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Department of Physiology and Biophysics (H.S., X.L., J.B., D.L.S.)
and Department of Pharmacology (K.N.) The University of
Iowa Iowa City, Iowa 52242
Department of Chemistry
(F.F.) University of Modena and Reggio Emilia 41100 Modena,
Italy
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Interestingly, L457R was completely unresponsive and the K- and H-substituted L457 hLHR mutants were significantly blunted in their cAMP responses to hCG stimulation. Cells expressing L457R were also unresponsive to hCG with regards to increased inositol phosphate production. Other substitutions of L457 were identified, though, that selectively permit the hormonal stimulation of only one of the two signaling pathways. These results suggest a pivotal role for L457 in hormone-stimulated signal transduction by the hLHR.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The LHR present in the ovaries and testes binds either LH, produced by the pituitary in postpubertal men and women, or the structurally related hormone hCG, produced by the placenta of pregnant females, and serves to regulate several key aspects of reproductive physiology and developmental biology. In nonpregnant females, LH is involved in the production of ovarian steroid hormones. The monthly midcycle surge of LH also mediates ovarian follicular maturation and ovulation. In males, the role of the LHR comes into play as early as fetal development. During that time, the LHR present on Leydig cells of the testes binds maternal hCG and stimulates the production of testosterone. Under the actions of this androgen, the external genitalia of the fetus differentiate into the male phenotype. After birth, the male is no longer exposed to maternal hCG and is not exposed to LH until the time of puberty. When puberty is reached, the hypothalamic-pituitary-gonadal axis matures, and the elevated levels of LH then stimulate testosterone production.
In recent years many naturally occurring loss-of-function and gain-of-function mutations of the hLHR have been described (see Ref. 21 for a review). The gain-of-function mutations have been identified in young boys with gonadotropin-independent precocious puberty, also called "testotoxicosis." These individuals have been found to have heterozygous mutations of the hLHR that cause it to constitutively elevate basal cAMP levels. Therefore, in the face of prepubertal low levels of pituitary LH, the testes of these boys constitutively secrete testosterone, which then elicits the physiological changes accompanying male puberty.
At this writing, 14 independent naturally occurring activating mutations of the human (h)LHR have been identified (21, 22). All of them have been identified in the carboxyl half of the receptor, with many of them clustering in TM VI. This may reflect the importance of this region of the hLHR in activating Gs (23, 24). It should also be pointed out, however, that many earlier studies focused only on this portion of the gene when searching for mutations. Indeed, as more recent studies have sequenced the entire carboxyl half of the gene from individuals thought to have activating hLHR mutations, substitutions causing activation have been found in other helices as well.
We had previously reported the identification of an activating mutation
of the hLHR in TM III (25). This mutation results in the substitution
of an arginine in place of a highly conserved leucine. This leucine,
designated Leu III.18 per the Baldwin model of the GPCR TM helices
(26), is conserved in 
70% of rhodopsin-like GPCRs. As one would
predict, cells transfected with the L457R mutant exhibited elevated
levels of basal cAMP. The basal levels of cAMP in cells expressing the
activating mutant, while greater than the basal levels of cells
expressing the wild-type hLHR, were less than the maximal levels of
cAMP observed in cells expressing the wild-type hLHR incubated with a
saturating concentration of hormone. However, whereas cells expressing
most activating mutants will respond with a further increase in cAMP
when challenged with hormone, cells expressing L457K are completely
unresponsive to further cAMP stimulation by hCG addition (25).
The present studies were undertaken to determine the molecular basis for the constitutive activation of the cAMP pathway by hLHR(L457R) and its inability to respond further to hormone. Results presented support the model that Leu III.18 plays a conserved role in stabilizing the inactive state of GPCRs, but that constitutive activity requires both the disruption of the bonds stabilized by this conserved leucine and the introduction of specific residues that promote interhelical interactions stabilizing the active state of the receptor.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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When transfected with maximal amounts of plasmid, the levels of cell
surface receptors for L457K were higher than those of wild-type
receptor. Those for L457R, L457H, and L457A were similar to the
wild-type receptor, and those of L457D were much lower (Table 1
). Since both basal and
hormone-stimulated second messenger production can be dependent upon
cell surface receptor number (27, 28), a correction for receptor
expression must be taken into account. Because second messenger
production was not linear with respect to receptor number over the
range of receptor numbers in these experiments, it was not possible to
correct a given response by dividing by the cell surface binding.
