M571I and T577I substitutions did not show increased activity. However, the peptide corresponding to the I575L substitution activated Gs to a greater extent that the helix 6 peptide corresponsing to the wild-type sequence. The ability of certain helix 6 peptides to exhibit greater Gs stimulatory activity does not readily correlate with the isoelectric point (pI) of the peptide or the relative contributions of polar, nonpolar, acidic, or basic residues to the peptide composition (Table 1Go). For example, whereas the I575L peptide is active and the M571I peptide is relatively inactive, the general physical properties of the two peptides are the same.



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Figure 5. Effect of Other hLHR KMAILIFTDFT Peptide Substitutions on 293 and S49-wt and cyc- Membrane Adenylyl Cyclase Activity

A, Membranes prepared from 293 cells that do not express the LHR were incubated for 20 min at 37 C with increasing concentrations of hLHR peptides containing substitutions corresponding to other activating mutations of helix 6 in the full-length hLHR. The sequences of the peptides are shown in Table 1Go. Basal adenylyl cyclase activity was assayed as previously described (21 ). Data shown are expressed as a percentage of basal cyclase activity in the absence of peptide (i.e. 100% represents no change from basal activity). Basal cyclase activity was 7.06 ± 1.77 pmol/min/mg protein. Data shown are the mean ± SEM of three experiments. B, Crude membranes were prepared from wt or cyc- S49 mouse lymphoma cells as previously described (21 ). S49-wt or cyc- membranes were incubated for 20 min at 30 C with a final concentration of 300 µM of the indicated hLHR peptides and assayed for basal adenylyl cyclase activity as previously described (21 ). Data shown are the mean ± range of duplicate determinations of a representative experiment of two such experiments. The control data for this experiment, expressed as the mean ± range for duplicate determinations of adenylyl cyclase activity in picomoles per min/mg of protein, were the following: for S49-wt membranes basal, 2.57 ± 0.02; NaF, 32.14 ± 2.18; forskolin, 54.08 ± 1.23; for S49 cyc- membranes basal, 0.28 ± 0.08; NaF, 0.22 ± 0.01; forskolin, 50.43 ± 6.47.

 
It was important to further determine whether the stimulatory effects of a hLHR juxtacytoplasmic region on Gs activation were limited to helix 6, or whether peptides corresponding to the juxtacytoplasmic portions of the other helices can also stimulate Gs. To address this question, peptides corresponding to the juxtacytoplasmic regions of all seven transmembrane helices (TM1, TM2, TM3, TM4, TM5, TM6, and TM7) were synthesized (Fig. 1Go and Table 3Go). Of these peptides, the peptide corresponding to helix 1 was insoluble and was not examined further. The effects of the other peptides on 293 membrane adenylyl cyclase activity were then examined. TM2 and TM3 peptides had no effect on 293 membrane cyclase activity (Fig. 6Go). As shown in Fig. 6Go, the TM4, TM5, TM6, and TM7 peptides had varied effects on 293 membrane adenylyl cyclase activity. The TM4 peptide stimulated 293 cyclase activity, but required much higher concentrations as compared with the TM6 peptide (Fig. 6Go). The TM5 and the TM7 peptides both stimulated cyclase activity at low concentrations but were strongly inhibitory at higher concentrations (Fig. 6Go). The effects of these transmembrane peptides were also examined in S49-wt membranes. TM2 and TM3 peptides did not stimulate S49 membrane cyclase activity at any concentration of peptide tested (Fig. 7AGo), consistent with their lack of cyclase stimulation in 293 membranes. However, at very high concentrations of TM2, there was a slight inhibition of S49-wt cyclase activity (Fig. 7AGo). As seen in 293 membranes, the TM4 peptide stimulated S49-wt cyclase activity, but again required much higher concentrations of peptide (Fig. 7AGo). Again, as observed in 293 membranes, low concentrations of the TM5 and TM7 peptides stimulated S49-wt cyclase activity, whereas high concentrations inhibited cyclase activity (Fig. 7AGo). Figure 7BGo further shows that whereas maximally stimulatory concentrations of each peptide stimulated cyclase activity in S49-wt membranes, they were without effect in S49 cyc- membranes. These data suggest that other hLHR transmembrane helices may be involved in direct interactions with Gs.


