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(Received for publication, July 13,
1995; and in revised form, August 16, 1995) From the
We have recently shown that addition of follitropin (FSH) or a
phorbol ester (phorbol 12-myristate 13-acetate (PMA)) to cells
expressing the recombinant follitropin receptor (FSHR) results in both
phosphorylation and uncoupling of the FSHR from adenylyl cyclase. In
the light of findings reported with other G protein-coupled receptors
we have proposed that phosphorylation of the FSHR mediates the
uncoupling from adenylyl cyclase. The experiments described herein
represent the first attempt to determine the location of the amino acid
residues that become phosphorylated in FSHR and to test the hypothesis
that phosphorylation is responsible for uncoupling of FSHR from
adenylyl cyclase. As a first step in identifying which residues may
be phosphorylated in response to hFSH and PMA, we constructed a mutant
of the FSHR cDNA in which the C-terminal cytoplasmic tail was truncated
at residue 635 (FSHR-t635), thus removing all but one of the potential
phosphorylation sites present in the C-terminal tail. Cells expressing
FSHR-t635 bind hFSH with the appropriate affinity and respond with
increases in cAMP and inositol phosphate accumulation. The maximal cAMP
and inositol phosphate responses of cells expressing FSHR-t635 are
higher than those of cells expressing the wild type FSHR, but the
concentration of hFSH required to elicit these responses is similar in
both cell lines. Immunoprecipitation of FSHR-t635 shows that the
truncated receptor is still effectively phosphorylated in response to
hFSH or PMA. Phosphoamino acid analysis reveals that, like the
wild-type FSHR, FSHR-t635 phosphorylation occurs on serine and
threonine residues. Peptide mapping suggests that the phosphorylated
residues in the FSHR and FSHR-t635 are located within the same areas of
the intracellular regions of the receptors. In addition to stimulating
phosphorylation of FSHR-t635, hFSH and PMA also effectively uncouple
the truncated receptor from adenylyl cyclase. Taken together, these
data show that hFSH and PMA can both phosphorylate and uncouple a FSH
receptor species with a cytoplasmic tail truncated at residue 635.
When target cells are exposed to a hormone their responsiveness
wanes with time, despite the continuous presence of hormone. This
phenomenon, referred to as desensitization, is due to regulatory events
that occur at the level of the hormone receptor as well as at
postreceptor steps. While postreceptor events may be specific for the
metabolic pathways that are activated in a given target cell, receptor
events are more general in nature and conserved for a given family of
receptors. There are at least two categories of regulatory events that
occur at the level of the receptor and contribute to the process of
desensitization. One of them, henceforth referred to as uncoupling, is
defined as a change in the functional properties of a constant number
of receptors resulting in a reduction in the ability of the receptor to
activate its effector system. The other, henceforth referred to as
down-regulation, is defined as a reduction in the density of cell
surface receptors. Uncoupling is generally faster than down-regulation
and is believed to be due to post-translational modifications of the
receptor. Down-regulation, on the other hand, is slower and could be
due to an increase in the rate of receptor
sequestration/internalization or receptor degradation and/or a decrease
in the rate of receptor externalization, processing, or synthesis. It is now generally accepted that the phosphorylation of the
The FSH-induced desensitization of the FSH-responsive
adenylyl cyclase has been studied in some detail in target cells
(Sertoli or granulosa cells) or membranes prepared
therefrom(12, 13, 14, 15, 16, 17) .
Moreover, since the agonist-induced desensitization of the
isoproterenol-responsive adenylyl cyclase of rat Sertoli cells was also
studied(15, 18) , it is relatively straightforward to
compare the properties of the homologous desensitization induced by
these two ligands in the same cell and to compare these data with what
we know now about the mechanisms of desensitization of the
Based on these findings, it is
reasonable to propose that phosphorylation of the FSHR is responsible
for the agonist-induced uncoupling of the FSHR. In fact, this
hypothesis was initially proposed by Hansson and co-workers in a paper
published 11 years ago(15) , but it has proven difficult to
test until now. It is also reasonable to propose that the
phosphorylation of the FSHR is involved in the heterologous regulation
of the actions of FSHR. A particularly relevant example of this
heterologous type of regulation of the actions of FSHR is that of
agonists that activate the inositol phosphate/diacylgylcerol pathway
(such as gonadotropin-releasing hormone in granulosa cells or
purinergic agonists in Sertoli cells) or pharmacological agents (such
as phorbol 12-myristate 13-acetate (PMA)) that activate protein kinase
C(21, 22, 23) . In Sertoli or granulosa
cells, the activation of this pathway results in an attenuation or
desensitization of FSH actions, which seems to be due to a decrease in
the functional properties (i.e. uncoupling) of the FSHR rather
than to the down-regulation of the
FSHR(21, 22, 23) . Recent studies from
