Involvement of G Protein-Coupled Receptor Kinases and Arrestins in Desensitization to Follicle-Stimulating Hormone Action
Carine Troispoux,
Florian Guillou,
Jean-Marc Elalouf,
Dmitri Firsov,
Luisa Iacovelli,
Antonio De Blasi,
Yves Combarnous and
Eric Reiter
INRA/CNRS URA 1291 (C.T., F.G., Y.C., E.R.) Station
de Physiologie de la Reproduction des Mammifères Domestiques
37380 Nouzilly, France
Département de Biologie
Cellulaire et Moléculaire (J.-M.E., D.F.) Service de Biologie
Cellulaire CEA Saclay 91191 Gif-sur-Yvette Cedex,
France
Consorzio Mario Negri Sud (L.I., A.D.B.) Istituto
di Ricerche Farmacologiche "Mario Negri" 66030 Santa Maria
Imbaro, Italy
 |
ABSTRACT
|
---|
FSH rapidly desensitizes the FSH-receptor (FSH-R)
upon binding. Very little information is available concerning the
regulatory proteins involved in this process. In the present study, we
investigated whether G protein-coupled receptor kinases (GRKs) and
arrestins have a role in FSH-R desensitization, using a mouse Ltk 7/12
cell line stably overexpressing the rat FSH-R as a model. We found that
these cells, which express GRK2, GRK3, GRK5, and GRK6 as well as
ß-arrestins 1 and 2 as detected by RT-PCR and by Western blotting,
were rapidly desensitized in the presence of FSH. Overexpression of
GRKs and/or ß-arrestins in Ltk 7/12 cells allowed us to demonstrate
1) that GRK2, -3, -5, -6a, and -6b inhibit the FSH-R-mediated signaling
(from 71% to 96% of maximal inhibition depending on the kinase,
P < 0.001); 2) that ß-arrestins 1 or 2 also
decrease the FSH action when overexpressed (80% of maximal inhibition,
P < 0.01) whereas dominant negative ß-arrestin 2
[319418] potentiates it 8-fold (P <
0.001); 3) that ß-arrestins and GRKs (except GRK6a) exert
additive inhibition on FSH-induced response; and 4) that FSH-R
desensitization depends upon the endogenous expression of GRKs, since
there is potentiation of the FSH response (2- to 3-fold,
P < 0.05) with antisenses cDNAs for GRK2, -5, and -6,
but not GRK3. Our results show that the desensitization of the
FSH-induced response involves the GRK/arrestin system.
 |
INTRODUCTION
|
---|
FSH plays a central role in mammalian reproduction. This hormone
is necessary for gonadal development and maturation at puberty and for
gamete production during the fertile phase of life. FSH acts by binding
to specific receptors localized exclusively in the male and female
gonads (reviewed in Ref. 1). The FSH receptor (FSH-R) is a complex
protein characterized by seven hydrophobic helices spanning the
membrane. The intracellular domain of the receptor is functionally
coupled to Gs (2). The binding of FSH to the receptor extracellular
domain initiates a cascade of events that ultimately leads to the
specific biological effects of the hormone (1).
As in many other hormonal systems, prolonged exposure of target cells
to FSH leads to a decreased response over time, a process called
desensitization (3, 4, 5, 6). The early desensitization step after FSH
binding and stimulation is the reduction of FSH-R function due to its
uncoupling from Gs (7, 8). Later, other mechanisms, such as the
increase of nucleotide-phosphodiesterase activities (9) or the
reduction of receptor number (down-regulation) (10, 11, 12), become
involved in the decreased cell response to prolonged FSH
stimulation.
The mechanisms governing receptor uncoupling have been extensively
studied at molecular level for rhodopsin and ß2-adrenergic receptors
(reviewed in Ref. 13). For these receptors, uncoupling occurs through
phosphorylation of the C-terminal intracellular domain. Two classes of
protein kinases may be involved in this process: 1) second
messenger-dependent kinases such as protein kinase A (PKA) or protein
kinase C (PKC), which are responsible for agonist-independent, or
heterologous, desensitization, and 2) G protein-coupled receptor
kinases (GRKs), which trigger the agonist-specific, or homologous
desensitization (13).
GRKs are serine/threonine kinases showing the unique property of
interacting only with agonist-occupied receptors while leaving
unaltered nonactivated receptors. To date, six distinct mammalian GRK
genes have been identified and classified into three subfamilies on the
basis of sequence and functional similarities (14). The first subfamily
includes GRK1 only (rhodopsin kinase). The second subfamily is composed
of GRK2 and GRK3 (ß-adrenergic receptor kinases 1 and 2), while GRKs
4, 5, and 6 represent the third subfamily. Expression of GRK1 is
preferentially confined to retinal photoreceptor cells where it
phosphorylates retinal opsins (13). Likewise, GRK4 is predominantly
expressed in testicular germ cells, but its receptor substrate is still
unknown (15, 16). In contrast, the other GRKs (i.e. GRKs 2,
3, and 5 and the two isoforms of rat GRK6) are ubiquitously expressed
(13, 17) and display broad and possibly overlapping substrate
specificities (13).
After phosphorylation of agonist-occupied receptors by GRKs, arrestins
bind to the intracellular domain of the receptors, thereby preventing
further signal transduction (reviewed in Ref. 18). Arrestins are also
known to interact with clathrin, providing a mechanism for
internalization or sequestration of the activated receptors (19). The
arrestin family includes visual arrestin, cone arrestin, ß-arrestin 1
(arrestin 2), and ß-arrestin 2 (arrestin 3), in addition to
arrestin D and arrestin E, which are only partially characterized.
Visual and cone arrestins are predominantly expressed in retinal
tissues where they regulate photoreceptor signaling. In contrast, the
other arrestins are ubiquitously expressed and possibly interact with a
wide variety of G protein-coupled receptors (18, 19).
The molecular mechanisms involved in the FSH-R uncoupling are still
poorly understood. Studies on embryonic kidney cell line (HEK 293)
permanently transfected with the rat FSH-R have shown that
phosphorylation of the receptor is induced within a few minutes either
by FSH or phorbol esters and does not involve PKA. This phosphorylation
occurs on both serine and threonine residues, which are probably
located upstream of amino acid 635 (20, 21). In addition, recent
studies by Nakamura et al. (22) have established that 1)
FSH-induced phosphorylation of the FSH-R maps to the first and third
intracellular loops and is involved in the processes of coupling with
Gs and internalization; 2) FSH-dependent phophorylation of the FSH-R is
increased by GRK2 overexpression (23); and 3) FSH-R internalization is
enhanced when ß-arrestin 2 and/or GRK2 are overexpressed (23). In the
natural FSH-R-bearing Sertoli cells, it has been demonstrated that both
PKA and PKC participate in desensitization of the FSH-induced response,
but that an additional PKA/PKC-independent uncoupling mechanism also
exists (24). Moreover, in rat granulosa cells overexpressing the FSH-R,
a specific staurosporine-sensitive receptor kinase may be responsible
for hormone-induced phosphorylation and uncoupling of the FSH-R
(25, 26).
Taken together, the available data suggest that, although the partial
involvement of PKA and/or PKC in the phosphorylation of the FSH-R is
possible, the early modulation of FSH action seems to be largely due to
specific receptor kinase(s). The experiments presented herein
demonstrate that the GRK/arrestin system is involved in homologous
desensitization of the FSH-R signaling in Ltk cells stably expressing
the rat FSH-R and producing cAMP in response to FSH action (27).
 |
RESULTS
|
---|
Demonstration of FSH-Induced Homologous Desensitization in Ltk 7/12
Cells
To evaluate FSH-induced desensitization in Ltk 7/12 cells that
stably overexpress the rat FSH-R, we first investigated the time course
of FSH-stimulated cAMP production in this cell line. First, we observed
that FSH-induced cAMP response reached a plateau within 30 min (Fig. 1a
). In addition, when the cells were
preincubated for 2 h with increasing concentrations of FSH and
then rechallenged for 30 min with a maximal concentration of FSH (100
ng/ml), cAMP production was significantly reduced at preincubation
concentrations >10 ng/ml (Fig. 1b
). These results suggest that FSH-R
undergoes desensitization in Ltk 7/12 cells. The time delay and the FSH
doses required to induce desensitization in Ltk 7/12 cells were higher
than those previously described for rat Sertoli cells (28). This is
probably a consequence of the higher density of FSH-R expressed in the
Ltk 7/12 cells [
10,000 receptors per Ltk 7/12 cell (27)] than in
primary rat Sertoli cells [
1,000 receptors per Sertoli cell (1)].
