Departments of Physiology and Biophysics (Y.-X.T., A.N.A., X.L., D.L.S.) and Pharmacology (K.N.) The University of Iowa Iowa City, Iowa 52242
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ABSTRACT |
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Because Leu III.18 is highly conserved in rhodopsin-like G protein-coupled receptors (GPCRs), we tested the effects of substitution of the comparable leucine in the human ß2-adrenergic receptor (hß2-AR). Substitution of L124 in the hß2-AR with arginine, lysine, or alanine resulted in constitutive activation as evidenced by increased basal levels of cAMP that could be attenuated by an inverse agonist. In all cases, isoproterenol-stimulated cAMP was unaffected.
Taken altogether, our data support a model whereby Leu III.18 may play a general role in GPCRs by stabilizing them in an inactive state. Constitutive activation may arise by both a disruption of Leu III.18 as well as the introduction of a specific residue that serves to stabilize the active state of the receptor.
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INTRODUCTION |
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In recent years many naturally occurring constitutively activating mutations of both the hTSHR (4) and hLHR (5) have been described. Those in the hTSHR have been identified in patients with hyperfunctioning thyroid adenomas and thyroid carcinomas and those in the hLHR have been seen in young boys with gonadotropin-independent precocious puberty (5) or Leydig cell tumors (6). Only one putative constitutively activating mutation of the hFSHR has been reported, a D567G substitution in the carboxyl portion of the third intracellular loop (i3) (7). This mutation was originally identified in a hypophysectomized male who exhibited normal testes volume after testosterone treatment, thus suggesting the patient carried an activating mutation of the hFSHR. In the initial report, the observed increase in basal cAMP in cells transfected with hFSHR(D567G) was quite low (<2-fold). Subsequent studies from two other laboratories were unable to demonstrate any elevation of basal cAMP in cells transfected with hFSHR(D567G) (8, 9). Therefore, it may be that the D567G substitution is a nonfunctional polymorphic mutation in the hFSHR, and a different, as yet unidentified, mutation may be causing the phenotype of the patient in the original study. Alternatively, the D567G mutation may cause a relatively small constitutive activation of the hFSHR that may be difficult to observe in a reproducible manner.
Other mutations in transmembrane (TM) VI of the hLHR that are known to cause constitutive activation of the hLHR were introduced by Kudo et al. (8) into the comparable residues of the hFSHR, and these, too, were found to be unable to induce constitutive activation of this receptor. These observations have led to the speculation that the hFSHR may be in a more highly constrained conformation and less susceptible in general to mutation-induced constitutive activation than the highly related hLHR and hTSHR. Alternatively, it is possible that the roles of TM VI and i3 in maintaining an inactive state of the hFSHR may be different from that of the hLHR and hTSHR. To address this question, we chose to introduce a mutation into the hFSHR in a region other than TM VI and i3 that would, based upon results obtained with the hLHR, be predicted to cause constitutive activation. The residue chosen for mutagenesis of the hFSHR was based on a previously reported mutation of L457 in TM III of the hLHR that was shown to cause constitutive activation (10). This leucine is conserved not only in the glycoprotein hormone receptors, but in >70% of all rhodopsin-like GPCRs and is defined as Leu III.18 by Baldwin et al. (11) in their model of GPCR transmembrane helices. Given its highly conserved nature, the strong constitutive activation observed in the hLHR(L457R) mutant, and the location of the leucine in a region other than TM VI and i3, we undertook the present studies to investigate the effects of substitution of the comparable TM III leucine (L460) within the hFSHR.
Our studies show that the substitution of L460 in TM III of the hFSHR does indeed cause a robust increase in basal levels of cAMP, consistent with it being constitutively active. The data presented further show that substitution of the comparable leucine residue in the human ß2-adrenergic receptor (hß2-AR) also causes constitutive activation. Leu III.18 is situated in a region of TM III postulated to be involved as a general activating switch region for GPCRs (12). Our data support this model and further implicate Leu III.18 as serving a conserved role in GPCRs in maintaining them in an inactive state. However, our data further suggest that constitutive activation only arises from a combination both of disruption of Leu III.18 as well as the introduction of specific residues that presumably stabilize the receptor in an active state.