Instead, we used an approach previously described (25, 29, 30) in which
we deliberately varied the plasmid concentrations used in the
transfections to yield cells with similar (no more than 2-fold
different) numbers of cell surface receptors. In our experience, a
2-fold difference in receptor number (within the range of expression
observed in these experiments) has not been of consequence. Therefore,
although the absolute numbers of cell surface receptors varied from
experiment to experiment, a given mutant was always compared within the
same experiment to a wild-type control with matched numbers of cell
surface receptors.
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, cells expressing hLHR(L457R) exhibit approximately
10-fold elevation in basal cAMP levels as compared with cells
expressing similar numbers of wild-type receptors, consistent with the
constitutive activation of this signaling pathway (31). Cells
expressing L457K also exhibited markedly increased basal levels of
cAMP, suggesting that the introduction of a positive charge at codon
457 is required for constitutive activation of the cAMP pathway by the
hLHR. Consistent with this conclusion is the observation that L457H
cells showed slightly elevated levels of basal cAMP and that the basal
levels of cAMP in L457A and L457D cells were not at all elevated.
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). It has been shown, however, that the
measurement of basal inositol phosphate levels does not always reveal
constitutive activation of this pathway due to the relatively poor
sensitivity of this assay (32). Rather, constitutive activation of this
pathway can more readily be observed using a more sensitive reporter
gene assay indicative of C kinase activation (32). Using the C
kinase-responsive reporter gene assay it was possible to detect small,
but reproducible, increases in basal C kinase activity in cells
expressing the K- and R-substituted mutants. Cells expressing L457K
exhibited approximately 20% increase in activity and cells expressing
L457R exhibited about 90% increase in activity. These results suggest
that substitutions of hLHR(L457) with positively charged residues cause
constitutive activation of both the cAMP and the inositol phosphate
pathways.
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).
Our data show that substitution of L467R in TM III of the hLHR with a
positively charged residue stabilizes the hLHR in a conformation that
can constitutively activate both the cAMP and the inositol phosphate
pathways. The following experiments were performed to examine what
effects substitutions of this highly conserved leucine residue have on
hormone-stimulated second messenger production. Looking at the hCG
responsiveness of the wild-type hLHR and the L457-substituted mutants,
there was little (L457K, L457H) or no (L457R) hCG-mediated stimulation
of cAMP in the mutants with a positively charged residue at III.18
(Fig. 1A). This is not because the basal
levels of cAMP induced by these mutants were already maximal. As shown
in Fig. 1B, the Rmax values of L457R, L457K, and L457H were about
one-half that of cells expressing the same numbers of wild-type hLHR.
On the other hand, the hCG responsiveness of cells expressing the
alanine or aspartate-substituted mutants was completely normal (Fig. 1, A and B). Therefore, with regard to the cAMP second messenger pathway,
there appears to be a correlation between the introduction of a
positive charge at III.18, the induction of constitutive activity, and
reduced hCG-mediated stimulation of cAMP.
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, binding assays to intact
cells revealed similar binding affinities for all the L457-substituted
mutants as compared with the wild-type hLHR. Another possibility
considered was whether the R, K, and H mutants might internalize hCG
more rapidly. If so, this may serve to terminate the signaling of cAMP
production by the hormone-occupied receptor. As shown in Fig. 2, we found that cells expressing L457R,
L457K, or L457H do internalize hCG at a faster rate than cells
expressing the wild-type hLHR. In contrast, cells expressing L457A or
L457D internalize hCG at a slower rate than cells expressing the
wild-type receptor.