Moreover, this high level of overexpression in a cell line that
normally does not express the FSH-R, could determine a stoichiometry
between the molecules involved in the coupling and in the
desensitization, which is different from a physiological system.

View larger version (10K):
[in this window]
[in a new window]
|
Figure 1. Homologous Desensitization of the FSH-R in Ltk 7/12
Cells
a, Time course of FSH-induced cAMP production. Ltk 7/12 cells were
stimulated with 100 ng/ml of FSH for indicated times. Intracellular
cAMP content was measured as described in Materials and
Methods. b, FSH-induced desensitization of the cAMP production.
Cells were preincubated for 2 h with increasing concentrations of
FSH. Cells were then washed with culture medium and stimulated with 100
ng/ml of FSH for 30 min. Intracellular cAMP content was measured. ***,
P < 0.001, significant statistical difference from
cells preincubated without FSH. These data represent the mean ±
SE of two independent experiments, each with four
replicates.
|
|
Expression of the GRKs and ß-Arrestins in the Ltk 7/12 Cells
We first investigated the expression of four GRKs (2, 3, 5, 6)
and of ß-arrestins 1 and 2, using RT-PCR analyses of total RNA in Ltk
7/12 cells (Fig. 2a
). GRKs 1 and 4 were
excluded from this study due to their tissue-specific expression
(i.e. retina for GRK1, male germ cells for GRK4). Specific
fragments of the four GRKs and of the two ß-arrestins were amplified,
as demonstrated by direct sequencing of the different PCR products.
These data indicate that the corresponding genes are transcribed in Ltk
7/12 cells. The efficiencies of the different RT-PCR reactions were not
compared in this study principally because all the primers were
designed from rat cDNA sequences that were the closest sequences
available for the GRKs and ß-arrestins when these experiments were
performed. As a consequence, this set of RT-PCR did not allow us to
compare the relative mRNA abundances for the different GRKs and
ß-arrestins in the Ltk 7/12 cells.

View larger version (27K):
[in this window]
[in a new window]
|
Figure 2. Expression of GRKs and ß-Arrestins in Ltk 7/12
Cells
a, RT-PCR analysis. Total RNAs (80 ng) extracted from Ltk 7/12 cells
were submitted to RT-PCR using primer pairs designed to amplify
fragments from GRK 2, 3, 5, 6 or from ß-arrestin (ß-Arr) 1 and 2
transcripts. Reactions were performed in the presence
of [ -32P]dCTP and were stopped after
25, 30, and 35 cycles. The expected sizes of the different amplified
products are indicated on the right. Positive controls
(+) were obtained by direct amplification of the appropriate expression
vector. Negative control reactions (-) were carried out in the absence
of reverse transcriptase (RT). Amplified products were resolved on a
2% agarose gel and detected by autoradiography. b, Specificities of
the anti-GRK antibodies. Cytosolic or membrane proteins from HEK293
cells transfected with one of the different GRKs or ß-arrestins were
prepared, electrophoresed on SDS-polyacrylamide gels, and transferred
to PVDF membranes. A monoclonal antibody recognizing both GRK2 and GRK3
and three rabbit polyclonal antibodies raised against GRK2, GRK5, and
GRK6 were used for GRK immunodetections. The blots were developed using
the Renaissance chemiluminescence detection system (NEN Life Science Products). The signals corresponding to GRKs are
indicated by arrows. Data are representative of three
separate experiments. c, Western blot analysis. Cytosolic or membrane
proteins from Ltk 7/12 cells and transfected positive controls were
electrophoresed on SDS-polyacrylamide gels, transferred to PVDF
membranes, and analyzed with the antibodies described above.
ß-Arrestins 1 and 2 were detected with a monoclonal antibody raised
against a epitope common to both isoforms. Colored standards allowed
determination of mol wt marker positions. The signals corresponding to
GRKs and ß-arrestins are indicated by arrows. Data are
representative of three separate experiments.
|
|
Western blot analyses of these different proteins
were also carried out on Ltk 7/12 preparations. The specificity of the
antibodies used was confirmed using different transfected preparations
as positive controls (Fig. 2b
). In addition, no signal was obtained for
GRK2 using the polyclonal antibody after preincubation with the
corresponding control peptide (data not shown). Immunoreactive bands of
the appropriate molecular weights, which comigrated with the positive
controls, were observed in Ltk 7/12 cytosolic extracts for GRK2, GRK3,
GRK5, and GRK6. GRK3 was more abundant than GRK2 in cytosolic extracts
(Fig. 2c
). However, in whole-cell extracts, GRK2 was clearly
predominant (Fig. 5c
). The monoclonal antibody F4C1 raised against an
epitope that is conserved in both ß-arrestins 1 and 2, was also
used. In Ltk 7/12 cytosolic preparations, two bands were detected with
apparent Mr of 45,000 and 50,000, corresponding to
ß-arrestin 2 and ß-arrestin 1 from transfected positive control,
respectively. The ß-arrestin 2 was more abundant than ß-arrestin 1
in these cytosolic extracts, but the ratio was found
inverted in whole cell extracts (data not shown). These results confirm
our RT-PCR experiments and definitely establish that Ltk 7/12 cells are
equipped with a potentially functional GRK/arrestin system. Thus, this
cellular model is suitable to study the involvement of these regulatory
proteins in desensitization of FSH-R.

View larger version (40K):
[in this window]
[in a new window]
|
Figure 5. Transient Overexpression of GRKs Antisense (AS)
Constructs in Ltk 7/12 Cells
a, Ltk 7/12 cells were cotransfected with pSOMLuc and with various GRK
AS constructs (1 µg GRK2 AS, 1 µg GRK3 AS, 2 µg GRK5 AS, 2 µg
GRK6 AS per well). Cells were stimulated with FSH (100 ng/ml) ( ) or
were not stimulated ( ).
Luciferase activity was determined in the cell lysate. Values were
expressed in percentage; the mean activity obtained for unstimulated
cells transfected with pSOMLuc and pCMV5 was taken as 100%. *,
P < 0.05, significant statistical differences from
the FSH-stimulated cells transfected with pSOMLuc and pCMV5. These data
are representative of two independent experiments, each with four
replicates. b, Cross-hybridization assay: unlabeled sense cRNAs
corresponding to the full-length sequences of GRK2, -3, -5, and -6A
were applied in 2-fold serial dilutions on nitrocellulose filters using
a slot blot apparatus. Four replicates were done and each of them was
hybridized with 15 x 106 cpm of a
32P-labeled antisense cRNA corresponding to GRK2, -3, -5,
or -6. c, Antisense-mediated inhibition of endogenous GRKs: Ltk 7/12
cells were transiently cotransfected with both an expression vector for
GFP and one of the antisense contruct or pCMV5 as control. GFP-positive
living cells were then sorted using fluorescence-activated cell
sorting. Twenty micrograms of whole-cell extracts from each sorted
population were analyzed by Western blotting as described above. The
transferred proteins were stained with Coomassie blue and scanned for
subsequent densitometric analysis. Antibodies specific for GRK2,
GRK2/3, GRK5, and GRK6 were used. The inhibition levels (values
presented between brackets) were determined by
densitometric analysis of the different immunoblots, corrected
according to gel loading and transfer efficiency as determined by
Coomassie blue staining of the PVDF membranes (data not shown). d,
Control of relative transfection efficiencies: Ltk 7/12 cells were transfected by
lipofection as described in Materials and Methods. Cells
were stimulated with FSH (100 ng/ml) for 4 h. Luciferase
activities and the amounts of plasmid transfected were determined in
parallel for each condition, luciferase activities being corrected
according to the relative transfection efficiencies. The results were
arbitrary expressed in percent, the pCMV5 control being taken as 100%.