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RESULTS |
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The data presented thus far suggest that, within the context of the
amino acids examined (arginine, lysine, alanine, and aspartic acid),
constitutive activation of the hFSHR by substitution of L460 is
restricted to the introduction of an arginine. If one examines the
ability of the different mutants to respond to FSH with increased cAMP
production, however, a different picture emerges. As shown in panel B
of Fig. 3, cells expressing hLHR(L460) mutants containing arginine,
lysine, alanine, or aspartic acid are blunted in their response to
hFSH, as indicated by the decreased Rmax of cells expressing each of
the mutants. The Rmax of L460R cells is approximately 25% that of
cells expressing wild-type hFSHR; however, this is due primarily to the
elevated levels of cAMP due to this mutants constitutive activity, as
opposed to the ability of the cells to respond to FSH (cf. panels A, B,
and C of Fig. 3
). The fold increases in cAMP elicited by FSH in L460K,
L460A, and L460D cells are greater than that of the L460R cells, but
they are still far less than observed for cells expressing the
wild-type hFSHR (Fig. 3C
). Thus, the substitution of L460 of the hFSHR
with any of the four residues examined results in a marked attenuation
of FSH responsiveness.
One possible cause of the decreased FSH responsiveness of cells
expressing arginine-, lysine-, alanine-, or aspartic acid-substituted
L460 mutants could be a decreased binding affinity of the mutants for
FSH. This possibility was examined directly by determining the binding
affinities of the cell surface form of each of the mutants. As shown in
Table 1, the L460A and L460D mutants
bound FSH with the same affinity as the wild-type receptor, while the
L460R and L460K mutants bound with a similar or slightly higher
affinity as the wild-type receptor. For each mutant, therefore, we can
clearly exclude a decrease in binding affinity as a cause for the
decreased FSH-stimulated cAMP production.
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Figure 5
summarizes the results of several experiments examining the basal and
isoproterenol-stimulated levels of cAMP in cells expressing
the L124-substituted hß2-AR mutants as compared
with cells expressing comparable numbers of cell surface wild-type
hß2-AR. Cells expressing
hß2-AR(L124R) exhibited a 9-fold elevation of
basal cAMP production relative to cells expressing the wild-type
receptor (Fig. 5A
). Thus, the effects of the L124R substitution in the
hß2-AR on basal cAMP production were similar to
those observed for hFSHR(L460R) (see Figs. 2
and 3
) and hLHR(L457R)
(10). Interestingly, unlike the hFSHR, cells expressing lysine and
alanine substitutions of Leu III.18 also displayed elevated basal cAMP
production relative to the wild-type receptor (Fig. 5A
). Furthermore,
the cells expressing L124R, L124K, or L124A
hß2-AR mutants each responded to isoproterenol
with a Rmax comparable to cells expressing the wild-type receptor (Fig. 5B
). Therefore, unlike the hLHR (10) or the hFSHR (Figs. 2
and 3
), in
which substitution of Leu III.18 impairs hormone-stimulated cAMP
production, substitution of Leu III.18 in the
hß2-AR does not affect agonist-stimulated cAMP
production.
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DISCUSSION |
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The FSHR, LHR, and TSHR form a subset of related GPCRs within the superfamily of rhodopsin-like receptors (1, 2, 3). In contrast to the hFSHR, numerous activating mutations of both the hLHR and hTSHR have been reported (see Refs. 5, 4 , respectively, for reviews). Furthermore, some mutations within i3 or TM VI of the hLHR that are known to cause constitutive activation have been reported to be without effect when introduced into the hFSHR (8, 9). These observations have led to the speculation that the hFSHR may be more resistant to mutation-induced constitutive activation than the hLHR or hTHSR. However, because the previous mutations that were ineffective in inducing constitutive activation of the hFSHR were located in i3 or TM VI, we chose to examine whether a mutation elsewhere could induce constitutive activation in this receptor. Previous studies had identified a leucine-to-arginine substitution in TM III of the hLHR that results in constitutive activity of the hLHR (10). Because this leucine is highly conserved in the superfamily of rhodopsin-like GPCRs (designated Leu III.18 per the numbering system of Baldwin et al. (11), and because this mutation is one of the more robust constitutively activating mutations of the hLHR (10), substitution of the comparable leucine in the hFSHR was chosen as a means to test whether the hFSHR could be made constitutively active.