this laboratory (24, 25, 26) have established
a transfected cell system that (i) faithfully reproduces the process of
FSH- or PMA-induced desensitization that has been documented in gonadal
cells bearing the FSHR, (ii) allows for biochemical analyses of the
phosphorylation of the FSHR, and (iii) can be used, in conjuction with
site directed mutagenesis of the FSHR cDNA, to conclusively determine
if FSHR phosphorylation is involved in the desensitization of FSHR. The
experiments described herein represent the first attempt to determine
the location of the amino acid residues that become phosphorylated in
FSHR and to test the hypothesis that phosphorylation is responsible for
uncoupling of FSHR from adenylyl cyclase. A wild-type rat FSH receptor cDNA (27) was subcloned
into the pcDNA1/Neo expression vector (Invitrogen, San Diego, CA). A
cDNA encoding for the FSH receptor truncated at residue 635 (designated
FSHR-t635) was constructed using the polymerase chain reaction (28) by introducing a stop codon after the codon for amino acid
residue 635. The identity of this construct was verified by sequencing
the entire open reading frame (29) The origin and handling of
the parental human embryonic kidney cells (293 cells) and the methods
used for transfection and isolation of clonal cell lines stably
transfected with the wild type or mutant FSH receptor cDNAs have been
described in detail elsewhere(11) . All transfected cells were
maintained in Dulbecco's modified Eagle's medium
supplemented with 10 mM HEPES, 10% new born calf serum, 50
µg/ml gentamicin, 700 µg/ml G418, pH 7.4, at 37 °C in a
humidified atmosphere containing 5% CO
At the end of the incubation, the
cells were scraped into ice-cold Buffer A (0.15 M NaCl, 20
mM Hepes, pH 7.4) containing various protease and phosphatase
inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 µM pepstatin A, 1 µM leupeptin, 5 mM EDTA, 3
mM EGTA, 50 mM
The peptides within the gel slices were then separated
using the discontinuous Tricine-urea SDS-polyacrylamide gel
electrophoresis system described by Schägger and
van Jagow (34) using a 16.5% acrylamide, 6 M urea
resolving gel. Ultralow molecular mass standards ((34) ;
700-2500 daltons) (Sigma) were included in each experiment to determine
the molecular weights of the generated peptides. Following
electrophoresis, the Tricine gels were fixed by incubating them in 50%
methanol, 10% acetic acid for 60 min followed by staining with 0.025%
Bromphenol Blue in 10% acetic acid and destaining with 10% acetic acid.
Autoradiograms of the wet gels were then obtained using intensifying
screens.
Figure 1:
Effect of increasing concentrations of
hFSH on the phosphorylation of FSHR in 293F(wt-10) cells.
Since truncations of the
C-terminal tails of several G protein-coupled receptors have provided
useful information about the location and functions of their
phosphorylation
sites(4, 5, 7, 11, 45) , we
constructed a mutant of the FSHR cDNA in which the C-terminal
cytoplasmic tail was truncated at residue 635 (designated FSHR-t635).
As shown in Fig. 2, this truncation removes all but one of the
potential phosphorylation sites (serine and threonine residues; see (25) ) present in the C-terminal cytoplasmic tail of the FSHR.
The possible phosphorylation of the remaining cytoplasmic tail serine
cannot be examined by C-terminal truncation of the receptor because
truncations done upstream of residue 635 generally result in mutant
receptors that do not localize to the cell surface (data not shown).
Figure 2:
Intracellular regions of the rat FSHR. The
three cytoplasmic loops and the C-terminal cytoplasmic tail of the rat
FSHR are shown(27) . Amino acid residues enclosed in squares are identical in the rat FSHR and the rat LHR (53) All potential phosphorylation sites (i.e. serine/threonine residues) are marked as being part of a consensus
sequence for protein kinase C (arrows) or as not being part of
any known consensus sequence (shaded squares or circles). The location of the truncation used in these studies
(t635) is also indicated.
Clonal, stably transfected cell lines expressing FSHR-t635 were
prepared and matched with a clonal cell line stably transfected with
the FSHR-wt expressing equivalent numbers of cell surface receptors. (Table 1). Quantitation of the FSH receptors expressed in these
cell lines by immunoprecipitation of the FSHR from
The
cAMP and inositol phosphate responses of 293F(wt-10) and
293F(t635-5) cells to FSH are shown in Fig. 3. The maximal
responses of 293F(t635-5) cells to FSH are 2-3-fold higher
than those of 293F(wt-10) cells, but there are no differences in the
EC
Figure 3:
Effects of increasing concentrations of
FSH on cAMP and inositol phosphate accumulation in 293F(wt-10) and
293F(t635-5) cells. Top panel, cells were preincubated
with 0.5 mM methylisobutylxanthine for 15 min and then
incubated with the indicated concentrations of hFSH for 15 min or with
500 ng/ml cholera toxin for 2 h. Total (i.e. intracellular
+ extracellular) cAMP levels were then measured using a
radioimmunoassay as described under ``Experimental
Procedures.'' Each point represents the mean ± S.E. of
three independent experiments. Basal levels of cAMP were 6.2 ±
1.3 and 11.2 ± 0.3 for 293F(wt-10) and 293F(t635-5) cells,
respectively. Bottom panel, cells that had been prelabeled
with [
Figure 4:
Effects of hFSH or PMA on the
phosphorylation of FSHR-wt or FSHR-t635-5.