*, P < 0.05; **, P < 0.01;
significant statistical differences from pCMV5 control cells.
|
|
A cAMP-Inducible Gene Reporter System to Measure the FSH Response
in Transfected Ltk 7/12 Cells
To examine the role of regulatory proteins in the FSH-R
desensitization mechanism, a variety of expression vectors for the GRKs
and ß-arrestins were transfected in Ltk 7/12 cells. As in a standard
transfection procedure, only a limited number of cells incorporate DNA,
we decided to use the cAMP-sensitive pSOMLuc construct as a reporter
system [i.e. the luciferase reporter gene driven by a part
of the somatostatin gene 5'-end region containing cAMP-responsive
elements (39)]. In Ltk 7/12 cells transiently transfected with
pSOMLuc, luciferase activity was induced within 10 min of FSH treatment
(Fig. 3a
), and a clear saturation of cell
response was observed within 2060 min. In this experiment, cells were
stimulated for the indicated time, washed, and then maintained in
serum-free medium for a total of 4 h after the beginning of the
stimulation to allow luciferase gene expression. These results, which
are similar to those of Fig. 1a
, demonstrate that the pSOMLuc reporter
gene system used here is suitable to measure the effects of potential
regulatory proteins on the FSH-R coupling. Figure 3b
shows that for
stimulation longer than 2 h, luciferase activity was
systematically higher in FSH-treated conditions than in untreated
controls. The maximum response intensity was observed after 4 h
and, on the basis of this experiment, 4-h treatments were used in
further experiments.

View larger version (12K):
[in this window]
[in a new window]
|
Figure 3. FSH-Induction of pSOMLuc Expression in Transiently
Transfected Ltk 7/12 Cells
a, Short-term induction of pSOMLuc expression by FSH. Ltk 7/12
cells were transiently transfected with 1 µg per well of pSOMLuc.
After 24 h of culture, cells were stimulated for the indicated
times with FSH (100 ng/ml). Then, cells were washed and maintained in
serum free medium to allow time for luciferase gene expression. Four
hours after the beginning of the stimulation, luciferase activity was
measured in the cell lysates. Values were expressed in percentage, with
the activity of the unstimulated control being taken as 100%. b,
Long-term induction of pSOMLuc expression by FSH. Ltk 7/12 cells were
transiently transfected with 1 µg per well of pSOMLuc. After 24
h of culture, cells were maintained for the indicated times under
control conditions ( ) or exposed to 100 ng/ml FSH ( ). At
the end of the incubation period, luciferase activity was measured in
the cell lysates. These data represent the mean ± SE
of two independent experiments, each with four replicates.
|
|
First, we used pCMVß-Gal as constitutively expressed standard to
monitor transfection efficiencies between plates. Early on, however, we
suspected that the expression of this construct was influenced by FSH
and indeed, when pCMVß-Gal was transfected alone and the cells
treated with either FSH or (Bu)2cAMP, ß-galactosidase
activity was significantly stimulated by both treatments (data not
shown). This suggests that cAMP response element-like sequences are
present in the pCMVß-Gal vector.
Transient Overexpression of Various GRKs: Attenuation of
the FSH-Stimulated Response
To investigate the possible role of different GRK subtypes in the
attenuation of FSH-R-induced response, rat GRKs 2, 3, 5, 6a, and 6b
were transiently overexpressed along with the pSOMLuc reporter gene in
Ltk 7/12 cells. We measured the effects of GRK overexpression on basal
and FSH-induced luciferase production (Fig. 4
, ae). Each of the tested rat GRKs was
shown to attenuate the luciferase response to FSH. The inhibition
levels were directly related to the amount of expression vector
transfected (P < 0.001). The maximum inhibition levels
recorded in these experiments were between 71.2 ± 0.9% (GRK5)
and 96.6 ± 0.2% (GRK3) in the presence of FSH (Table 1
). The relative transfection
efficiencies were determined for each plate by hybridization of DNA
extracts prepared from half of the transfected cells, with a luciferase
cDNA probe. The CaPO4 precipitate method was not used in
this control experiment as the transfection levels are too low to allow
direct detection of the transfected plasmids. This control experiment
confirmed that all the GRKs decreased the FSH response (Fig. 4f
). The
fact that similar results were obtained using two different
transfection methods demonstrate that our results are independent from
possible Ca++- or liposome-induced alterations of cell fate
and/or signaling. The observation that the five GRKs analyzed were all
capable of reducing the FSH response is not surprising. Indeed,
numerous reports in the literature have shown that when overexpressed,
these kinases often lose their substrate specificities (reviewed in
Ref. 13). These results provide evidence that GRKs are able to interact
with FSH-R. However, these data are not sufficient to definitely
demonstrate the involvement of the GRKs in the desensitization to FSH
action. It is interesting, however, to note that the basal levels of
luciferase activity were also inhibited after GRK overexpression. This
finding is unexpected as GRKs are known to interact only with
agonist-occupied receptors. It can be hypothesized that when
overexpressed, FSH-R may display constitutive activity in the absence
of agonist as already described for the ß2-adrenergic receptor (30).
GRKs could then be able to desensitize the constitutively active FSH-R.
Another explanation could be that the overexpressed GRKs interact with
other Gs-coupled receptors activated by autocrine effectors or by
residual serum factors (Ltk 7/12 cells are cultured with 10% serum
before transfection and a 48-h starvation period).

View larger version (32K):
[in this window]
[in a new window]
|
Figure 4. FSH-R Uncoupling in Ltk 7/12 Cells by Transiently
Overexpressed GRKs
Ltk 7/12 cells were cotransfected with pSOMLuc and various quantities
of the following: panel a, pCMV5-GRK2 (0.5, 1, and 2 µg); panel b,
pCMV5-GRK3 (0.25, 1, and 2 µg); panel c, pCR3-GRK5 (0.5, 1, and 2
µg); panel d, pCB6-GRK6A (1, 2, and 3 µg); or panel e, pCB6-GRK6B
(0.5, 1, and 2 µg). Calcium phosphate precipitations were used for
transfections. Cells were stimulated ( )or not
( ) with FSH (100 ng/ml)
for 4 h. Luciferase activity was measured in the cell lysate.
Values were expressed in percentage, the activity of the unstimulated
control was taken as 100%. Panel f, 1 µg of each GRK expression
vector was transfected by lipofection as described in Materials
and Methods. Cells were stimulated with FSH (100 ng/ml) for
4 h. Luciferase activities and the amounts of plasmid transfected
were determined in parallel for each condition, luciferase activities
being corrected according to the relative transfection efficiencies.
The results were arbitrary expressed in percent, the control being
100%. These data are representative of two independent experiments,
each with four replicates. *, P < 0.05,
significant statistical differences from pCMV5 control cells.
|
|
Effects on FSH-Stimulated Response of the Specific Inhibition of
Endogenously Expressed GRKs Using Antisense cDNAs
To test whether endogenous GRKs in Ltk 7/12 cells were able to
regulate FSH-R function, we investigated the effects of specific
inhibition of GRK subtypes. Vectors containing GRK cDNAs in antisense
orientation were used as specific inhibitors. The cotransfection scheme
was as follows: one antisense construct of either rat GRK2, -3, -5, or
-6 was transfected along with the pSOMLuc reporter gene in Ltk 7/12
cells, and its effect on the FSH-induced luciferase production was
measured. As shown in Fig. 5a
, strong
increases in the cell response to FSH treatment were recorded in cells
transfected with antisense for GRK2, -5, and -6 [2- to 3-fold over the
FSH-treated control, P < 0.05; see (Table 2
)]. The antisense for GRK3 did not
affect the FSH-induced response of the cells. However, the ability of
this GRK3 antisense construct to induce increased cell response to an
agonist was observed in another cellular model (C. Troispoux, F.
Guillou, Y. Combarnous, and E. Reiter, unpublished data). Parallel to
the attenuation of basal luciferase activity after the overexpression
of sense GRKs (Fig. 4
), the four antisense constructs tested (Fig. 5a
)
significantly increased the basal levels by 2- to 4-fold
(P < 0.05, Table 2
). The antisense for GRK3 also
increased the basal level and did not affect FSH-stimulated response,
suggesting that basal luciferase activity is due to the activation of
receptor(s) other than the FSH-R by residual serum factor(s) or by an
autocrine mechanism. In addition, this confirms that the GRK3 antisense
construct effectively inhibits GRK3 action.