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Table 3. Peptides Corresponding to the Juxtacytoplasmic Regions of the hLHR Transmembrane Helices

 


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Figure 6. Effect of Peptides Corresponding to the Juxtacytoplasmic Regions of the Transmembrane Helices of the hLHR on 293 Membrane Adenylyl Cyclase Activity

Membranes prepared from 293 cells that do not express the LHR were incubated for 20 min at 37 C with increasing concentrations of hLHR peptides shown in Table 3Go and assayed for basal adenylyl cyclase activity as previously described (21 ). Data shown are expressed as a percentage of basal cyclase activity in the absence of peptide (i.e. 100% represents no change from basal activity). A representative experiment of two such experiments is shown and all points represent the mean of duplicate determinations. Basal cyclase activity for these experiments was 4.24 ± 0.21 pmol/min/mg protein.

 


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Figure 7. Effect of Peptides Corresponding to the Juxtacytoplasmic Regions of the Transmembrane Helices of the hLHR on S49-wt and S49 cyc- Membrane Adenylyl Cyclase Activity

Crude membranes were prepared from S49-wt or S49 cyc- mouse lymphoma cells as previously described (21 ). A, S49-wt membranes were incubated for 20 min at 30 C with increasing concentrations of the indicated hLHR peptides and assayed for basal adenylyl cyclase activity as previously described (21 ). A representative experiment of two such experiments is shown, and all points represent the mean of duplicate determinations. Basal adenylyl cyclase activity in this experiment was 2.76 ± 0.08 pmol/min/mg protein. B, S49-wt or cyc- membranes were incubated for 20 min at 30 C with the indicated hLHR peptides at a concentration that was found to be maximally activating in S49-wt membranes (TM4, 300 µM; TM5, 30 µM; TM6, 100 µM; TM7, 56 µM) and assayed for basal adenylyl cyclase activity as previously described (21 ). Data points represent the mean ± range of duplicate determinations of a representative experiment of either two or three experiments. The control data for this experiment, expressed as the mean ± range for duplicate determinations of adenylyl cyclase activity in picomoles per min/mg of protein, were the following: for S49-wt membranes basal, 2.76 ± 0.08; NaF, 34.55 ± 0.13; forskolin, 52.93 ± 3.27; for S49 cyc- membranes basal, 0.26 ± 0.04; NaF, 0.24 ± 0.01; forskolin, 51.49 ± 7.55.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
G protein-coupled receptors comprise an extremely large (>1,000) superfamily of proteins involved in numerous, diverse biological systems (see Refs. 37, 38 for recent reviews). Much research has focused on understanding the mechanism of activation of these receptors with the goal of using this information to target them for potential drug development. Recently, numerous constitutively active receptors characterized by agonist-independent activity have been identified (3, 39, 40, 41, 42, 43, 44, 45, 46). Some of these mutations have been identified through site-directed mutagenesis studies, whereas many others represent naturally occurring mutations discovered in patients with several endocrine disorders (see Refs. 3, 31, 41, 46 for examples). The mechanism by which mutations produce constitutively active receptors has been difficult to determine. Some mutations are predicted to alter a direct interaction site of the receptor with the G protein, whereas other mutations are thought to alter the general conformation of the receptor through changes in interhelical interactions or allosteric changes in the conformation of the receptor that expose interaction sites for the G protein (15, 20, 44).

The overall goal of this study was to examine the mechanisms by which mutations in the hLHR cause the constitutive activity of the hLHR. In a previous report, we identified a peptide KMAILIFT corresponding to the juxtacytoplasmic region of helix 6 of the LHR that stimulates Gs (21). Located just C-terminal to the KMAILIFT sequence is Asp-578, the residue most frequently mutated in patients with MPP (3, 4, 8). Based on the location of Asp-578 and the effect of mutations of Asp-578, we hypothesized that some mutations of this residue may alter the direct activation of Gs by helix 6 of the hLHR. To test this hypothesis, a peptide KMAILIFTDFT with three additional residues including Asp-578 was examined. The Gs-stimulatory activity of the KMAILIFTDFT peptide was reduced compared with the original KMAILIFT peptide. Substitution of the aspartate in the wild-type KMAILIFTDFT peptide with either glycine or tyrosine, corresponding to substitutions of the full-length hLHR that cause constitutive activation (15), resulted in peptides with even greater activity than the original shorter KMAILIFT peptide. These data suggest that it must be the aspartate in the DFT sequence, not the phenylalanine or threonine, that is responsible for the decreased activity of the KMAILIFTDFT peptide as compared with the original KMAILIFT peptide.