Another interpretation of the finding that FSHR-t635 is
phosphorylated to about the same extent as FSHR-wt is that truncation
of the cytoplasmic tail of FSHR may artifactually expose new
phosphorylation site(s) that are not accessible for phosphorylation in
the full-length FSH receptor. In order to assess this possibility, two
additional experiments were performed. In one experiment, we compared
the phosphoamino acid analysis of FSHR-wt and FSHR-t635. Since we have
previously shown that FSHR-wt is phosphorylated on serine and threonine
residues upon FSH or PMA stimulation(25) , we reasoned that if
phosphorylation of FSHR-t635 were occurring at different sites, it
would be possible for the phosphoamino acid analysis of FSHR-t635 to be
different from that of FSHR-wt. Both receptor species were found to be
phosphorylated in serine and threonine residues, however (data not
shown). To more stringently analyze potential differences in
phosphorylation sites between these two receptor species, we generated
peptide maps of FSHR-wt and FSHR-t635 phosphorylated in response to FSH
or PMA. Peptide maps were obtained by chemical cleavage of the
immunoprecipitated receptor with N-chlorosuccinimide, a
reagent that cleaves polypeptides at tryptophan
residues(32, 33) . Fig. 5shows that the
phosphopeptide maps generated upon cleavage of the full-length or
truncated FSH receptor isolated from FSH-stimulated cells are
indistinguishable from each other. In addition, the phosphopeptide maps
of the full-length and truncated FSHR isolated from PMA-stimulated
cells were also the same, and were very similar to those obtained from
FSH-stimulated cells (data not shown).
Figure 5:
Phosphopeptide maps of FSHR-wt and
FSHR-t635. The
Taken together, these data
suggest that the residues that become phosphorylated in FSHR-wt or
FSHR-t635 are located within the same areas of the intracellular
regions of the receptor. Therefore, removal of the terminal 40 amino
acids of the cytoplasmic tail of the FSHR does not remove serine or
threonine residues that are phosphorylated in response to the ligand or
to PMA, nor does it artifactually introduce additional phosphorylation
sites.
The
results summarized in Fig. 6(left panel) show that
preincubation of 293F(wt-10) or 293F(t635-5) cells with hFSH
results in a decrease in the cAMP response of these cells to a further
stimulation with hFSH. Importantly, the data in Fig. 6also show
that the time course and magnitude of this phenomenon are very similar
in both cell lines. Under these experimental conditions, however, the
blunting of FSH-stimulated cAMP accumulation following preincubation
with FSH could result from reduced numbers of cell surface receptors
(receptor down-regulation) or from receptor uncoupling. To quantitate
the contribution of these two pathways to the blunting of the cAMP
response, we also measured the extent of FSH-induced FSHR
down-regulation in cells incubated under identical conditions. As can
be seen in the right panel of Fig. 6, the extent of
FSHR down-regulation is similar in both cell lines, and the magnitude
of down-regulation is rather small compared with the magnitude of the
loss of cAMP responsiveness. For example, a 15-min preincubation with
hFSH reduced
Figure 6:
Time course of hFSH-induced
desensitization and down-regulation. Left panel, cells were
preincubated with 100 ng/ml hFSH at 37 °C for the times indicated.
The free and receptor-bound hormones were then removed by washing at
neutral and acid pH as described under ``Experimental
Procedures,'' and the cells were divided into two groups and
incubated with or without 100 ng/ml hFSH for 15 min at 37 °C. At
the end of this incubation the medium was removed, and the
intracellular cAMP was measured. The amount of cAMP present in the
cells incubated without hFSH was then subtracted from that present in
the cells incubated with hFSH. Finally, this response was then
expressed as the percentage of the response of cells treated in an
identical fashion but preincubated for the same length of time without
hFSH. Each point represents the mean ± S.E. of three independent
experiments. Right panel, cells were preincubated with 100
ng/ml hFSH at 37 °C for the times indicated. The free and
receptor-bound hormones were then removed by washing at neutral and
acid pH, respectively, as described under ``Experimental
Procedures.'' Residual FSH receptors were then measured during an
overnight incubation at 4 °C in the presence of 100 ng/ml
In additional experiments,
we showed that preincubation of 293F(wt-10) or 293F(t635-5) cells
with hFSH induced a similar reduction in maximal FSH-stimulated cAMP
accumulation between the two cell lines, (40 and 54%, respectively) but
had no effect on the concentration of hFSH required to elicit a
half-maximal response (Fig. 7).