View this table:
[in this window]
[in a new window]
|
Table 2. Stimulation of the FSH Response in Ltk 7/12
Cells Transfected with Antisense cDNAs for the Different
GRKs
|
|
To assess the specificity of the antisense experiments above, slot blot
hybridizations were carried out using both sense and antisense cDNAs
corresponding to each GRK subtype (Fig. 5b
). The only significant
cross-hybridizations were obtained between GRK2 and GRK3. These results
were expected as GRK2 and GRK3 display the closest homologies among
GRKs. GRK2/3 cross-hybridizations were less than 10% and, more
importantly, GRK2 antisense triggered a significant increase in
FSH-induced response while GRK3 antisense did not. To check the
specificity and the efficiency of antisense activities on GRK protein
synthesis, cells were transiently cotransfected with both an expression
vector for green fluorescent protein (GFP) and one of the antisense
constructs. Doubly-transfected cells were selected by
fluorescence-activated cell sorting, using the GFP fluorescence and
expression of the different GRK proteins was assessed by Western
blotting (Fig. 5c
). The antisense-specific decreases in protein levels
were 22% for GRK2, 17% for the GRK2/3 ratio, 51% for GRK5, and 11%
for GRK6. No cross-inhibition between different GRKs was observed in
the different populations of sorted GFP-positive cells (data not
shown). Not surprisingly, using our antisense constructs, only partial
inhibition were observed, as it is well known that the use of
full-length cDNA in antisense orientation is not the most efficient
antisense strategy. In our model, we showed that pronounced biological
effects can be obtained at low inhibition rates for GRK2 and GRK6. The
relative efficiencies of the antisense treatments depend probably also
on the abundance of each kinase in the Ltk 7/12 cells. When pSOMLuc
responses were corrected with the relative transfection efficiencies,
pronounced potentializations of the FSH response were demonstrated for
GRK2AS, GRK6AS, and, to a lesser extent, for GRK5AS (Fig. 5d
).
Transient Overexpression of ß-Arrestins 1 and -2 and Dominant
Negative ß-Arrestin 2 [319418] in Ltk 7/12 Cells
To investigate the possible role of arrestins in
the homologous desensitization of FSH-R-stimulated-response, Ltk
7/12 cells were transiently cotransfected with the pSOMLuc reporter
gene along with either control or ß-arrestin 1 or ß-arrestin 2
expression vectors. Significant signal blunting was achieved by
ß-arrestin 1 or ß-arrestin 2 overexpression (Fig. 6a
), this effect being proportional to
the quantity of plasmid DNA transfected. Similarly, the overexpression
of ß-arrestin 2 [319418], a potent dominant negative inhibitor of
ß-arrestins (19), strongly potentiates FSH action (Fig. 6b
). These
results were not due to differential transfection efficiencies as shown
in Fig. 6c
. It is well established that arrestins bind to the
intracellular domain of the agonist-occupied receptors after their
phosphorylation by GRKs, thereby desensitizing signal transduction to
heterotrimeric G proteins (18). Thus, this experiment strongly suggests
that endogenously expressed GRKs, probably GRK2, GRK5, and GRK6 as
indicated by the results with the antisense constructs, might
phosphorylate agonist-occupied FSH-R in Ltk 7/12 cells. To ascertain
whether the overexpressed arrestins were indeed able to potentiate the
effects of GRKs, we assessed the ability of the different GRK/arrestin
combinations to desensitize FSH-R. Transient cotransfections were
carried out using a submaximal plasmid concentration to detect whether
the effects of GRKs and arrestins were additive (Fig. 7
). The trend of the results was quite
similar for both arrestins, but stronger inhibition was consistently
observed with ß-arrestin 2. GRK3, GRK6b, and, to a lesser extent,
GRK2 generated additional signaling inhibition with the two arrestins.
In contrast, the combination of GRK6a with ß-arrestin 1 or
ß-arrestin 2 displayed the same signal dampening as arrestins alone.
GRK5 raised the inhibitory effects of ß-arrestin 2 but not
ß-arrestin 1. These data suggest that some GRK/ß-arrestin
combinations are more efficient than others in the inhibition of FSH-R
coupling.

View larger version (22K):
[in this window]
[in a new window]
|
Figure 6. Transient Overexpression of ß-Arrestins 1, 2, and
Dominant Negative ß-Arrestins 2 [319418] in Ltk 7/12 Cells
a, Ltk 7/12 cells were transfected with pSOMLuc and either
ß-arrestin 1 (ß-arr1) or ß-arrestin 2 (ß-arr2) expression
vectors. Two quantities (0.25 and 0.5 µg per well) of the
ß-arrestin plasmids were tested. All the cells were stimulated by
FSH. *, P < 0.05; **, P <
0.01; significant statistical differences from control cells. b, Ltk
7/12 cells were transfected with pSOMLuc and ß-arrestin 2
[319418] expression vectors. Two quantities (1 and 2 µg per well)
of the ß-arrestin 2 [319418] were tested, the quantities of DNA
being normalized using the pCMV5 empty vector. All the cells were
stimulated by FSH. *, P < 0.05; ***,
P < 0.001; significant statistical differences
from control cells. c, Control of relative transfection efficiencies:
Ltk 7/12 cells were transfected using lipofection as described in
Materials and Methods. Cells were stimulated with FSH
(100 ng/ml) for 4 h. Luciferase activities and the amounts of
plasmid transfected were determined in parallel for each condition,
luciferase activities being corrected according to the relative
transfection efficiencies. The results were arbitrary expressed in
percent; the pCMV5 control was taken as 100%. *, P
< 0.05; **, P < 0.01; significant statistical
differences from pCMV5 control cells.
|
|

View larger version (39K):
[in this window]
[in a new window]
|
Figure 7. FSH-R Uncoupling in Ltk 7/12 cells by Transient
Overexpression of Combinations of ß-Arrestins and GRKs
a, ß-Arrestin 1 plus GRKs. Ltk 7/12 cells were cotransfected with
pSOMLuc and 0.5 µg of each GRK construct alone, or together with 0.25
µg per well of pCMV5-ß-arr1. b, ß-Arrestin 2 plus GRKs. Ltk 7/12
cells were cotransfected with pSOMLuc and 0.5 µg of each GRK
construct alone, or together with 0.25 µg per well of pCMV5-ß-arr2.
Values were expressed in percentage. The activity measured for cells
transfected with pSOMLuc and pCMV5 in nonstimulated conditions ( )
was taken as 100%. Significantly different from cells transfected with
the ß-arrestin 1 or 2 construct alone: *, P <
0.05; **, P < 0.01; ***, P <
0.001. These data are representative of two independent experiments,
each with four replicates.
|
|
The GRK/ß-Arrestin System Is Involved in the Modulation of the
FSH-Induced cAMP Response
The luciferase gene reporter did not allow measurement of
short-term stimulation by FSH. For this reason, we measured the rapid
(15 min) FSH-induced cAMP generation in Cos-7 cells (which do not
express the FSH-R) transiently cotransfected with both rat FSH-R
expression vector and GRK2, ß-arrestin 2, or GRK2AS constructs. Both
GRK2 and ß-arrestin 2 severely blunted the FSH-stimulated cAMP
production when compared with controls (Fig. 8
). On the contrary, the antisense
construct for GRK2 significantly enhanced cAMP accumulation after
short-term (15 min) FSH stimulation. This experiment suggests that the
GRK/ß-arrestin system is involved in the control of the acute
FSH-promoted cAMP accumulation.

View larger version (19K):
[in this window]
[in a new window]
|
Figure 8. Effect of GRK2, ß-arrestin 2, and GRK2AS on the
Acute FSH-Promoted cAMP Accumulation in Cos-7 Cells
Cos-7 cells were transiently cotransfected with the rat FSH-R and with
either pCMV5, GRK2, ß-arrestin 2, or GRK2AS. After 48 h
transfection, cells were stimulated with 100 ng/ml of FSH for 15 min.
Intracellular cAMP content was measured as described in
Materials and Methods. Values were expressed in
percentage. The FSH-dependent cAMP induction measured for cells
transfected with the FSH-R and pCMV5 was taken as 100%. These data
represent the mean ± SE of two independent
experiments, each with four replicates. **, P <
0.01; ***, P < 0.001; significant statistical
difference from control conditions.
|
|
 |
DISCUSSION
|
---|
The gonadotropin receptors (LH and FSH-R) are members of the
rhodopsin-like subfamily of G protein-coupled receptors (GPCRs) and
bind their ligands with high affinities. Both LH and FSH-Rs as well as
the TSH-receptor (TSH-R), are characterized by very large ectodomains
(31). Like other GPCRs, LH-R and FSH-R undergo homologous
desensitization (reviewed in Ref. 1). They become rapidly
phosphorylated on serine and threonine residues in response to agonist
stimulation, and this phosphorylation facilitates agonist-induced
functional uncoupling and internalization (21, 22). However, despite
the key role of gonadotropin receptors in the control of reproductive
functions, very little is known concerning the regulatory proteins
involved in their agonist-induced desensitization process. Premont
et al. (32) have shown that transient cotransfection of HEK
293 cells, with rat LH-R and either GRK2 or one of the four human GRK4
isoforms, dampened the cAMP response to a subsequent LH treatment.