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). The basal adenylyl cyclase activity
of L457R membranes was elevated more than 2-fold above that of
wild-type hLHR membranes. The basal activities of L457K and L457H
membranes, however, showed little or no increase though. With regard to
the hCG-stimulated cyclase activity in membranes containing the
L457R-substituted hLHR mutants, the maximal response of L457K and L457H
membranes in response to hCG was similar to that of the wild-type hLHR
membranes (Fig. 3). However, L457R membranes showed no detectable
increase in adenylyl cyclase activity in response to hCG. These results
suggest that the increased rate of internalization of hCG by L457R
cells cannot account for the inability of cells expressing this mutant
to respond to hCG with increased cAMP production. Rather, there must be
a structural modification of the L457R mutant per se that
impairs its ability to stimulate adenylyl cyclase when occupied by
agonist.
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).
The L457R mutant, however, is the only mutant in which there is a
correlation between hCG-stimulated cAMP and hCG-stimulated inositol
phosphate responses. Cells expressing L457H responded to hCG with as
great a fold stimulation of inositol phosphates as cells expressing the
wild-type receptor (Fig. 4). This, one should note, is in contrast to
hCG-stimulated cAMP production, which is blunted in the L457H cells
(cf. Fig. 1). Similarly, cells expressing L457K responded to hCG with
increased inositol phosphate production (albeit not as robustly as
wild-type cells or L457R cells; see Fig. 4), whereas they showed little
hCG stimulation of cAMP production (cf. Fig. 1). Cells expressing L457A
also exhibited a moderate response to hCG with regard to inositol
phosphate production (Fig. 4), in contrast to a fully normal response
with regard to cAMP production (cf. Fig. 1). Interestingly, L457D cells
did not show any detectable increases in inositol phosphates in
response to hCG (Fig. 4) in spite of their responding normally to hCG
with increased cAMP (cf. Fig. 1). Taken altogether, the data presented
in Figs. 1 and 4 demonstrate that substitution of L457 of the hLHR with
aspartate causes the receptor to respond to hCG with the selective
stimulation of the cAMP pathway, whereas substitution with histidine
causes the receptor to respond to hCG with the selective stimulation of
the inositol phosphate pathway.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The revised ternary model for GPCR activation predicts that a given receptor exists in the plasma membrane in an equilibrium between inactive R state and an active R* state (see Ref. 1 for a review). The binding of agonist shifts the equilibrium toward the active state, thereby stabilizing this pool of receptor. Constitutively activating mutations of GPCRs are thought to also stabilize the active state of the receptor. Whether a mutation-induced R* conformation is identical to the agonist-induced R* conformation is not yet clear. This issue is further compounded by observations suggesting that there may be multiple activated states for a given GPCR, some intermediary in nature (designated R') (1, 34, 35, 36). As such, any one of a number of intermediary R' or fully active R* states may in turn be stabilized by a given mutation causing constitutive activation.
Leucine 457 of the hLHR represents a highly conserved leucine in TM III in GPCRs (26). The studies presented herein show that substitution of L457R of the hLHR with arginine, lysine, or histidine, but not with alanine or aspartate, causes the receptor to constitutively activate the cAMP pathway, with the greatest activation observed with an R substitution and the least with the H substitution. We could not detect any elevations in inositol phosphate levels in cells expressing these mutants. However, since it was previously shown by Jinsi-Parimoo and Gershengorn (32) that whereas constitutive activation of the inositol phosphate/C kinase pathway by isoforms of the TRH receptor could not be detected by classical measurements of inositol phosphate production but could be readily discerned using a more sensitive C kinase-responsive reporter gene assay, we also examined activation of this pathway using the same C kinase-responsive reporter gene construct. Under these conditions, modest (20% and 90%) increases in basal C kinase activity were observed for cells expressing L457K and L457R, respectively. Although reports differ in the identification of G proteins mediating the stimulation of the inositol phosphate pathway by the LHR (16, 20), it is clear that Gs is not involved and it is likely that Gi is involved. Therefore, our results suggest that substitutions of L457 of the hLHR with positively charged residues stabilize the receptor in a conformation capable of constitutively activating both Gs and Gi.