Figure 7:
Cyclic AMP responses of 293F(wt-10) or
293F(t635-5) cells preincubated with hFSH. Cells were
preincubated with or without 100 ng/ml hFSH for 30 min at 37 °C as
indicated. The free and receptor-bound hormones were then removed by
washing at neutral and acid pH, respectively, as described under
``Experimental Procedures,'' and the cells were incubated
with buffer only or with the indicated concentrations of hFSH for 15
min at 37 °C. At the end of this incubation, the medium was removed
and the intracellular cAMP was measured. The amount of cAMP present in
the cells incubated with buffer only was then subtracted from that
present in the cells incubated with the increasing concentrations of
hFSH. Each point represents the mean of two independent
experiments. The bars extend to the individual values obtained
in each experiment.
Last, the data summarized in Fig. 8show that preincubation of 293F(wt-10) or
293F(t635-5) cells with PMA effectively reduces the
FSH-stimulated cAMP accumulation. In both cell lines, the blunting of
FSH-stimulated cAMP accumulation by PMA was particularly evident with
low stimulatory concentrations of FSH. In fact, a preincubation with
PMA either had no effect or enhanced the stimulatory effects of high
concentrations of FSH on cAMP accumulation ( Fig. 8and (25) ). This stimulatory effect of PMA, however, is not
specific for the FSH receptor, as it can also be detected when cAMP
synthesis is activated with any concentration of cholera toxin (25) .
Figure 8:
Effect of PMA on the FSH responses of
293F(wt-10) or 293F(t635-5) cells. Cells were preincubated for 15
min at 37 °C with 0.5 mM methylisobutylxanthine. PMA (200
nM) or vehicle was then added, and the incubation was
continued for another 30 min at 37 °C. At the end of this
incubation the cells received buffer only or the indicated
concentrations of hFSH, and the incubation was continued for an
additional 15 min at 37 °C. Total cAMP (i.e. intracellular
+ extracellular) was measured as described under
``Experimental Procedures.'' Each bar represents the
mean ± S.E. of three independent experiments. The numbers above each pair of bars depict the response of
the cells treated with PMA expressed as the percentage of the response
of the cells treated without PMA.
Taken together, these data show that hFSH or PMA can
both phosphorylate and uncouple a FSH receptor species with a
cytoplasmic tail truncated at residue 635. We have previously shown that the recombinant FSH receptor
expressed in mammalian cells becomes phosphorylated on serine and
threonine residues when cells are exposed to FSH or PMA(25) .
The experiments presented herein were designed as an initial attempt to
identify the location of the phosphorylated residues and to test the
hypothesis that phosphorylation is responsible for the uncoupling of
FSHR from adenylyl cyclase. Since 13 of the 25 serine/threonine
residues present in the intracellular regions of the FSHR are located
in the C-terminal tail (Fig. 2), a mutant FSHR cDNA with a
truncated C-terminal cytoplasmic tail was constructed. This mutant cDNA
encodes for a protein (designated FSHR-t635) in which 12 of the 25
intracellular serine/threonine residues were removed. The analysis of
more severe truncations was not possible due to the lack of expression
of those truncated receptors at the cell surface. FSHR-t635 is fully
functional, in that cells expressing this truncated receptor species
bind hFSH with high affinity (Table 1) and respond to the bound
hormone with the expected increases in cAMP and inositol phosphate
accumulation (Fig. 3). In fact, while the potency of hFSH is
similar in cells expressing FSHR-wt or FSHR-t635, the efficacy of hFSH
is 2-3-fold higher in cells expressing FSHR-t635 (Fig. 3).
These results are similar to those reported for several C-terminal
truncations of the closely related LHR (11, 46) . FSHR-t635 appears to be phosphorylated normally in response to FSH
or PMA stimulation (Fig. 4). The simplest interpretation of
these results is that the 12 serine/threonine residues removed in this
truncated receptor are not phosphate acceptors. Another more complex
interpretation is that the truncation of the C-terminal cytoplasmic
tail resulted in the exposure of phosphorylation sites that are not
accessible in the full-length receptor. In this scenario, FSHR-wt and
FSHR-t635 would be phosphorylated to the same extent but on different
sites. The detection of phosphoserine and phosphothreonine residues in
the full-length and truncated receptors as well as the similarity of
the phosphopeptide maps generated when either receptor is cleaved with N-chlorosuccinimide (Fig. 5), however, strongly suggest
that FSHR-wt and FSHR-t635 are phosphorylated in the same sites. The
finding that FSHR-t635 is phosphorylated normally in response to FSH
stimulation is interesting because C-terminal truncations of many other
GPCRs(4, 5, 7, 45) , including the
closely related LHR(11) , have been previously shown to abolish
(or at least drastically diminish) agonist-induced phosphorylation. In
fact, to the best of our knowledge, there are only two other GPCRs that
become phosphorylated upon agonist stimulation, in which the
phosphorylated residues are not localized in the C-terminal tail. These
are the human m2 muscarinic receptor(47) , where the
phosphorylation sites appear to be located in the third intracellular
loop, and the human The finding that truncation of the C-terminal tail of the
FSHR does not affect the agonist- or PMA-induced uncoupling (Fig. 6Fig. 7Fig. 8) is in agreement with our
hypothesis that phosphorylation is responsible for uncoupling.