Recent studies have also demonstrated the involvement of the
GRK/arrestin regulatory proteins in the homologous desensitization of
the structurally related TSH-R (33, 34). Nakamura et al.
(23) have reported that the overexpression of GRK2 enhances
agonist-dependent FSH-R phosphorylation. These authors have also shown
that ß-arrestin 2 overexpression stimulates FSH-R internalization.
However, so far, it has not been proven that GRKs and arrestins promote
the functional uncoupling of the FSH-R in a way similar to that of the
ß2-adrenergic receptor and rhodopsin models (13). In
contrast, since gonadotropin hormones dissociate very slowly from their
receptors, it has been proposed that LH-R and FSH-R may utilize unusual
mechanisms for deactivation (22).
In this study, we have used a cAMP-sensitive reporter gene to
indirectly monitor the coupling between G
s and the FSH-R. The
results obtained using this reporter system and many data of the
literature suggest that GRKs and ß-arrestins participate to the
functional uncoupling of the FSH-R. However, direct measurement of
early signaling events (i.e. receptor phosphorylation, G
s
activation, and adenylate cyclase activity) will be necessary to
confirm that the mechanisms described for the
ß2-adrenergic receptor are applicable to the homologous
desensitization of the FSH-R (13). This study describes three distinct
aspects of the relationships between GRKs, ß-arrestins, and the
rat FSH-R in Ltk 7/12 cells: 1) individual GRKs inhibit FSH-R-mediated
signaling as demonstrated by the diminished response to FSH in cells
transfected with specific GRK subtypes; 2) ß-arrestins quench FSH-R
signal transduction when transiently overexpressed, and this effect is
potentiated by certain GRKs; and 3) FSH-R desensitization depends on
endogenous GRKs and ß-arrestins; in fact, transient overexpression of
antisense constructs for the different GRKs and of a dominant negative
construct for ß-arrestins enhances FSH stimulation. All the tested
GRKs were able to dampen the FSH-induced response. Since high levels of
overexpressed proteins were reached in Ltk 7/12 cells for both receptor
and GRKs, a problem of specificity was raised. In fact, it is possible
that, by mass action, even GRK subtypes with low affinity for the
agonist-bound receptor may phosphorylate it in heterologous system but
not in physiological conditions.
To investigate further the interaction between the FSH-R and the
different GRKs, we used GRK antisense constructs to selectively inhibit
the different kinases. Increased signaling responses were recorded with
the antisense cDNA constructs for GRK2, GRK5, and GRK6, but not GRK3.
These four kinases are detected in Ltk 7/12 cells using RT-PCR and
Western blotting. We were confident about the specificity of the
antisense approach as we could show that GRK3 antisense had no effect
in a cell line expressing this kinase. On the basis of our control
experiments, the possibility of cross-reactivities between GRK2, GRK5,
and/or GRK6 could be excluded. As assessed by the increase in signal
stimulation in the presence of antisense GRK constructs, the FSH-R
appears to interact with three of the four GRKs tested. The regulation
of FSH-R by one or more GRKs in vivo is further validated by
our previous finding that GRK2, -3, -5, and -6 are expressed in rat
Sertoli cells (C. Troispoux, F. Guillou, Y. Combarnous, and E. Reiter,
unpublished data). It could be anticipated, however, that in a
physiological model, the specificity of the FSH-R/GRK interactions may
be different and probably more stringent. Until now, only GRK5 in
thyroid cells (33) and GRK2 in both CHO and A-431 human epidermoid
carcinoma cells (35) have been selectively inhibited using antisense
cDNA or oligodeoxynucleotides. Here, we demonstrate that the use of
antisense cDNAs is sufficient to selectively inhibit virtually any GRK
allowing us to study the substrates of these kinases.
Previous work has demonstrated that overexpression of ß-arrestin
augments the desensitization of various GPCRs (34, 36). In agreement
with these reports, we demonstate that FSH-R signaling is blunted by
transiently overexpressed ß-arrestins and is strongly potentiated by
the dominant negative form of this molecule. These data demonstrate
that endogenous ß-arrestins could play a role in the
desensitization of FSH-R. Both ß-arrestins potentiate the GRK-induced
uncoupling of the FSH-occupied receptors, and the degree of inhibition
obtained varies according to the types of the cotransfected
GRK/ß-arrestin couples. In spite of the limits of this transfection
approach (see discussion above), these data suggest that preferential
GRK/ß-arrestin associations might exist in vivo for the
interaction with the FSH-R. Interestingly, GRK6a completely fails to
enhance the effects of ß-arrestin 1 or 2, while the GRK6b isoform
displays marked additive action with both ß-arrestins. This is the
first direct evidence of a functional difference between the two
isoforms of GRK6, whose expression patterns in the rat are different:
the GRK6b is the predominant subtype in most tissues, including the
testis, while GRK6a is the main isoform expressed in the brain
(17).
By analogy with other receptors shown to be GRK substrates, serine and
threonine residues present in the intracellular parts of the FSH-R
would most likely be sites for GRK phosphorylation (13). In a recent
work, Nakamura et al. (22) have shown that the
agonist-induced phosphorylation of the rat FSH-R maps on residues
located in the first (T369, T370, S371, and T376) and third (T536,
T541, S544, S545, S546, S547, and T549) intracellular loops. When three
of the four putative phosphorylation sites present in the first loop
were mutated, the FSH-induced, but not PMA-induced, phosphorylation
of the receptor was almost completely abolished. Moreover, this mutated
receptor was found significantly less uncoupled after a 15-min
pretreatment with FSH than the wild-type or a third loop mutant of
FSH-R (the phosphorylation sites of the third loop were substituted
into alanine). The first loop mutant was also significantly less
internalized than the wild-type FSH-R or the third loop mutant. These
data suggest that the first intracellular loop of the agonist-occupied
FSH-R could be important for the interaction with GRKs.
In summary, the present study demonstrates that overexpressed GRKs or
ß-arrestins, probably by interacting with agonist-occupied FSH-R, can
inhibit receptor signaling. Antisense cDNAs, selectively inhibiting the
synthesis of each endogenous GRK, were found to potentiate FSH
action.This demonstrates the involvement of GRK2/6 and, to a lesser
extent, of GRK5 in the homologous desensitization of the rat FSH-R
expressed in Ltk 7/12 cells. To appreciate the specificity of
GRK/arrestin interactions with the FSH-R in physiological conditions
(i.e. very low levels of both receptor and GRK), primary
cultures of Sertoli or granulosa cells should be studied in future
works.
 |
MATERIALS AND METHODS
|
---|
Materials
Cell Culture
MEM, geneticin, trypsin-EDTA, and FCS were purchased from Life Technologies, Inc. (Gaithersburg, MD). Penicillin, streptomycin,
4-methyl-umbelliferyl-ß-D-galactopyranoside, and
(Bu)2 cAMP were purchased from Sigma Chemical Co. (St. Louis, MO). Porcine FSH (pFSH) was purified in our
laboratory (CY 1737 III: 41x NIH FSH P1 in homologous porcine RRA).
RT-PCR
MMLV reverse transcriptase was from Life Technologies, Inc. Taq DNA polymerase was from Pharmacia Biotech (Uppsala, Sweden). Radiolabeled
[
-32P]dCTP (3000 Ci/mmol) was purchased from
Amersham Pharmacia Biotech (Arlington Heights, IL).
Plasmid Constructs
pCMVßgal was from CLONTECH Laboratories, Inc. (Palo
Alto, CA). The cAMP-sensitive reporter construct pSOMLuc was kindly
donated by Dr. B. Peers (Liège, Belgium). pCMV5-rat GRK2,
pCMV5-rat GRK3, pCMV5-rat ß-arrestin 1, and pCMV5-rat ß-arrestin 2
were generous gifts from Dr. R. J. Lefkowitz (Durham, NC) (37, 38). pCR-ratGRK5 and pCR-ratGRK5 antisense were kindly donated by Dr.