Recently, a theoretical model for mutation-induced constitutive activation of the hLHR with regard to stimulation of cAMP production via Gs activation was proposed (33). The comparative MD analyses showed that the hLHR sites susceptible to activating mutations lie mainly at interhelical positions close to highly conserved amino acids. Constitutively active hLHR mutants were characterized by the opening of a crevice between i2 and i3 that allows solvent exposure of the intracellular extensions of helices III and VI. This presumably allows greater accessibility of Gs to the regions of the hLHR involved in G protein activation. In agreement with the conclusions of the modeling studies, a peptide corresponding to the juxtacytoplasmic region of helix VI has been shown to be able to activate Gs directly (23, 24).
Similar computer simulations on the L457-substitutions of the hLHR
predict conformations of the R, K, and H-substituted mutants as being
active (Table 1) and further suggest a mechanism underlying their
constitutive activation. According to the theoretical model, L457 is
close to D578 in helix IV (VI:16) as well as to N615 and N619 in helix
VII (VII:17), where N619 belongs to the highly conserved NPXXY motif.
The introduction of a positively charged amino acid at position 457
generates an attractive effect on D458 (VI:16), thus inducing
perturbations in helix III-helix VI and helix III-helix VII interaction
patterns. The local perturbations introduced by these amino acid
replacements culminate in the opening of a solvent-accessible site in
the cytosolic domains potentially involved in Gs recognition.
To provide further insight into the structural features of the L457
mutants, a new hLHR model has been built (F. Fanelli, manuscript in
preparation) by comparative modeling using the recently determined
crystal structure of rhodopsin (37). Preliminary computer simulations
on the wild-type and the L457 mutants with this homology model agree
with the ab initio model in that positively charged amino
acids in position L457 are predicted to induce perturbations in helix
III-helix VI and helix III-helix VII interactions. As shown in Fig. 5, these rearrangements result from the
formation of new interactions between the replacement amino acid at
III.18, on the one hand, and D578 (VI:16), N615 (VII:13), and N619
(VII:17), on the other. Similarly to the ab initio model,
the homology model predicts that the local perturbations induced by
these activating mutations enhance the solvent exposure of the
cytosolic extensions of helices III and VI.
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),
but constitutive activation of the hFSH was observed only with an
arginine substitution (30). Constitutive activation of the
hß2-AR was unrelated to the insertion of
positive residues since arginine, lysine, or alanine caused activation
(in this case the histidine replacement was not examined and an
aspartate substitution caused lack of cell surface expression) (30).
The mutagenesis studies of Leu III.18 in these five GPCRs further
complement other studies suggesting that helix III may serve as a
critical component of the switch for GPCR activation (1, 39, 40, 41, 42, 43). The wild-type LHR can activate both the cAMP and the inositol phosphate second messenger pathways (18, 44). Our studies show that substitution of L457 with arginine renders the hLHR unable to stimulate either the cAMP or the inositol phosphate pathway. However, the other hLHR(L457) substitutions examined herein showed no correlation between an attenuation of hCG-stimulated cAMP and hCG-stimulated inositol phosphate production. Notably, certain substitutions of L457 were found to confer selectivity with respect to the second messenger pathway stimulated by hCG occupancy. For example, in response to hCG, a histidine substitution of L457 causes activation of the inositol phosphate pathway, but not the cAMP pathway. In contrast, an aspartate substitution of L457 causes hCG-stimulated activation of the cAMP pathway, but not the inositol phosphate pathway. Previous studies by Ji and colleagues (45) showed that certain substitutions of K583 in the third extracellular loop of the rat LHR (rLHR) caused the receptor to respond to hCG with increased inositol phosphate production, but not cAMP production, suggesting a divergence in the signaling by the LHR to these two pathways. The present studies now show that not only can certain substitutions of the LHR cause selective agonist-induced activation of the inositol phosphate pathway, but also others can confer selective agonist-induced activation of the cAMP pathway. The ability of substitutions of L457 of the hLHR to abrogate the agonist stimulation of one or both second messenger pathways suggests a key role for this residue in mediating the hormonal activation of the hLHR.