As shown herein, a L460R substitution in TM III of the hFSHR causes an increase in basal levels of cAMP in cells expressing the mutant hFSHR, consistent with the mutation causing constitutive activity. It should be noted that because there are no known inverse agonists for either the hLHR or the hFSHR, verification of constitutive activation of these receptors by the attenuation of increased basal activity by an inverse agonist cannot be ascertained. Consequently, constitutive activation of the gonadotropin receptors has been defined routinely by an observed increase in basal levels of cAMP in transfected cells under conditions where the cell surface expression of the mutant receptor is comparable to (or in some cases less than) that of the wild-type receptor (5, 7, 19). Our results clearly show that the hFSHR is not entirely resistant to mutation-induced constitutive activation. Of four amino acid substitutions tested (arginine, lysine, alanine, and aspartate), constitutive activation of the hFSHR was only observed by substitution of Leu III.18 with arginine. Therefore, the introduction of a positive charge (i.e. lysine) into the III.18 position is not sufficient for conferring constitutive activity.
Having previously shown that a L460R substitution of the hLHR causes constitutive activation (10) and now showing that the comparable L457R substitution of the hFSHR causes constitutive activation, we wished to explore the generality of the substitution of Leu III.18 in causing constitutive activation of GPCRs. Toward this end, we investigated the effects of substituting L124 of the hß2-AR. Indeed, substitution of L124 with arginine, lysine, or alanine caused constitutive activation of the hß2-AR, as determined by increased levels of basal cAMP in transfected cells that could be attenuated by the inverse agonist ICI 118,551, properties previously shown to be associated with constitutively activating mutants of the hß2-AR (13, 14). While our studies were in progress, two other reports were published showing that substitutions of Leu III.18 in the rat m1 muscarinic acetylcholine receptor (rm1AChR) and the C5a receptor (C5aR) cause constitutive activation (12, 20). Both studies used alanine scanning mutagenesis and thus were not focusing on Leu III.18 per se. However, for both GPCRs, it was found that one of several alanine substitutions that resulted in constitutive activation included that of the Leu III.18. As of this writing, therefore, constitutive activation by substitution of Leu III.18 has now been shown for five different GPCRs (the hLHR, hFSHR, hß2-AR, rm1AChR, and C5aR) which couple to at least two different second messenger pathways. Of these, however, the amino acid requirements for conferring constitutive activation were only examined in the hFSHR and the hß2-AR. Whereas any of three amino acids tested (arginine, lysine, alanine) replacing Leu III.18 of the hß2-AR resulted in constitutive activation, only one (arginine) of four amino acids tested (arginine, lysine, alanine, and aspartate) replacing Leu III.18 of the hFSHR resulted in constitutive activation. Consequently, alanine scanning mutagenesis of TM III of the hFSHR would not have identified Leu III.18 as a residue whose substitution would cause constitutive activation.
The first report of a mutation causing constitutive activation of a
GPCR showed that substitution of an alanine residue in the third
intracellular loop of the 1B-AR with any other
amino acid resulted in constitutive activation of the inositol
phosphate second messenger pathway (21). It has since been reported
that any residue substituted in place of an aspartate within the highly
conserved E/DRY motif at the cytoplasmic end of TM III also results in
constitutive activity of this receptor (22). Substitution of these two
residues does not, therefore, appear to be selective in that any other
residue introduced in their place confers constitutive activity. This
has led to the hypothesis that constitutive activation of GPCRs results
from the disruption of interhelical bonds that stabilize the receptor
in an inactive state. However, in many other studies examining
constitutive activation of GPCRs (including the hLHR) as a result of
different amino acid substitutions of a given residue, there appears to
be a selectivity as to which newly introduced residues can confer
constitutive activity (see Refs. 23, 24, 25, 26 for examples). In these cases,
as with Leu III.18 of the hFSHR, some, but not all substitutions of a
given residue result in constitutive activation. The hypothesis that
constitutive activation arises solely as a result of disruption of
interhelical bonds that stabilize the receptor in an inactive state
cannot fully account for these observations. Rather, in these cases, a
more likely hypothesis is that constitutive activity arises by the
disruption of interhelical bonds stabilizing the inactive state as well
as formation of other bonds involving the newly introduced residue,
which can stabilize an active state of the receptor. It should further
be considered that whereas different substitutions causing constitutive
activation may stabilize the same activated state, it is also possible
that different substitutions impart different interhelical
interactions, which in turn stabilize different activated states. An
additional point that needs to be taken into consideration is that in
the saturation mutagenesis studies of the
1B-AR by Cotecchia and colleagues (21, 22) it was observed that the wild-type receptor did not exhibit any
basal inositol phosphate production above that seen in mock transfected
cells. This lack of basal activity by the wild-type receptor is unusual
and may contribute to the lack of selectivity reported in these
studies.