Other investigators have identified peptides that directly stimulate Gs (17, 47). For example, Munch et al. (47) identified a peptide corresponding to eight residues from the C-terminal portion of the third intracellular loop and four residues from helix 6 of the avian ß-adrenergic receptor that stimulated cyclase activity. However, this stimulation required 100 µM concentrations of peptide and produced only a 30% increase in cyclase activity. The studies presented herein reveal a strong stimulation of cyclase activity (~75% of that produced by LHR-expressing 293 membranes in response to a maximally stimulatory concentration of hCG) by helix 6 peptides such as KMAILIFT, KMAILIFTGFT, and KMAILIFTYFT at peptide concentrations as low as 10 µM.

Further experiments were performed to address whether there was any specificity for amino acids with a given property to confer Gs-stimulatory activity in D578-substituted hLHR helix 6 peptides. The data presented suggest that the decreased activity of the KMAILIFTDFT peptide is not simply due to the negative charge of the aspartate, as substitution of the aspartate with a negatively charged glutamate resulted in a peptide with strong stimulatory activity. These results are consistent with a recent report showing that substitution of Asp-578 with glutamate in the full-length hLHR resulted in a receptor displaying constitutive activation (15). Therefore, the increased activity of the helix 6 peptides with D578G, D578Y, or D578E substitutions correlates well with the reported constitutive activity of the full-length hLHR harboring these substitutions (15). However, there is a discrepancy between the helix 6 D578N peptide, which displays increased activity, and the D578N substitution of the full-length hLHR, which does not cause constitutive activity (15).

One possible explanation for the effects of the aspartate on the KMAILIFTDFT peptide is that this peptide is able to bind to Gs, but is a poor activator of Gs. This explanation is supported by experiments in which increasing concentrations of the KMAILIFTDFT peptide inhibited the stimulation of cyclase activity by the KMAILIFTYFT peptide. These experiments suggest that the KMAILIFTDFT peptide may compete with the KMAILIFTYFT peptide for the interaction with Gs. In a previous study, Wade et al. (24) demonstrated the ability of some {alpha}2-adrenergic peptides to dimerize, with dimerized peptides possessing even greater potency than the monomeric peptides. The possibility that the KMAILIFTDFT peptide may form heterodimers with the KMAILIFTYFT peptide, and in doing so inhibit the association of the KMAILIFTYFT peptide with Gs, cannot be excluded by our experiments. We performed additional experiments to examine whether the KMAILIFTDFT peptide could inhibit hCG-stimulated adenylyl cyclase activity in membranes from 293 cells expressing the LHR. Inhibition of hCG-stimulated cyclase was observed but only at relatively high concentrations of peptide (600 µM), and under these conditions forskolin-stimulated cyclase was inhibited as well. Therefore, we were precluded from making any meaningful conclusions from these experiments.

Taken altogether, the data showing the greater activity of the shorter KMAILIFT helix 6 peptide, as compared with the longer KMAILIFTDFT peptide containing Asp-578, and the data on the D578-substituted peptides suggest that the juxtacytoplasmic region of helix 6 has the potential to directly interact with and activate Gs, but that Asp-578 normally constrains this activity. The data further suggest that one mechanism by which mutations of Asp-578 cause constitutive activation of the full-length hLHR may be by relieving the constraint imposed by Asp-578 in the Gs-activating properties of helix 6. This hypothesis does not preclude other additional mechanisms, such as disruption of interhelical interactions, that may also be involved in the constitutive activation of the hLHR by mutation of Asp-578. However, our data do suggest a hitherto unreported potential role of helix 6 in the activation of Gs per se. It should be pointed out that a recent mutagenesis study on the related TSH receptor also suggests a role for helix 6 of the TSH receptor in activating Gs (48, 49).

Mutations of several residues within helix 6 of the hLHR have been reported to cause constitutive activation. Therefore, we also examined the properties of hLHR helix 6 peptides containing either M571I, I575L, or T577I substitutions. Of these three peptides, only one (I575L) showed increased activity. Thus, in addition to the correlation of the activating properties of D578G, D578Y, and D578E peptides with the corresponding activating mutations of the full-length hLHR, there is a correlation between peptides and the full-length receptor with an additional mutation located elsewhere in helix 6 (I575L). However, there is a lack of correlation between the helix 6 M571I and T577I peptides and the corresponding mutations in the full-length hLHR. One possible hypothesis to explain this apparent discrepancy is that in the context of the full-length receptor, M571 and T577 may not be constraining the Gs-stimulatory activity of helix 6. Rather, they may be inhibiting the Gs-stimulatory activity of the receptor via holding it in a particular conformation through interhelical interactions. This is, however, speculative and awaits further investigation.