The mutants in which a positively charged R, K, or H was inserted at position 457 were both constitutively active and unresponsive to further hormonal stimulation of cAMP production. It should be noted that although these mutants caused basal levels of cAMP to be elevated, they were not elevated to the same magnitude as cells expressing the wild-type hLHR incubated with a saturating concentration of hormone. Therefore, the lack of hormonal responsiveness is not simply a consequence of the mutants causing maximal elevation of cAMP under basal conditions. Indeed, these observations suggest that the active states of constitutively active hLHR mutants are probably intermediate between the inactive state and the fully activated state stabilized by hormone. Further studies to explore the potential causes of the lack of hCG-stimulated cAMP production by these mutants revealed several interesting features. First, the lack of hCG responsiveness is not due to a decreased binding affinity of the mutants for hCG since the binding affinities of all were normal. Second, it was observed that cells expressing the R, K, and H-substituted L457 mutants all internalized hCG at a significantly higher rate than the wild-type hLHR. Since the internalized hormone-occupied LHR no longer stimulates cAMP production (46), the increased rate of internalization by these mutants could conceivably account for their decreased hCG responsiveness if internalization was the primary means of terminating the signaling to Gs. Indeed, when the rat LHR (rLHR) containing a mutation of Leu III.18 to arginine was examined, it was found to constitutively elevate basal cAMP production, to be unresponsive to hCG with regard to further cAMP increases, and to internalize hCG at a faster rate than the wild-type rLHR (47). When isolated membranes (which, by definition, are devoid of any internalization activity) derived from cells expressing either the wild-type or the mutant rLHR were examined for basal and hCG-stimulated cAMP production, it was found that the membranes with mutant rLHR had an elevated basal adenylyl cyclase activity (consistent with the mutant’s constitutive activity), but responded similarly to wild-type rLHR membranes when incubated with hCG (47). These observations supported the hypothesis that the increased internalization of hCG by the rLHR mutant was responsible for the decreased ability of intact cells to respond to further hormonal stimulation of the cAMP pathway.
We, therefore, also measured the basal and hCG-stimulated adenylyl cyclase activity of membranes isolated from cells expressing the wild-type hLHR or the mutants L457R, L457K, or L457H. Although all three of these mutants, when analyzed in the context of intact cells, exhibited constitutively elevated levels of basal cAMP, only L457R showed a significant increase in basal cyclase activity above that of wild-type hLHR membranes. It is likely, however, that the inability to detect a measurable increase in the basal cyclase activity of the L457K and L457H membranes is due to the reduced sensitivity of the measurement of adenylyl cyclase activity in membranes as compared with cAMP accumulation in intact cells. Since the constitutive activity (as measured by cAMP accumulation) of the L457R mutant is greater than that of the L467K and L457H mutants, its increased basal activity, as determined in cyclase assays, may be more readily detectable. With regard to agonist-stimulated cyclase activity, the L457K and L457H membranes responded to hCG with increased adenylyl cyclase activity, suggesting that the inability of these mutants to respond in the context of intact cells is due to their more rapid internalization of hCG and/or to other properties of the receptors that are present in intact cells but not in membranes. However, the L457R membranes remained completely unresponsive to hCG stimulation. These data contrast with the observations reported on the rLHR containing the comparable substitution and suggest a different mechanism underlying the lack of hCG-stimulated cAMP production by cells expressing the human vs. rat LHR in which Leu III.18 is substituted by arginine.1 The lack of hCG responsiveness of membranes containing hLHR(L457R) suggests that there is a modification of this mutant that makes it unable to further activate Gs when occupied by hCG. This change may reflect a structural alteration or posttranslational modification of the receptor and/or an association of the mutant receptor with other proteins. Whatever it may be, it must be preserved during the preparation of membranes from intact cells, and it prevents the receptor from undergoing the transition from one of constitutively active in an intermediate state to one of full activation stabilized by hormone.