Molecular modeling of the GPCR helices based upon the known structure of rhodopsin predicts that Leu III.18 lies approximately midway in the third transmembrane helix between the predicted cytoplasmic and extracellular boundaries and that it faces inward toward the crevice formed by the transmembrane helices (11). Studies using different experimental approaches have suggested that both the ligand-induced and mutation-induced activation of GPCRs involves the movement of helices III and VI relative to each other (16, 27, 28, 29, 30). Based on the data from these studies, the hypothesis has been put forth that helices III and VI serve as switches for GPCR activation (12). The conserved nature of Leu III.18, coupled with its position in a region postulated to be a switch region for GPCR activation, suggests that it may serve a conserved function in GPCRs by being one of several residues on this face of TM III that serve to help stabilize the receptor in an inactive conformation. Interestingly, residues in TM III of the TSHR predicted to lie on the same face as Leu III.18 have also been shown to cause constitutive activation. For example, naturally occurring mutations resulting in the substitution of S505 with either arginine or asparagine or the substitution of S509 with alanine result in TSHR that constitutively activates the cAMP pathway (31, 32, 33).
Whereas the L460R substitution in the hFSHR causes constitutive activation, it also renders the receptor unresponsive to further hormonal stimulation, in spite of the fact that the mutant binds hFSH with the same affinity as the wild-type receptor. Because cells expressing the mutant hFSHR have elevated levels of basal cAMP, the ability of the mutant to activate cAMP synthesis is at least partially intact. Therefore, it is likely that the impairment in FSH-stimulated cAMP production by hFSHR(L460R) is restricted to the transduction of agonist binding to cAMP accumulation. Whether this impairment is at the level of the receptor itself or reflects a postreceptor event remains to be elucidated. Interestingly, substitutions of hFSHR(L460) with either lysine, alanine, or aspartic acid also resulted in decreased FSH-stimulated cAMP. Unlike the arginine substitution, however, they did not also cause constitutive activation. Although we cannot yet conclude whether or not the different substitutions mediate decreased hormonal responsiveness by the same mechanism, the data thus far suggest that Leu460 may play a critical role in FSH-stimulated cAMP production by the hFSHR. It has previously been shown that the comparable L457R mutant of the hLHR, which is constitutively active, is also relatively unresponsive to further hormonal stimulation (10). However, as shown herein, when Leu III.18 in the hß2-AR was substituted with arginine, lysine, or alanine, the resulting mutants were both constitutively active and fully responsive to additional stimulation by isoproterenol. Therefore, in contrast to its apparent role in stabilizing the inactive state of GPCRs, the role of Leu III.18 in hormone-stimulated cAMP production is not universal among all GPCRs. Rather, it appears to be restricted to the gonadotropin receptors (or possibly even the glycoprotein hormone receptors if the TSHR is found to behave similarly).