Y. Nagayama (Nagasaki, Japan) (33), while pCB6-rat GRK6a and pCB6-rat
GRK6b were previously described (17). The pCMV5-GRK2 antisense
construct was generated by digesting pCMV5-GRK2 vector with
BglII/HindIII, blunting the ends, inserting the
GRK2 full-length cDNA into SmaI-linearized pCMV5 vector, and
selecting a clone with the appropriate antisense orientation. The
pCMV5-GRK3 antisense was constructed by cutting pCMV5-GRK3 with
EcoRI. The 2.4-kb fragment was then ligated with
EcoRI-linearized pCMV5. A clone displaying the appropriate
antisense orientation was selected. The pCMV5-GRK6 antisense construct
was obtained by cutting pBSSK-GRK6B with HindIII and
EcoRI enzymes and inserting the fragment into the
HindIII/EcoRI-digested pCMV5. The pRK-FSHR/3
plasmid was a kind gift from Dr. R. Sprengel (Heidelberg, Germany). The
ß-arrestin 2 [319418] expression vector was kindly donated by Dr.
J. L. Benovic (Philadelphia, PA).
Cell Culture and Transfection
Mouse Ltk cell line permanently transfected with the rat FSH-R
(Ltk 7/12 FSHR) was a kind gift from Dr. E. Nieschlag (Münster,
Germany) (27). These cells were cultured in MEM supplemented with 10%
heat-inactivated FCS and 200 mg Geneticin per 500 ml culture medium.
Cos-7 cells were cultured in DMEM supplemented with 10%
heat-inactivated FCS, glutamine, and antibiotics. Cells were maintained
at 37 C in a humidified atmosphere of 5% CO2.
Cells, plated at a density of 2.5 x 105 per well in
12-well culture plates (Corning, Inc., Corning, NY), were
transfected 24 h later with various quantities of the appropriate
plasmid mixtures. The calcium phosphate precipitation method was used:
62 µl CaCl2 (2 M) were mixed with the
constructs and the volume adjusted to 500 µl with water. A DNA
precipitate was formed by adding this mixture dropwise to 500 µl
HEPES-buffered saline (HBS), after which 120 µl of precipitate were
added per well followed by incubation for 4 h. The transfection
medium was then aspirated and 300 µl glycerol (15% in HBS) was added
for 1 min. Cells were rinsed with MEM and were incubated without serum
for 40 h. Alternatively, in some experiments, transfections were
carried out using Transfast liposomes (Promega Corp.,
Madison, WI) according to the manufacturers instructions. Cells were
stimulated with either 100 ng/ml FSH or saline for 4 h (without
phosphodiesterase inhibitor), and then cells were then collected to
determine luciferase activity. In all the experiments, the empty vector
was added to keep the transfected DNA quantities constant.
Cotransfections of the pSOMLuc reporter gene alone with the empty
vector were used as control. Each transfection was repeated at least
four times, with at least two different DNA preparations for each
construct. Results were expressed in percentage as relative light
unit values are directly related to cell density at the moment
of transfection. Relative variations were highly reproducible among all
experiments. Repeated transfections were performed to measure
efficiencies of transfection between precipitates. The variabilities
between precipitates were always lower than 25% (data not shown).
In some experiments, cells were trypsinized and divided in two
equivalent pools: the first to measure luciferase activities and the
second to determine the relative amounts of plasmid transfected.
Briefly, frozen cell pellets were digested overnight at 37 C under
gentle agitation in 100 mM Tris-HCl (pH 8.0), 200
mM NaCl, 5 mM EDTA, 0.2% SDS, and 7.5 mg/ml of
proteinase K. The digested mixtures were extracted by phenol-chloroform
and were then digested with RNAse A for 1 h at 37 C. The different
DNA samples were immobilized on Hybond-N+ (Amersham Pharmacia Biotech) membranes using a slot blot apparatus and standard
denaturation/neutralization protocols. The membranes were hybridized
with a random priming labeled cDNA probe corresponding to
Photinus pyralis luciferase coding sequence.
Prehybridizations and hybridizations were performed for 1 and 16
h, respectively, at 65 C in mixtures recommended by the manufacturer.
Quantifications were done using ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA) after autoradiography using a
phosphorscreen (Phosphorimager, Molecular Dynamics, Inc.).
RT-PCR Analysis of GRKs and ß-Arrestins
Total RNA was extracted from Ltk 7/12 cells by the single-step
guanidium-phenol-chloroform method described by Chomczynski and Sacchi
(29). The expression of the GRK2, -3, -5, and -6 and ß-arrestin 1 and
2 genes in Ltk 7/12 cells was studied using RT-PCR assays.
One microgram of total RNA was reverse-transcribed into single-strand
cDNA by MMLV reverse transcriptase (Gibco BRL Europe, Ghent, Belgium).
Reverse transcription was carried out in a 50 µl reaction volume
containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl,
3 mM MgCl2, 200 µM of each
deoxynucleotide triphosphate, 10 mM dithiothreitol, 100
pmol of oligo[dT]1218 (Pharmacia Biotech), and 200 U
of reverse transcriptase. After completion of RT (45 min at 42 C), the
temperature was raised to 70 C for 15 min to inactivate the enzyme.
The reverse transcribed first-strand cDNAs were amplified with a set of
primer pairs designed to specifically amplify parts of the different
GRK and ß-arrestin transcripts: GRK2 sense primer
(5'-TCCAGTCGGTGGAAGAGACACA-3') and antisense primer
(5'-GCTGAATCAGTGGCACCTTGCT-3') corresponded to bases 19601981 and
21692190 of the rat GRK2 cDNA, respectively. GRK3 sense
primer (5'-CATGTCTGTGGAGGAGACCCAA-3') and antisense primer
(5'-CAGATGAATATTCAATTCCAC-3') corresponded to bases 18211842 and
20502072 of the rat GRK3 cDNA, respectively. GRK5 sense
primer (5'-CAAGGAGCTGAATGTGTTCGGAC-3') and antisense primer
(5'-GCTGCTTCCAGTGGAGTTTGAAT-3') corresponded to bases 17461768 and
19311953 of the rat GRK5 cDNA, respectively. GRK6 primers
have been already described and they generated a 252-bp insert (15).
ß-Arrestin1 sense primer (5'-GTCAAAGTGAAGCTGGTGGTGTC-3')
and antisense primer (5'-CCATCATCCTCTTCGTCCTTGTC-3') corresponded to
bases 10021024 and 12391262 of the rat ß-arrestin 1 cDNA,
respectively. ß-Arrestin 2 sense primer
(5'-TACAGGGTCAAGGTGAAGCTGGT-3') and antisense primer
(5'-GGTCATCACAGTCGTCATCCTTC-3') corresponded to bases 11581180 and
13911413 of the rat ß-arrestin 2 cDNA, respectively.
PCRs were carried out on 4 µl of RT in 100 µl reaction volumes
containing 10 mM Tris-HCl (pH 9.0), 50 mM KCl,
1.5 mM MgCl2, 200 µM of each
deoxynucleotide triphosphate, 10 pmol of each primer, 0.5 µCi/nmol
[
-32P]dCTP, and 1.25 U of Taq polymerase
(Pharmacia Biotech). The samples were overlaid with
mineral oil and processed for 25, 30, or 35 PCR cycles (95 C, 1 min; 60
C, 1 min; 72 C, 1 min); the last extension was for 10 min at 72 C. The
DNA fragments were separated by electrophoresis through 2% agarose
gels. The gels were fixed in 10% acetic acid, dried, and submitted to
x-ray film autoradiography.
Immunoblotting
Ltk 7/12 cells were washed twice in PBS and homogenized at 0 C
in lysis buffer (10 mM Tris-Cl, pH 7.4, 2 mM
EDTA with the following protease inhibitors: 0.1 mM
phenylmethylsulfonyl fluoride; 10 µg/ml benzamidine, leupeptin, and
soybean trypsin inhibitor; 5 µg/ml aprotinin; 1 µg/ml pepstatin A).