It is intriguing to note that certain substitutions of Leu III.18 of the hFSHR also cause this receptor to exhibit little or no response to FSH with regard to further increases in cAMP (30). The demonstration that certain substitutions of Leu III.18 of either the hLHR or hFSHR cause decreases in hormone-stimulated activation of the cAMP pathway and/or inositol phosphate pathway suggests that Leu III.18 plays a key role in the transduction of hormone binding to the activation of G proteins in these two related GPCRs. Because substitutions of Leu III.18 of the hß2-AR receptor do not cause decreased hormone responsiveness (30), it suggests that the role of Leu III.18 in hormone-stimulated signal transduction may be restricted to the gonadotropin receptors, or possibly the glycoprotein hormone receptors if it is found to play a similar role in the closely related TSHR.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Plasmids and Cells
The wild-type hLHR cDNA was kindly provided by Ares Advanced
Technology (Ares-Serono Group, Randolph, MA) and was subcloned into
pcDNA 3.1 (Invitrogen, San Diego, CA). Mutagenesis was
performed using the PCR overlap extension method (49, 50). The entire
region amplified by PCR, as well as the sites of ligation, were
sequenced to ensure that there were no unintended mutations of the
amplified cDNA. DNA sequencing was performed either within our
laboratory or by automated sequencing within the DNA Core of the
Diabetes and Endocrinology Research Center of the University of
Iowa.
Human embryonic 293 cells were obtained from the American Type Tissue Collection (CRL 1573) and were maintained at 5% CO2 in growth media consisting of high-glucose DMEM containing 50 µg gentamicin, 10 mM HEPES, and 10% newborn calf serum. For most experiments, cells were plated onto 35-mm wells that had been precoated for 1 h with 0.1% gelatin in calcium and magnesium-free PBS, pH 7.4. Cells were transiently transfected when they were 50–70% confluent following the protocol of Chen and Okayama (51) except that the overnight precipitation was performed in a 5% CO2 atmosphere. Differing concentrations of plasmids were used to achieve the same cell surface expression of a given mutant with the wild-type hLHR. Cells were then washed with Waymouth’s MB752/1 media modified to contain 50 µg gentamicin and 1 mg/ml BSA, after which fresh growth media were added. The cells were used for experiments 24 h later.
Standardization of Cell Surface Receptor Numbers
In all experiments where the signaling properties of cells
expressing the hLHR(wt) were compared with cells expressing a mutant,
293 cells were transiently transfected with varying plasmid
concentrations such that within each experiment the number of cell
surface receptors for a given mutant was matched to cells expressing
comparable numbers of wild-type receptors. For each experiment the
ratio of 125I-hCG binding to cells expressing a
given mutant vs. the matched controls was determined, and
the experiment was used only if the ratio was within the range of
0.5–2.0 (i.e. within a 2-fold difference). For the
experiments measuring basal and hormone-stimulated cAMP production
(Table 1 and Fig. 1) the means ± SEM of the
ratios of cell surface 125I-hCG binding to mutant
vs. wild-type hLHR-expressing cells were the following:
0.98 ± 0.16 for L457R (n = 8); 1.30 ± 0.18 for L457K
(n = 7); 0.92 ± 0.06 for L457H (n = 5); 1.19 ±
0.15 for L457A (n = 7); and 1.17 ± 0.15 for L457D (n =
9). For the experiments measuring basal (Table 2) and hCG-stimulated
inositol phosphate production (Fig. 4) the means ±
SEM of the ratios of cell surface
125I-hCG binding to mutant vs.
wild-type hLHR-expressing cells from three experiments were the
following: 0.81 ± 0.13 for L457R; 0.97 ± 0.30 for L457K;
0.1.18 ± 0.10 for L457H; 1.09 ± 0.19 for L457A; and
1.19 ± 0.24 for L457D. For the experiments measuring basal C
kinase-responsive luciferase activity (Table 2) the means ±
SEM of the ratios of cell surface
125I-hCG binding to mutant vs.
wild-type hLHR-expressing cells were the following: 1.13 ± 0.11
for L457R (n = 9); 1.17 ± 0.12 for L457K (n = 11);
1.09 ± 0.12 for L457H (n = 11); 0.81 ± 0.08 for L457A
(n = 13); and 0.96 ± 0.014 for L457D (n = 6).