Admittedly, however, these results do not provide a rigorous test for
our hypothesis, and additional experiments will be needed to
conclusively determine if this hypothesis is correct. The main
functional difference between FSHR-t635 and FSHR-wt is that the former
is a better transducer of at least two FSH-induced signals: cAMP and
inositol phosphate generation. Thus, while the concentrations of hFSH
required to elicit half-maximal responses are similar in 293F(wt-10)
and 293F(t635-5) cells, the maximal cAMP and inositol phosphate
responses are higher in 293F(t635-5) cells (Fig. 3). At
first glance these results may appear contradictory to the finding that
the FSH-induced uncoupling of FSHR-t635 is normal because the increased
responsiveness could be due to a loss of desensitization. Another
interpretation that accommodates both sets of results, however, is that
the C-terminal tail of the FSHR contains sequences that restrain the
interaction of this receptor with G proteins. Thus, when these
sequences are removed a more productive interaction takes place, and
hormonal responsiveness is enhanced even if desensitization is normal.
Enhanced hormonal responsiveness upon truncation of the C-terminal tail
of GPCRs is, in fact, a rather common
finding(4, 9, 11, 50, 51, 52) ,
which does not always correlate with an impairment of desensitization.
The best example of this lack of correlation is the closely related
LHR, where truncations at residues 653 and 631 result in an enhanced
human CG responsiveness but phosphorylation and desensitization are
impaired only in LHR(t631) and not in LHR(t653) ((11) ). ( Last, it is interesting to note that the removal of the
C-terminal tail of the FSHR did not affect either the agonist or the
PMA-induced phosphorylation (Fig. 4) and that the phosphoamino
acid analysis (not shown) and peptide maps of the FSHR phosphorylated
in response to FSH or PMA (not shown) are very similar. Thus, it
appears that regardless of the stimulus used (i.e. FSH or
PMA), the FSHR becomes phosphorylated on serine/threonine residues
present in one or more of the three cytoplasmic loops and/or in
Ser In summary, then, the data presented herein have
eliminated 12 of the 25 serine/threonine residues present in the FSHR
as potential phosphorylation sites of agonist- or PMA-induced
phosphorylation and as mediators of agonist- or PMA-induced uncoupling
of FSHR from adenylyl cyclase. Additional studies will be needed to
determine the location of the phosphorylation sites, to determine if
the same sites are phosphorylated in response to FSH- or
PMA-stimulation, and to ascertain the functional impact of
phosphorylation on the functions of FSHR.
Volume 270,
Number 44,
Issue of November 3, 1995 pp. 26683-26689
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-adrenergic receptor that results as a consequence of
agonist binding is an important event in the agonist-induced uncoupling
of this receptor(1, 2, 3) . Although the
phosphorylation of other G protein-coupled receptors (GPCRs) (
)has not been studied in as much detail, it is rather clear
that phosphorylation plays a central role in the regulation of the
functions of many other GPCRs. Thus, in all other members of this
family that have been studied so far, the removal or mutation of
phosphorylation sites leads to a decrease in agonist-induced
phosphorylation and an impairment in the process of agonist-induced
uncoupling (see (4, 5, 6, 7, 8, 9, 10, 11) for a few
examples).-adrenergic receptor. These studies showed that the
time course, magnitude, and other biochemical properties of the
agonist-induced desensitization of the FSH receptor (FSHR) and
-adrenergic receptor are very similar. Although in
both cases one can demonstrate that uncoupling and down-regulation are
involved in desensitization, uncoupling seems to be quantitatively more
important than down-regulation(13, 19) . Last, in
agreement with what was reported for the agonist-induced
desensitization of -adrenergic receptor in turkey
erythrocyte membranes(20) , the FSH-induced desensitzation of
FSHR in Sertoli cell membranes was reported to require Mg and ATP (15) .
. Experimental
cultures were plated on day 0 and used on day 3 or 4.Immunoprecipitation of the FSH Receptor from
Metabolic labeling of cells and
subsequent immunoprecipitation of the FSH receptor was achieved as
described previously(24, 25) . Cells plated in 100-mm
plates were labeled using 3.5 ml of phosphate-free Dulbecco's
modified Eagle's medium containing 100-400 µCi/ml
[P-Labeled Cells
P]orthophosphate for 3 h. All incubations were
done at 37 °C under 5% CO
. Unless otherwise stated in
the figure legends, the various hormones and pharmacological agents
were then added directly to the medium, and the cells were further
incubated for the indicated times.-glycerophosphate, 10 mM sodium fluoride, 0.1 mM sodium orthovanadate, 100 nM okadaic acid, and 1 nM cypermethrin), pelleted by
centrifugation, and lysed by vortexing in Buffer A containing 0.5%
Nonidet P-40 and the aforementioned inhibitors. The detergent lysates
were clarified by centrifugation at 100,000 
g for 30
min, and the protein content of the supernatants was assessed by the
method of Bradford(30) . Equal amounts of lysate protein were
then immunoprecipitated with a polyclonal antibody to the rat FSH
receptor as described before(24, 25) .