Furthermore, there does not appear to be a correlation between pI or percent composition of polar, nonpolar, acidic, and basic residues among the active or inactive helix 6 peptides (Table 1Go). Clearly, the active peptides do not share similar physical properties (as defined by these broad categories). It is possible, however, that the secondary structure adapted by the active peptides is distinct from that of the inactive peptides, and studies are underway to address this question.

To examine the potential role of other hLHR transmembrane regions in interactions with Gs, the effects of the juxtacytoplasmic portions of the other helices were examined. The precise location of the transmembrane boundaries of the hLHR are unknown. Because of the strong homology between the rat and human LHRs, transmembrane peptides were synthesized based on hydropathy plots of the rLHR (2). While this manuscript was in preparation, a computer model of the hLHR based on the two-dimensional electron density map of bovine rhodopsin was published (50). The transmembrane boundaries suggested by this model differ somewhat from the boundaries used for our transmembrane peptide studies (Fig. 1Go) (50). Importantly, in both models, the LHR peptides we used were located within the proposed transmembrane regions (Fig. 1Go and Table 3Go). Peptides corresponding to the juxtacytoplasmic portions of helices 4, 5, 6, and 7 possessed the ability to stimulate Gs to various extents. As summarized in Table 3Go, there does not appear to be a correlation between the gross physical properties of activating transmembrane peptides vs. inactive transmembrane peptides. The stimulation of Gs by peptides corresponding to helices 4, 5, 6, and 7 is consistent with the hypothesis that multiple transmembrane regions of the hLHR may form a binding pocket for interaction with Gs.

Studies with other G protein-coupled receptors support the general model that activation of the receptors is associated with increased movement of the helices (reviewed in Ref. 38). This, in turn, is thought to open up the cytoplasmic cleft exposing specific sites for G protein interaction and activation (51, 52, 53). Data presented herein is consistent with the cleft of the hLHR forming a binding pocket for Gs. Our data further suggest that the pocket formed by the helices of the hLHR for the binding and activation of Gs may be deeper than that previously suggested for other receptors.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Supplies
Highly purified hCG (CR-127) was provided by the National Hormone and Pituitary Agency of the NIDDK/NIH. Alumina WN3 and reagents for adenylyl cyclase assays were obtained from Sigma (St. Louis, MO). AG50W-X4 resin was obtained from Bio-Rad (Richmond, CA), and [{alpha}32P]ATP was purchased from New England Nuclear (Boston, MA). Tissue culture reagents and plasticware were purchased from Life Technologies, Inc. (Grand Island, NY and Corning, NY, respectively).

Peptides
LHR peptides were synthesized as C-terminal amides and were purified by reverse-phase HPLC to >95% purity by the Mayo Protein Core Facility, Mayo Foundation (Rochester, MN). Peptides were initially dissolved in dimethylsulfoxide and diluted further with water to yield a 10% dimethylsulfoxide stock solution. The physical properties of each of the peptides were calculated using the computer program MacVector.

Cells
Human embryonic kidney 293 cells were obtained from American Type Culture Collection (ATCC CRL 1573; Rockville, MD). The clonal cell line rLHR-wt-16 expressing approximately 9,000 rLHRs per cell was kindly donated by Mario Ascoli (University of Iowa). S49-wt and S49 cyc- mouse lymphoma cell lines were obtained from Dr. Henry Bourne (University of California, San Francisco). S49 cyc- cells, which lack G{alpha}s (33), are a clonal derivative of S49-wt cells (35). All cell lines were maintained as described previously (21).

Preparation of Membranes
293 membranes were prepared as described previously (21). S49-wt and cyc- membranes were prepared as described by Ross et al. (54).