Therefore, the mutants were well matched to the controls and what
little deviation existed varied equally between mutants being expressed
2-fold higher vs. 2-fold lower density than the
wild-type hLHR.
Binding Assays to Intact Cells Expressing the hLHR
HEK 293 cells were plated onto gelatin-coated 35-mm wells and
transiently transfected as described above. On the day of the
experiment cells were placed on ice for 15 min and washed two times
with cold Waymouth’s MB752/1 containing 50 µg/ml gentamicin and 1
mg/ml BSA but no sodium bicarbonate. To determine the maximal binding
capacity, the cells were then incubated overnight at 4 C in the same
media containing a saturating concentration of
125I-hCG (500 ng/ml) with or without an excess of
unlabeled crude hCG (50 IU/ml). To determine the binding affinity, the
cells were incubated overnight at 4 C with increasing concentrations of
125I-hCG in the presence or absence of unlabeled
hCG. To terminate the assay, the cells were placed on ice. The contents
of each well were scraped into a plastic tube on ice and combined with
a 1 ml wash using cold HBSS modified to contain 50 µg/ml gentamicin
and 1 mg/ml BSA. The tubes were centrifuged at 4 C and the pellets
resuspended in 2 ml of the same wash media. After a second
centrifugation, the supernatants were aspirated and the pellets counted
in a 
counter. Apparent binding affinities were determined as the
concentrations of 125I-hCG yielding half-maximal
binding as calculated by the DeltaGraph software Deltapoint (Monterey,
CA) when the data were fit to a sigmoidal equation (52).
Measurement of cAMP or Inositol Phosphate Production
The levels of cell surface receptors were measured within the
same experiment, and only those experiments in which the numbers of
cell surface receptors for wild-type vs. mutant receptors
differed by no more than 2-fold (see above) were used for second
messenger analyses. HEK 293 cells were plated on gelatin-coated 35-mm
wells and transfected as described above.
For cAMP determinations, cells were washed on the day of the experiment twice with warm Waymouth MB752/1 media containing 50 µg/ml gentamicin and 1 mg/ml BSA and placed in 1 ml of the same medium containing 0.5 mM isobutylmethylxanthine. After 15 min at 37 C, a saturating concentration of hCG (100 ng/ml final concentration) or buffer only was added, and the incubation was continued for 60 min at 37 C. The cells were then placed on ice, the media were aspirated, and intracellular cAMP was extracted by the addition of 0.5 N perchloric acid containing 180 µg/ml theophylline and then measured by RIA. All determinations were performed in triplicate.
For inositol phosphate determinations, after the transfection, the cells were washed with inositol-free DMEM containing 1% newborn calf serum, 20 mM HEPES, and 50 µg/ml gentamicin and placed in 1 ml of the same media supplemented with 2 µCi/ml of [2-3H]myo-inositol for 24 h. On the day of the experiment, the cells were washed twice with warm Waymouth MB752/1 media modified to contain 50 µg/ml gentamicin, 1 mg/ml BSA, and 20 mM LiCl and then placed in this medium. After 15 min at 37 C, a saturating concentration of hCG (100 ng/ml final concentration) or buffer only was added, and the incubation was continued for 60 min at 37 C. To terminate the assay, the cells were placed on ice, and cold 0.5 N perchloric acid was added to each well. The inositol phosphates were extracted and assayed as described previously (53). All determinations were performed in triplicate.