Immunoprecipitates were resolved on 7.5% sodium dodecyl
sulfate-polyacrylamide gels in the presence of thiol-reducing agents as
described previously(31) . Autoradiograms of the dried gels
were obtained using intensifying screens, and the autoradiograms were
scanned using a Bio-Rad Molecular Imaging System. All of the
autoradiograms shown here are reproductions from the scanned images.
Receptor phosphorylation was quantitated by densitometry using the
software included with this imaging system.Peptide Mapping
FSHR immunoprecipitated from P-labeled cells was detected by autoradiography of dried
gels and excised. The dried gel piece was then rehydrated with water
for 60 min, and the paper backing was removed. The receptor polypeptide
within the gel slice was then chemically cleaved at tryptophan residues
with N-chlorosuccinimide using a modification of the method
described by Lischwe and Ochs (32) as follows. The receptor
protein was first denatured by incubating the gel slices in 4 M guanidine hydrochloride in 80% acetic acid (33) for 20
min, with one change of denaturing solution. The gel slices were then
incubated in the presence of 50 mMN-chlorosuccinimide in the same denaturing solution for
1-2 h, washed twice with 20 ml of water for 10 min each time, and
equilibrated in 1.5 ml of Tricine gel sample buffer (12% glycerol
(w/v), 4% sodium dodecyl sulfate (w/v), 2% mercaptoethanol (v/v), and
0.01% Bromphenol Blue (w/v) in 50 mM Tris-HCl, pH 6.8) for 90
min. This sample buffer was changed three times during the 90-min
incubation.
FSH-induced Desensitization of the FSH-responsive
Adenylyl Cyclase
Cells were plated in 35-mm wells that had been
coated with a fibronectin-like engineered protein polymer (5 µg/ml)
according to the manufacturer's instructions. Two to three days
after plating, the cells were washed twice with 2-ml portions of warm
assay medium (Waymouth MB 752/1 containing 1.12 g/liter NaHCO3, 50
µg/ml gentamicin, 20 mM HEPES, and 1 mg/ml bovine serum
albumin, pH 7.4) and incubated at 37 °C in 1 ml of assay medium in
the absence or presence of hFSH for various time periods. The free
hormone was removed by placing the wells on ice and washing the cells
three times with 2-ml portions of ice-cold Hanks' balanced salt
solution containing 1 mg/ml bovine serum albumin. The receptor-bound
hormone was then removed during a 4-min incubation with 2 ml of cold
100 mM NaCl, 50 mM glycine, pH 3, followed by a 2-ml
wash with the same cold acid buffer(35) . Finally, the cells
were washed twice with 2-ml portions of warm assay medium, and 1 ml of
warm assay buffer was added to each well. The cells were then incubated
in the presence or absence of various concentrations of hFSH for 15 min
at 37 °C. At the end of this incubation the wells were placed on
ice, the medium was aspirated, and the cells were washed once with a
2-ml aliquot of cold assay medium containing 0.5 mM
isobutylmethylxanthine. The washing solution was removed, and the
intracellular cAMP was extracted by adding 1 ml of 1 N perchloric acid containing 180 µg/ml theophylline. The samples
were then processed, and the cAMP was assayed as described
previously(35) .FSH-induced Down-regulation of the FSHR
Cells were
plated in coated dishes and treated as described above. Following a
preincubation with or without hFSH for increasing periods of time at 37
°C, the free and receptor-bound hormones were removed as described
above. The binding of I-FSH was then measured during an
overnight incubation at 4 °C as described
previously(24, 25) .
PMA-induced Desensitization of the FSH-responsive
Adenylyl Cyclase
Cells (plated in 35-mm wells) were washed twice
with 2-ml portions of assay medium. The cells were then incubated in 1
ml of assay medium containing 0.5 mM isobutylmethylxanthine
for 15 min at 37 °C. PMA (200 nM) or vehicle was then
added, and the incubation continued for 30 min. At this time, the cells
were further incubated in the absence or presence of various
concentrations of hFSH for 10 min. The samples were then processed and
assayed for total cAMP (i.e. intracellular and extracellular)
as described previously(11) .Other Methods
The different parameters that
describe the concentration-response curves were determined using the
curve-fitting routine of the computer program Deltagraph and the
equations initially described by DeLean et al.(36) .
In the experiments measuring inositol phosphate accumulation, cells
were metabolically labeled with [
H]inositol in
myo-inositol-free Dulbecco's modified Eagle's medium as
described previously (11, 25) and incubated with
various concentrations of hFSH or with 100 µM UTP for 60
min in the presence of 20 mM LiCl. The methods used for the
measurement of phosphatidylinositols and inositol phosphate levels have
also been described elsewhere(37) . Phosphoamino acid analysis
was performed as described previously (38) except that the
hydrolyzed phosphoamino acids were separated by two-dimensional thin
layer electrophoresis(39) .Hormones and Supplies
Purified hFSH (AFP-5720D)
was kindly provided by the National Hormone and Pituitary Agency of the
National Institute of Diabetes and Digestive and Kidney Diseases.