The supernatants of a 1000 x g spin were subsequently
centrifuged for 1 h at 150,000 x g. The
supernatants of this second centrifugation were designated as the
cytosolic fractions, while the membrane fractions were the 150,000
x g pellets resuspended in lysis buffer. Cytosolic and/or
membrane fractions were applied to 10% SDS-polyacrylamide gels.
Colored mol wt markers (Amersham Pharmacia Biotech) were
simultaneously loaded on the gels. Membrane or cytosol extracts from
HEK 293 cells transfected with a single GRK or arrestin were used as
positive controls. Western blots were prepared by electrophoretically
transferring the proteins (2 h at 250 mA using 25 mM Tris,
192 mM glycine, pH 8.3, and 20% methanol) onto
polyvinylidene fluoride (PVDF) transfer membranes (NEN Life Science Products, Boston, MA). The membranes were blocked
for 2 h at room temperature with buffer A (Tris-HCl, pH 8.0, 150
mM NaCl, 0.05% Tween 20, 5% instant nonfat dry milk).
Next, they were incubated for 2 h at room temperature in buffer A
containing immune serum. Rabbit polyclonal antibody raised against GRK2
(epitope: amino acids 675689 of human GRK2) and the corresponding
control peptide were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The GRK2/GRK3 monoclonal antibody C5/1,
raised against the C terminus of GRK2, was kindly donated by Dr.
R. J. Lefkowitz. Rabbit polyclonal antibodies raised against GRK5
(N-terminal peptide) and GRK6 (C-terminal peptide) were kind gifts
from Dr. F. Boulay. The monoclonal antibody F4C1, raised against the
highly conserved epitope DGVVLVD, identical in ß-arrestins, was
kindly provided by Dr. L. A. Donoso. After this, each membrane was
washed three times for 5 min with buffer B (Tris-HCl, pH 8.0, 150
mM NaCl, 0.05% Tween 20). The blots were incubated for
1 h in buffer A containing a peroxidase-conjugated second
antibody. Each was then washed three times for 5 min with buffer B and
once for 5 min with buffer C (Tris-HCl, pH 8.0, 150 mM
NaCl). Finally, the blots were developed using the ECL
chemiluminescence detection system (Amersham Pharmacia Biotech).
Cross-Hybridization Assay
The full-length GRK2, -3, -5, and -6A cDNAs were subcloned in
pBluescript SK, and each was linearized at both ends in the polylinker.
Complementary RNAs (cRNAs) corresponding to the different GRKs were
then synthesized in both sense and antisense orientations using T3 and
T7 RNA polymerases (Promega Corp.). The antisense cRNAs
were synthesized in the presence of [
-32P]CTP
(NEN Life Science Products) while the sense cRNAs were
unlabeled.
The sense cRNAs were denatured at 50 C for 15 min in 10x SSC
(1xSSC = 0.15 M NaCl, 0.015 M sodium
citrate, pH 7) and 5% formaldehyde (vol/vol). The material was applied
to nitrocellulose filters (Schleicher & Schuell, Inc.,
Dassel, Germany) in serial 2-fold dilutions using a slot blot
apparatus. The filters were baked 2 h at 80 C and were then
hybridized with one of the purified antisense probes (15
106 cpm). Prehybridizations and hybridizations were
performed for 2 and 16 h, respectively, at 60 C in the
manufacturers recommended mixtures. Quantifications were done using
ImageQuant software (Molecular Dynamics, Inc., Sunnyvale,
CA) after autoradiography using a phosphorscreen.
Sorting and Analysis of Antisense Transfected Cells
Ltk 7/12 cells were transiently cotransfected with both an
expression vector for GFP (CLONTECH Laboratories, Inc.
Palo Alto, CA) and one of the antisense constructs described above.
Transfast liposomes were used in a 1:1 ratio; 2.5 µg of
GFP expression vector and 40 µg of either pCMV5 or antisense
construct were used for each 75-cm2 culture flask. After
48 h, GFP positive living cells were than selected using a FACStar
Plus cell sorter (Beckton Dickinson and Co., Franklin
Lakes, NJ). To avoid contaminations with untransfected cells, only
1520% of cells presenting the more intense GFP signal (
50% of
the cells were GFP positive) were sorted for subsequent analysis. About
3 x 105 cells were obtained for each condition.
Whole-cell extracts were prepared using the lysis buffer described
above plus 0.5% NP40. Solubilization was carried out for 1 h on
ice. The extracts were then cleared by centrifugation, and the amount
of protein was determined. Twenty micrograms of the different extracts
were loaded on gels and blotted as described above. The blots were
stained with Coomassie blue and scanned to allow quantifications. The
densitometric measurement of the blots was achieved using NIH Image
software.
cAMP Assay
For cAMP assay, 2-day cultured cells were stimulated by FSH for
various times without phosphodiesterase inhibitor. Cells were frozen
and lysed and the intracellular cAMP content was determined. The
intracellular cAMP content was assayed using a RIA kit (Sanofi Pharmaceuticals, Inc., Pasteur, France).
Luciferase Activity Measurement
The luciferase activity was measured using the luciferase assay
system supplied by Promega Corp. Briefly, cells were
scraped in 100 µl of reporter lysis buffer. The cell lysate was
centrifuged (12,000 x g, 2 min, 4 C), and the
supernatant was collected. Each sample (40 µl) was mixed with 100
µl of luciferase assay reagent, containing the substrate. The light
produced was measured in a luminometer (Lumat LB 9507, EG&G Berthold,
Turku, Finland) and expressed in relative light units.
Statistics
Statistical analysis of the data was performed using a single
mean Students t test (Statview, Abacus Concepts, Berkeley,
CA).
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs. R. J. Lefkowitz (Durham, NC), J. L.
Benovic (Philadelphia, PA), E. Nieschlag (Münster, Germany), Y.
Nagayama (Nagasaski, Japan), B. Peers (Liège, Belgium), R.
Sprengel (Heidelberg, Germany), and F. Boulay (Grenoble, France) for
their kind gifts of materials. We are grateful to Drs D. Kerboeuf and
Y. Le Vern (Nouzilly, France) for cell sorting analysis. Thanks are
also due to Mrs. C. Barc and Mr. S. Marion for technical assistance
with the Western blot analyses and to Mr. A. Beguey for the
photography.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Eric Reiter, INRA/CNRS URA 1291, Station de Physiologie de la Reproduction des Mammifères Domestiques, 37380 Nouzilly, France.
Carine Troispoux was recipient of a doctoral fellowship from the
Ministère de la Recherche et de lEducation Nationale.
Received for publication June 24, 1998.
Revision received May 27, 1999.
Accepted for publication June 2, 1999.
 |
REFERENCES
|
---|
-
Simoni M, Gromoll J, Nieschlag E 1997 The follicle
stimulating hormone receptor: biochemistry, molecular biology,
physiology and pathophysiology. Endocr Rev 18:739773[Abstract/Free Full Text]
-
Sprengel R, Braun T, Nikolics K, Segaloff DL, Seeburg PH 1990 The testicular receptor for follicle stimulating hormone: structure and
functional expression of cloned cDNA. Mol Endocrinol 4:525530[Abstract]
-
Verhoeven G, Cailleau J, de Moor P 1980 Desensitization of
cultured rat Sertoli cells by follicle-stimulating hormone and by
L-isoproterenol. Mol Cell Endocrinol 20:113126[CrossRef][Medline]
-
Le Gac F, Attramadal H, Jahnsen T, Hansson V 1985 Studies on
the mechanism of follicle-stimulating hormone-induced desensitization
of Sertoli cell adenylyl cyclase in vitro. Biol Reprod 32:916924[Abstract]
-
Sanchez-Yagüe J, Hipkin W, Ascoli M 1993 Biochemical
properties of the agonist-induced desensitization of the
follicle-stimulating hormone and luteinizing hormone/chorionic
gonadotropin-responsive adenylyl cyclase in cells expressing the
recombinant gonadotropin receptors. Endocrinology 132:10071016[Abstract]
-
Laurent-Cadoret V, Guillou F, Combarnous Y 1994 Heterologous
and homologous desensitization of the plasminogen activator response of
rat Sertoli cells by FSH and isoproterenol. Endocr J 2:805812
-
Ford KA, LaBarbera AR 1988 Autoregulation of acute
progesterone and adenosine 3',5'-monophosphate responses to
follicle-stimulating hormone (FSH) in porcine granulosa cells: effects
of FSH, cholera toxin, forskolin and pertussis toxin. Endocrinology 123:23672373[Abstract]
-
Grasso P, Reichert Jr LE 1989 Follicle-stimulating
hormone (FSH) induces G protein dissociation from FSH
receptor-G protein complexes in reconstituted proteoliposomes.