One possible mechanism by which substitutions of L460 in the hFSHR might lead to an impairment of FSH-stimulated cAMP production in intact cells is the potential increased internalization of the hormone-receptor complex by the mutants. Indeed, when Leu III.18 of the rat LHR (rLHR) is substituted with arginine, the resulting receptor internalizes hCG at a much faster rate than the wild-type receptor (34). Interestingly, although cells expressing this rLHR mutant do not respond to hCG with increased cAMP, isolated membranes derived from these cells are fully responsive to hCG (34). These data suggest that, for the rLHR, mutation of Leu III.18 to arginine does not impair hCG-induced coupling of the receptor to Gs, but rather it induces a faster rate of internalization of the hormone-receptor complex, thus terminating the signal. However, for the hFSHR, our data clearly show a lack of correlation between decreased FSH-stimulated cAMP production and increased rates of internalization of the hormone-receptor complex in cells expressing Leu III.18-substituted hFSHR mutants. Thus, whereas arginine, lysine, alanine, and aspartate substitutions of hFSHR(L460) resulted in decreased hFSH responsiveness, only the mutants with arginine and lysine substitutions internalized hCG at a faster rate. Whether the correlation between decreased hormonal responsiveness and increased rates of internalization in the rLHR and the lack thereof in the hFSHR are due to a difference between species (i.e. human vs. rat) and/or LHR vs. FSHR remains to be determined.
In summary, these studies unequivocally show that the hFSHR can be made constitutively active. The observations that substitutions of Leu III.18 cause constitutive activation of several GPCRs, but that the substitutions rendering constitutive activity are receptor specific, suggest that Leu III.18 contributes to maintaining rhodopsin-like GPCRs in an inactive state. However, specific substitutions are required to stabilize an active conformation. Lastly, Leu III.18 also is involved in the agonist-mediated activation of the cAMP pathway of the gonadotropin receptors, but not other GPCRs. Further studies identifying the rearrangements of interhelical bonds that occur as a result of the substitutions of Leu III.18 should provide a more detailed understanding for the mechanisms of mutation-induced vs. hormone-induced activation of the gonadotropin receptors.
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MATERIALS AND METHODS |
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Human embryonic 293 cells were obtained from the American Type Tissue Collection (CRL 1573) and were maintained at 5% CO2 in a culture medium 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. For competition binding experiments on cells expressing the hß2-AR, cells were plated on uncoated 100-mm dishes. Cells were transiently transfected when they were 5070% confluent following the protocol of Chen and Okayama (37) except that the overnight precipitation was performed in a 5% CO2 atmosphere. Cells were then washed with Waymouths 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.
Binding Assays to Intact Cells Expressing the hFSHR
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 washed two times with warm Waymouths MB752/1
containing 50 µg/ml gentamicin and 1 mg/ml BSA. To determine the
maximal binding capacity, the cells were then incubated 1 h at 37
C in the same media containing a saturating concentration of
125I-hFSH (500 ng/ml final concentration) with or
without an excess of unlabeled PMSG (480 IU/ml final concentration). To
determine the binding affinity, the cells were incubated with
increasing concentrations of 125I-hFSH in the
presence or absence of unlabeled PMSG. The assay was finished by
washing the cells three times with cold HBSS modified to contain 50
µg/ml gentamicin and 1 mg/ml BSA. The cells were then solubilized in
100 µl of 0.5 N NaOH and transferred to plastic test
tubes with cotton swabs. Apparent binding affinities were determined as
the concentrations of 125I-hFSH yielding
half-maximal binding as calculated by the Deltapoint software
(DeltaGraph, Monterey, CA) when the data were fit to a sigmoidal
equation (38).
Admittedly, the binding of 125I-hFSH was not performed under equilibrium conditions. Although ideally the binding of hormone should be performed at 4 C, conditions in which no internalization occurs, the binding of 125I-hFSH at 4 C results in unacceptably high levels of nonspecific binding. The conditions described (1 h at 37 C), however, were found to yield reasonable levels of nonspecific binding (i.e. <20% of total counts per min bound). In contrast to hFSH binding to the hFSHR, hCG binding to the hLHR can readily be measured at 4 C. Therefore, we compared the binding of 125I-hCG overnight at 4 C vs. 1 h at 37 C to cells expressing the hLHR(wt) or hLHR(L457R) mutant, which like the hFSHR(L460R) mutant internalizes hormone at a faster rate. Although the absolute numbers differed between the two assay conditions, the relative amount of binding to wild-type vs. mutant receptor was the same. By extrapolation, we feel it reasonable to assume that the relative amounts of wild-type vs. mutant hFSHR determined by the bindings at 1 h at 37 C are a good approximation.