[I]FSH was prepared as described previously (40) . Materials for cell culture, electrophoresis, Western
blotting, and metabolic labeling were obtained as described
elsewhere(24, 25) . The antibody to the FSH receptor
has recently been described(24) . The fibronectin-like
engineered polymer, PMA, leupeptin, pepstatin A, phenylmethylsulfonyl
fluoride, N-chlorosuccinimide, and protein A-agarose were
obtained from Sigma. Guanidine HCl was purchased from Mallincklrodt
Chemical, Inc. (Paris, KY). Okadaic acid and cypermethrin were
purchased from LC Laboratories (Woburn, MA). Phosphate- and
myo-inositol-free Dulbecco's modified Eagle's medium were
purchased from ICN Biomedicals (Irvine, CA), while
[
P]orthophosphate and
[
H]myo-inositol were obtained from DuPont NEN.
Preparation and Functional Properties of
FSHR(t635)
We have previously established that addition of hFSH
or PMA to stably transfected 293 cells expressing the FSHR results in
an increase in FSH receptor phosphorylation(25) . However, in
these initial experiments, pharmacological concentrations of the hFSH
(EC = 500 ng/ml) were required to
increase receptor phosphorylation during a 15-min incubation. As it is
highly unlikely that such high concentrations of FSH occur in
vivo, we attempted to improve the sensitivity of our assay by
increasing the length of the incubation with FSH to 1 h by using
subconfluent cell cultures and by omitting the lectin purification step
that was used prior to receptor immunoprecipitation (25) . As
shown in Fig. 1, these changes have now allowed us to detect an
increase in FSHR phosphorylation with 3 ng/ml hFSH, a half-maximal
increase with about 30 ng/ml hFSH, and a maximal increase with 100
ng/ml hFSH. While the maximal increase in receptor phosphorylation
detected under these conditions is similar to that detected
previously(25) , the FSH concentration range that elicits
receptor phosphorylation is now similar to the concentration range
required to detect FSH-induced increases in cAMP, estradiol, or
c-fos mRNA levels in Sertoli cells (41, 42, 43) as well as FSH-induced increases
in cAMP accumulation in mammalian or insect cells transfected with the
FSHR(25, 44) . The reported EC
values for
these responses vary between 2 and 30 ng/ml, and maximal responses are
attained at 100-500 ng/ml hFSH.
P
-labeled 293F(wt-10) cells were incubated
with the indicated concentrations of hFSH for 1 h at 37 °C. The P-labeled FSHR was then immunoprecipitated with AntiF and
resolved on SDS gels as described under ``Experimental
Procedures.'' The top panel shows a scan of the relevant
portion of the autoradiogram, and the bottom panel shows the
densitometric quantitation of the data. The results of a representative
experiment are presented.
S-labeled cells or by immunoblotting support the
conclusion that 293F(wt-10) and 293F(t635-5) cells express
equivalent numbers of the mature 74-kDa FSHR (data not shown).
values for these responses between the two cells lines.
The EC
values for cAMP and inositol phosphate accumulation
in both cell lines are 2-5 ng/ml and 150-250 ng/ml hFSH,
respectively. Since the cAMP response to cholera toxin and the inositol
phosphate response to UTP are similar for these two cell lines (Fig. 3), it can be safely concluded that the increased efficacy
of hFSH in the 293F(t635-5) cells is not due to clonal variation.
H]inositol were incubated with the
indicated concentrations of hFSH or UTP in the presence of 20 mM LiCl for 60 min. The medium was removed, and total inositol
phosphates and phosphatidylinositols were measured as described under
``Experimental Procedures.'' The amount of inositol
phosphates detected are presented as a percentage of the sum of the
phosphatidylinositol + inositol phosphates detected. Each point represents the mean ± S.E. of three independent
experiments. Basal levels of inositol phosphates were 3.7 ± 0.7
and 4.2 ± 1.0 for 293F(wt-10) and 293F(t635-5) cells,
respectively.
Phosphorylation of FSHR-wt and
FSHR-t635
Immunoprecipitation of the FSHR from P-labeled 293F(wt-10) and 293F(t635-5) cells
revealed that FSH and PMA each markedly increased phosphorylation of
both the full-length and the truncated FSHR. (Fig. 4). In
addition, the magnitude of the FSH- or PMA-induced phosphorylation of
FSHR-t635 was equivalent to that of FSHR-wt, suggesting that none of
the 12 serines and threonines located downstream of residue 635 in the
C-terminal cytoplasmic tail are phosphorylated in response to FSH or
PMA. It is also important to note that although the mass of FSHR-t635
is expected to decrease by about 5 kDa, the resolution of our
polyacrylamide gels is such that it does not resolve this small
difference in mass. This phenomenon was also noted in analogous studies
with several C-terminal truncations of the LHR (11) .