Biochem Biophys Res Commun 162:12141221[Medline]
-
Conti M, Toscano MV, Petrelli L, Geremia R, Stefanini M 1983 Involvement of the phosphodiesterase in the refractoriness of the
Sertoli cell. Endocrinology 113:18451853[Abstract]
-
Saez JM, Jallard C 1986 Processing of follitropin and its
receptor in cultured pig Sertoli cells. Effects of monensin. Eur J
Biochem 158:9197[Abstract]
-
Themmen AP, Blok LJ, Post M, Hoogerbrugge JW, Parmentier M,
Vassart G, Grootegoed JA 1991 Follitropin receptor down-regulation
involves a cAMP-dependent post transcriptional decrease of receptor
mRNA expression. Mol Cell Endocrinol 78:R7R13
-
Monaco L, Foulkes NS, Sassone-Corsi P 1995 Pituitary
follicle-stimulating hormone (FSH) induces CREM gene expression in
Sertoli cells: involvement in long-term desensitization of the FSH
receptor. Proc Natl Acad Sci USA 92:1067310677[Abstract]
-
Freedman NJ, Lefkowitz RJ 1996 Desensitization of G
protein-coupled receptors. Recent Prog Horm Res 51:319353[Medline]
-
Premont RT, Inglese J, Lefkowitz RJ 1995 Protein kinases that
phosphorylate activated G protein-coupled receptors. FASEB J 9:175182[Abstract/Free Full Text]
-
Sallese M, Mariggio S, Collodel G, Moretti E, Piomboni
P, Baccetti B, De Blasi A 1997 G-protein coupled receptor kinase GRK4:
molecular analysis of the four isoforms and ultrastructural
localisation in spermatozoa and germinal cells. J Biol Chem 272:1018810195[Abstract/Free Full Text]
-
Virlon B, Firsov D, Cheval L, Reiter E, Troispoux C, Guillou
F, Elalouf JM 1998 Rat G protein-coupled receptor kinase GRK4:
identification, functional expression, and differential tissue
distribution of two splice variants. Endocrinology 139:27842795[Abstract/Free Full Text]
-
Firsov D, Elalouf JM 1997 Molecular cloning of two rat GRK6
splice variants. Am J Physiol 273:C953C961
-
Ferguson SSG, Zhang J, Barak LS, Caron MG 1996 G
protein-coupled receptor kinases and arrestins: regulators of G
protein-coupled receptor sequestration. Biochem Soc Trans 24:953959[Medline]
-
Krupnick JG, Goodman OB, Keen JH, Benovic JL 1997 Arrestin/clathrin interaction. Localization of the clathrin binding
domain of non-visual arrestins in the carboxyl terminus. J Biol
Chem 272:1501115016[Abstract/Free Full Text]
-
Quintana J, Hipkin WR, Sanchez-Yagüe J, Ascoli M 1994 Follitropin (FSH) and a phorbol ester stimulate the phosphorylation of
the FSH receptor in intact cells. J Biol Chem 269:87728779[Abstract/Free Full Text]
-
Ascoli M 1996 Functional consequences of the phosphorylation
of the gonadotropin receptors. Biochem Pharmacol 52:16471655[CrossRef][Medline]
-
Nakamura K, Hipkin WR, Ascoli M 1998 The agonist-induced
phosphorylation of the rat follitropin receptor maps to the first and
third intracellular loops. Mol Endocrinol 12:580591[Abstract/Free Full Text]
-
Nakamura K, Krupnick JG, Benovic JL, Ascoli M 1998 Signaling
and phosphorylation impaired mutants of the rat follitropin receptor
reveal an activation- and phosphorylation-independent but
arrestin-dependent pathway for internalization. J Biol Chem 273:2434624354[Abstract/Free Full Text]
-
Laurent-Cadoret V, Guillou F, Combarnous Y 1994 Protein
kinases and protein synthesis are involved in desensitization of the
plasminogen activator response of rat Sertoli cells by
follicle-stimulating hormone. FEBS Lett 352:1923[CrossRef][Medline]
-
Keren-Tal I, Dantes A, Amsterdam A 1996 Activation of
FSH-responsive adenylate cyclase by staurosporine: role for protein
phosphorylation in gonadotropin receptor desensitization. Mol Cell
Endocrinol 116:3948[CrossRef][Medline]
-
Selvaraj N, Amsterdam A 1997 Modulation of FSH receptor
phosphorylation correlates with hormone-induced coupling to the
adenylate cyclase system. Endocrine 6:179185[Medline]
-
Guderman T, Brockmann H, Simoni M, Gromoll J, Nieschlag E 1994 In vitro bioassay for human serum follicle-stimulating
hormone (FSH) based on L cells transfected with recombinant rat FSH
receptor: validation of a model system. Endocrinology 135:22042213[Abstract]
-
Rommertz FFG, van Loenen HJ, Schipper I, Fauser BCJM 1997 FSH-receptor interaction and signal transduction: an alternative
view. In: Fauser BCJM (ed) FSH Action and Intraovarian Regulation.
Studies in Profertility Series. The Parthenon Publishing Group, London,
vol 6:6181
-
Chomczynski P, Sacchi N 1987 Single-step method of RNA
isolation by acid guanidium thiocyanate-phenol-chloroform extraction.
Anal Biochem 162:156- 159[CrossRef][Medline]
-
Milano CA, Allen LF, Rockman HA, Dolber PC, McMinn TR, Chien
KR, Johnson TD, Bond RA, Lefkowitz RJ 1994 Enhanced myocardial
function in transgenic mice overexpressing the ß2-adrenergic
receptor. Science 264:582586[Medline]
-
Segaloff DL, Ascoli M 1993 The lutropin/chorionic gonadotropin
(LH/CG) receptor ... 4 years later. Endocr Rev 14:324347[Abstract]
-
Premont RT, Macrae AD, Stoffel RH, Chung N, Pitcher JA,
Ambrose C, Inglese J, MacDonald ME, Lefkowitz RJ 1996 Characterization
of the G protein-coupled receptor kinase GRK4. Identification of four
splice variants. J Biol Chem 271:64036410[Abstract/Free Full Text]
-
Nagayama Y, Tanaka T, Hara T, Namba H, Yamashita S, Taniyama
K, Niwa M 1996 Involvement of G protein-coupled receptor kinase 5 in
homologous desensitization of the thyrotropin receptor. J
Biol Chem 271:1014310148[Abstract/Free Full Text]
-
Iacovelli L, Franchetti R, Masini M, De Blasi A 1996 GRK2 and ß-arrestin1 as negative regulators of thyrotropin
receptor-stimulated response. Mol Endocrinol 10:11381146[Abstract]
-
Shih M, Malbon CC 1994 Oligodeoxynucleotides antisense to
mRNA encoding protein kinase A, protein kinase C, and
ß-adrenergic receptor kinase reveal distinctive cell-type specific
roles in agonist-induced desensitization. Proc Natl Acad Sci USA 91:1219312197[Abstract/Free Full Text]
-
Pippig S, Andexinger S, Daniel K, Puzicha M, Caron MG,
Lefkowitz RJ, Lohse MJ 1993 Overexpression of ß-adrenergic
receptor kinase augments desensitization of ß-adrenergic
receptors. J Biol Chem 268:32013208[Abstract/Free Full Text]
-
Arriza JL, Dawson TM, Simerly RB, Martin LJ, Caron
MG, Snyder SH, Lefkowitz RJ 1992 The G protein-receptor kinases
ß-ARK1 and ß-ARK2 are widely distributed at synapses in rat brain.
J Neurosci 12:40454055[Abstract]
-
Attramadal H, Arriza JL, Aoki C, Dawson TM, Codina J, Kwatra
MM, Snyder SH, Caron MG, Lefkowitz RJ 1992 ß-Arrestin2, a novel
member of the arrestin/ß- arrestin gene family. J Biol Chem 267:1788217890[Abstract/Free Full Text]
-
Gonzalez GA, Montminy MR 1989 Cyclic AMP stimulates
somatostatin gene transcription by phosphorylation of CREB at serine
133. Cell 59:675680[Medline]