Measurement of C Kinase Activation Using a C Kinase-Responsive
Reporter Gene Assay
Plasmid containing a C kinase-responsive AP-1-fos-Luc reporter
gene construct was a gift from Dr. Marvin Gershengorn (NIDDK/NIH). HEK
293 cells were plated onto gelatin-coated 35-mm wells and transiently
cotransfected as described above with the C kinase-responsive reporter
gene construct, pcDNA3.1/neo containing the cDNA encoding the hLHR(wt)
or L457-substituted mutant, and empty pcDNA3.1/neo. The concentrations
of plasmid encoding the wild-type and mutant receptors were varied so
that a given mutant could be matched with a parallel set of cells
expressing the same level of wild-type receptor (see above). Because
luficerase activity was observed to be affected by the concentration of
pcDNA3.1/neo in the 293 cells, empty vector was utilized to adjust the
total concentration of pcDNA3.1/neo to be equal in all the cells.
Measurement of Adenylyl Cyclase Activity
The levels of cell surface receptors were measured within the
same experiment, and only those experiments in which the numbers of
cell surface receptors for wild-type vs. mutant receptors
differed by no more than 2-fold (see above) were used to determine
adenylyl cyclase activity. Membranes were prepared from 293 cells as
described previously (23). Adenylyl cyclase assays, based on the
procedure of Salomon (54), were performed as described previously (23)
except that GTP was not added to the assay. This change was
incorporated into the protocol because we observed a greater fold
stimulation of adenylyl cyclase activity in response to hCG when the
exogenous GTP was omitted.
Internalization of Receptor-Bound hCG
The hLHR-mediated internalization of hCG was measured following
the protocol described by Ascoli and colleagues (55). Transiently
transfected cells in 35-mm wells were preincubated in 1 ml Waymouth
MB752/1 media containing 1 mg/ml BSA and 20 mM HEPES, pH
7.4, for 30 min at 37C. 125I-hCG was then added
to give a final concentration of 40 ng/ml (with or without an excess of
unlabeled hCG to correct for nonspecific binding), and the cells were
incubated for 9 min at 37 C. The cells were then washed twice with cold
HBSS modified to contain 50 µg/ml gentamicin and 1 mg/ml BSA. The
surface-bound 125I-hCG was released by incubating
the cells on ice in 1 ml of cold 50 mM glycine, 150
mM NaCl, pH 3, for 4 min and rinsing them with 1 ml of the
acidic buffer (56). The acid washes from each well were combined and
counted to determine the amount of surface-bound
125I-hCG. Each well of acid-treated cells was
then solubilized in 0.5 N NaOH and counted to determine the
amount of internalized radioactivity. The results of these experiments
are expressed as an internalization index, which is defined as the
ratio of internalized vs. surface-bound
125I-hCG (57). Under the experimental conditions
used herein, the internalization index accurately reflects the rate of
internalization (55, 57). The rate of internalization is a first order
rate constant (55, 57) and is, therefore, independent of the
concentration of receptor or hormone. Therefore, for these experiments,
no effort was made to standardize the number of cell surface receptors
between the wild-type vs. mutant-expressing cells. In
addition, a subsaturating concentration of hormone was used to conserve
125I-hCG.
Molecular Modeling of the hLHR Mutants of L457
The initial structures of the L457 hLHR mutants were obtained by
replacing the target amino acid in the wild-type hLHR input structure
previously built after an ab initio approach (33). Energy
minimization and molecular dynamics simulations of the mutants were
performed using the program CHARMm (Molecular Simulations, Inc,
Waltham, MA) following the computational protocol previously described
(33).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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These studies were supported by NIH Grant HD-22196 (to D.L.S.). The services and facilities of the University of Iowa Diabetes and Endocrinology Research Center, supported by NIH Grant DK-25295, are also acknowledged.
1 We have replicated the previously reported
results showing that membranes expressing the rLHR containing a Leu
III.18 to arginine substitution respond with increased adenylyl cyclase
activity using the same conditions as those used to assay hLHR(L457R).
Therefore, we conclude that the differences observed are indeed due to
differences between the two species of receptor and not to
methodological differences. 
Received for publication November 7, 2000. Revision received February 21, 2001. Accepted for publication March 19, 2001.
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