P
-labeled cells were incubated with no
additions, 1000 ng/ml hFSH, or 200 nM PMA for 15 min. Cell
lysates were prepared, and identical amounts of lysate were
immunoprecipitated with AntiF and resolved on SDS gels as described
under ``Experimental Procedures.'' The data presented are
from a representative experiment showing the scanned image of the
relevant portion of the autoradiogram.
P-labeled FSHR-wt or FSHR-t635 was isolated
from pre-labeled cells that had been stimulated with 1000 ng/ml hFSH
for 15 min at 37 °C. Cell lysates were prepared, immunoprecipitated
with AntiF, and resolved on SDS gels as described under
``Experimental Procedures.'' The relevant portion of the gels
was cut, digested with N-chlorosuccinimide, and subjected to
electrophoresis on Tricine-urea-SDS gels as described under
``Experimental Procedures.'' The results (densitometric scan
of an autoradiogram) of a representative experiment are shown. The
apparent molecular weights shown were calculated based on the migration
of molecular weight standards.
Desensitization of FSHR-wt and FSHR-t635
By
analogy with what is known about other G protein-coupled receptors, we
have previously proposed that phosphorylation of the FSHR is
responsible for the uncoupling of the FSHR from adenylyl cyclase that
is observed when cells are incubated with FSH or PMA(25) .
Having established that the truncated FSH receptor expressed in the
293F(t635-5) cells is phosphorylated in response to FSH or PMA,
we next assessed whether incubation with either of these stimuli would
uncouple the truncated receptor species from adenylyl cyclase.I-FSH binding to only 91 ± 2% and 87
± 3% of control levels in 293F(wt-10) and 293F(t635-5)
cells, respectively. In contrast, under the same conditions,
hFSH-stimulated cAMP accumulation was reduced to 60 ± 7% and 55
± 2% of control levels in 293F(wt-10) and 293F(t635-5)
cells, respectively. Therefore, the FSH-induced blunting of the
FSH-sensitive adenylyl cyclase appears to be due primarily to to FSHR
uncoupling rather than down-regulation.
I-hFSH as described under ``Experimental
Procedures.'' Results are expressed as the percentage of the
I-hFSH binding detected in cells preincubated for the
same length of time, but without hFSH. Each point represents
the mean ± S.E. of three independent
experiments.
2-adrenergic receptor, where the
phosphorylation sites cannot be located in the C-terminal tail simply
because this receptor does not have any serine/threonine residues in
its C-terminal tail(48, 49) . Based on the amino acid
sequence of the rat FSHR(27) , it can be predicted that N-chlorosuccinimide cleavage should generate three
phosphopeptides: a 22-kDa peptide containing the third intracellular
loop and the C-terminal cytoplasmic tail, a 7.4-kDa peptide containing
the first intracellular loop, and a 2.96-kDa peptide containing the
second intracellular loop. As shown in Fig. 5, however, there
are at least four detectable peptides that are generated upon N-chlorosuccinimide cleavage, and their molecular weights do
not correspond to the predicted molecular weights of the three
theoretical peptides. These findings could be reconciled by assuming
that the cleavage conditions used may not be optimal and by the
knowledge that there is a fair amount of uncertainty in the molecular
weight estimates derived from the separation system used (34) .
Thus, it is possible that the 18- and 8.8-kDa peptides shown in Fig. 7correspond to the 22- and 7.4-kDa peptides predicted
above, and the 14.4- and 12-kDa peptides may be incomplete degradation
products. If this interpretation is correct, the data presented in Fig. 5suggests that phosphorylation occurs in the first and
third intracellular loops. Clearly, however, additional studies
utilizing peptide mapping and/or site-directed mutagenesis will be
needed to provide a definitive identification of the phosphorylation
sites.), the only potential phosphorylation site that remains
in the C-terminal tail of FSHR-t635 (c.f. Fig. 2). These
findings also suggest that at least some of the serine/threonine
residues phosphorylated in response to either stimuli are the same.
While this conclusion also remains to be confirmed, it is interesting
to note that in the closely related LHR, the PMA- or agonist-induced
phosphorylation maps to the same four serine residues in the C-terminal
tail(11) . In contrast to the similarities in the phosphoamino
acid analysis and phosphopeptide maps of the FSHR isolated from cells
stimulated with PMA or FSH, there is a rather obvious difference in the
effects of FSH or PMA on the desensitization of the FSHR. A
preincubation of cells with FSH leads to a reduction in the FSH-induced
cAMP accumulation at all concentrations of FSH tested (Fig. 7),
while a preincubation of cells with PMA leads to a reduction in the
FSH-induced cAMP accumulation only at low concentrations of FSH (Fig. 8). This and other differences between the functional
effects of PMA and FSH (25) suggest that the sites
phosphorylated in response to PMA stimulation are not identical to
those phosphorylated in response to FSH stimulation. This suggestion is
also supported by the finding that protein kinase C is only partially
responsible for mediating the FSH-induced phosphorylation of the FSHR (25) .
))
We thank Cheri B. Whisler and Kari Kruel for expert
technical assistance and Dr. Deborah L. Segaloff for reading the
manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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