Binding Assays to Intact Cells Expressing the
hß2-AR
To determine cell surface receptor numbers, bindings were
performed to intact cells. Dishes were placed on ice for 10 min and
subsequently washed twice with ice-cold Waymouths MB752/1 medium
supplemented with 1 mg/ml BSA and 50 µg/ml gentamicin but lacking
bicarbonate. Dishes were incubated overnight with a saturating
concentration of [125I]-pindolol (200 pM,
final concentration) and either cold buffer A (150 mM NaCl,
20 mM HEPES, 2.1 mM ascorbic acid, pH 7.4) to
measure total binding, a saturating concentration of propranolol
dissolved in cold buffer A (1 µM, final concentration) to
measure nonspecific binding, or CGP 12177 dissolved in cold buffer A
(0.3 µM, final concentration) to determine cell surface
expression. Dishes were incubated overnight in the dark at 4 C. The
following day, cells were scraped, rinsed, and transferred into 12
x 75 tubes. Bindings were terminated by rapid filtration over filters
(Whatman, Clifton, NJ) preincubated for 1 h in 3%
BSA dissolved in PBS. Filters were washed five times with iced 1% BSA
in PBS, dried, and counted for 1 min with a counter. All points
were in duplicate. Specific binding was calculated as the difference
between total and nonspecific binding. Cell surface binding was
calculated from the percentage of total binding displaced by the
hydrophilic ligand CGP-12177.
Measurement of cAMP Production
In each experiment in which cAMP production was measured, the
levels of cell surface receptors were measured within the same
experiment. Only those experiments in which the numbers of cell surface
receptors for wild-type vs. mutant receptors differed by no
more than 2-fold were used for cAMP analyses. 293 cells were plated on
gelatin-coated 35-mm wells and transfected as described above. On the
day of the experiment, cells were washed 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 hormone was added (hFSH at 100 ng/ml
final concentration, isoproterenol at 1 µM
final concentration, or ICI 118, 551 at 10 µM,
final concentration) or buffer only and the incubation was continued
for 60 min at 37C. 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.
Internalization of hFSH
The hFSHR-mediated internalization of hFSH was measured
following the protocol described by Ascoli and colleagues (39).
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 37 C. 125I-hFSH was
then added to give a final concentration of 40 ng/ml (with or without
an excess of PMSG 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-hFSH 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 (40, 41). The acid washes from each well were
combined and counted to determine the amount of surface-bound
125I-hFSH. 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-hFSH (42). Under the experimental conditions
used herein, the internalization index accurately reflects the rate of
internalization (39, 42). The rate of internalization is a first order
rate constant (39, 42) and is, therefore, independent of the
concentration of receptor or hormone. Therefore, for these experiments
no effort was made to standardize the numbers of cell surface receptors
between the wild-type vs. mutant-expressing cells. In
addition, a subsaturating concentration of hormone was used to conserve
125I-hFSH.
Hormones and Supplies
Purified hFSH and PMSG were provided by NIDDKs National
Hormone and Pituitary Program and A. F. Parlow. hFSH was iodinated
following the procedure described for the iodination of hCG (43).
125I-cAMP and cell culture media were obtained
from the Iodination Core and the Media and Cell Production Core,
respectively, of the Diabetes and Endocrinology Research Center of the
University of Iowa. Tissue culture reagents were purchased from
Life Technologies, Inc. (Gaithersburg, MD) and
plasticwares (Corning, Inc., Corning, NY) were obtained
from Fisher Scientific (Pittsburgh, PA).
[125I]-pindolol was purchased from NEN Life
Science Products (Boston, MA). ICI 118,551 and CGP 12177 were
from RBI (Natick, MA). General chemicals, including (-) isoproterenol,
were purchased from Sigma (St. Louis, MO).
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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.) Y.-X.T. and A.N.A. were supported by Training Grants DK-07018 and HL-07121, respectively, from the NIH. The services and facilities of the University of Diabetes and Endocrinology Research Center, supported by DK-25295, are also acknowledged.
1 Y.-X.T. and A.N.A. should be considered co-first authors.
Y.-X.T. was responsible for the studies on the hFSHR and A.N.A for
those on the hß2-AR.
Received for publication November 15, 1999. Revision received April 14, 2000. Accepted for publication April 27, 2000.
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REFERENCES |
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