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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
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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).
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). 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.
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). 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.
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). 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.
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), and a clear saturation of cell
response was observed within 20–60 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.
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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, a–e). 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).
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, 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.
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). 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 [319–418] 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 [319–418], 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.
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). 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.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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, Gs
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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 [319–418] 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 manufacturer’s 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]12–18 (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 1960–1981 and 2169–2190 of the rat GRK2 cDNA, respectively. GRK3 sense primer (5'-CATGTCTGTGGAGGAGACCCAA-3') and antisense primer (5'-CAGATGAATATTCAATTCCAC-3') corresponded to bases 1821–1842 and 2050–2072 of the rat GRK3 cDNA, respectively. GRK5 sense primer (5'-CAAGGAGCTGAATGTGTTCGGAC-3') and antisense primer (5'-GCTGCTTCCAGTGGAGTTTGAAT-3') corresponded to bases 1746–1768 and 1931–1953 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 1002–1024 and 1239–1262 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 1158–1180 and 1391–1413 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 675–689 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 manufacturer’s 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
15–20% 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 Student’s t test (Statview, Abacus Concepts, Berkeley,
CA).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Carine Troispoux was recipient of a doctoral fellowship from the Ministère de la Recherche et de l’Education Nationale.
Received for publication June 24, 1998. Revision received May 27, 1999. Accepted for publication June 2, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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) 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.
). 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.
)
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.