Adenylyl Cyclase Activity Assay
Adenylyl cyclase activity assays were performed as described previously (21). Briefly, for 293 membranes, 10 µg of membrane in 10 µl of membrane buffer (125 mM Tris-Cl, 5 mM EDTA, pH 7.4) were preincubated with increasing concentrations of peptides (dissolved in 10 µl of 10% Me2SO) for 15 min at 37 C followed by 15 min at 4 C. After preincubation, the following components were added: 10 µl of 75 µg/ml hCG in 150 mM NaCl, 20 mM HEPES, and 1% BSA, pH 7.4, or buffer only; 10 µl of 100 µM GTP; and 10 µl of reaction mixture containing 0.5 mM ATP, 20 mM MgCl2, 5 mM cAMP, 100 mM phosphocreatine, 200 U/ml creatine kinase, 200 U/ml myokinase, 3 µCi/ml [3H]cAMP, and 140 µCi/ml [{alpha}-32P]ATP. These components were incubated 20 min at 37 C, after which reactions were terminated by the addition of stop solution (40 mM ATP, 10 mM cAMP, 1% SDS) and boiling for 3 min. [32P]cAMP was isolated and counted as previously described (21). For S49 membranes, cyclase assays were peformed essentially as described above for 293 membranes with the following modifications: 10 µg of S49-wt or cyc- membranes in HME buffer (20 mM HEPES, 2 mM MgCl2, 1 mM EDTA, pH 8.0) were preincubated with increasing concentrations of LHR peptides for 15 min at 30 C with shaking followed by 15 min at 4 C. After preincubation, the following components were added: 10 µl of 230 mM HEPES and 4 mM EDTA, pH 8.0; 10 µl of 250 µM GTP; and 10 µl of the reaction mixture described above except that the reaction mixture contained 50 mM MgCl2 instead of 20 mM MgCl2. These components were incubated for 20 min at 30 C with shaking, after which reactions were terminated; [32P]cAMP was isolated and counted as described above. For each experiment in which peptide-stimulated cyclase activity in S49-wt and cyc- membranes was compared, basal, NaF-, and forskolin-stimulated cyclase activities were determined to verify the absence or presence of Gs{alpha} and the presence of comparable levels of functional adenylyl cyclase. For each adenylyl cyclase assay, background [32P]cAMP formation (0.95 ± 0.10) was measured and subtracted from each sample.

It should also be pointed out that the conditions for assaying cyclase activity differ in incubation temperature (37 C vs. 30 C), in the concentrations of GTP (20 µM vs. 50 µM), and in the concentrations of MgCl2 (4 mM vs. 10 mM) for 293 and S49 membranes, respectively. Because the isoforms of adenylyl cyclase in S49 membranes are rapidly inactivated by heating (55), it is technically not possible to examine S49 membrane cyclase activity at the same incubation temperature used to examine 293 membrane cyclase activity. Additionally, measurement of cyclase activity in S49 membranes requires higher concentrations of GTP and MgCl2 as compared with those for 293 membranes (55). Our measurements of cyclase activity in 293 membranes under conditions used for S49 membranes resulted in a 5 to 10-fold elevation of basal cyclase activity. This increase in basal cyclase activity of 293 membranes incubated with higher concentrations of MgCl2 and GTP is consistent with previous reports of activation of Gs by higher concentrations of MgCl2 and/or GTP in 293 membranes (56) and other cell types (57). Membranes from 293 cells assayed under S49 adenylyl cyclase assay conditions were stimulated by LHR peptides. However, the peptide-stimulated activity was reduced by 40–50% as compared with that observed using 293 assay conditions, demonstrating the decreased sensitivity of the 293 membranes under the conditions used for S49 membranes.


    ACKNOWLEDGMENTS
 
We thank Drs. Mario Ascoli and Nikolai Artemyev for helpful discussions and Dr. Ascoli for critically reading the manuscript. We also gratefully acknowledge Harinder Kaur for her technical support. All peptides were synthesized and purified by the Mayo Protein Core Facility, Mayo Clinic/Mayo Foundation.


    FOOTNOTES
 
Address requests for reprints to: Deborah L. Segaloff, Ph.D., Department of Physiology and Biophysics, The University of Iowa College of Medicine, Iowa City, Iowa 52242. E-mail: deborah-segaloff{at}uiowa.edu

These studies were supported by NIH Grant HD-22196 (to D.L.S.). During the course of these studies D.L.S. was a recipient of NIH Research Career Development Award HD-00968. The services and facilities provided by the University of Iowa Diabetes and Endocrinology Research Center Grant DK-25295 are gratefully acknowledged.

1 Although our previous study examined the coupling of the rLHR to Gs, the amino acid sequence of helix 6 is identical between the rat and the human LHRs (2 32 ). This complete identity allows us to extend our conclusions from the previous study of the rLHR on the role of helix 6 in the direct activation of Gs to the hLHR. Back

2 There are both biological and technical reasons that could account for the differences in cyclase activity between the 293 and S49-wt cell membranes. Thus, the two cell types may have differential expression of long and short forms of {alpha}s, different concentrations and/or isoforms of adenylyl cyclase, or may vary in the degree of compartmentalization of signaling molecules. Furthermore, as explained in Materials and Methods, the two cell types require different conditions for the assaying of adenylyl cyclase activity. Back

Received for publication March 11, 1998. Revision received August 4, 1998. Accepted for publication August 28, 1998.


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