Department of Pharmacology (M. de F.M.L., X.L., K.N., M.A.) The
University of Iowa College of Medicine Iowa City, Iowa 52242
Department of Microbiology and Immunology (J.L.B.) Kimmel
Cancer Institute Thomas Jefferson University Philadelphia,
Pennsylvania 19107
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ABSTRACT |
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INTRODUCTION |
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While this paradigm has been derived mostly from the study of the
ß2-adrenergic receptor, recent studies have now shown that it is not
fully applicable to all GPCRs. For example, the coated pit-mediated
internalization of the µ-opioid receptor is stimulated by some, but
not all, agonists (4, 5). The removal of the phosphorylation sites of
the -opioid receptor (6) or the mutation of the phosphorylation
sites of the PTH receptor (7) does not affect the agonist-induced
internalization of these receptors. Moreover, in the absence of
receptor phosphorylation, the overexpression of nonvisual arrestins can
rescue the agonist-induced internalization of the ß2-adrenergic
receptor (8) and the rat FSH receptor (rFSHR) (9), but it cannot
enhance the morphine-induced internalization of the µ-opioid receptor
(5).
Recent studies from this laboratory have shown that the rFSHR
becomes phosphorylated upon agonist activation (10, 11, 12). While the
GRK-catalyzed phosphorylation of the majority of GPCRs occurs in the
C-terminal tail, the rFSHR belongs to a small subfamily of GPCRs that
are exclusively phosphorylated in the intracellular loops. Like the
2A-adrenergic (13) and the m2 muscarinic (14, 15) receptors, the
agonist-induced phosphorylation of the rFSHR occurs in the third
intracellular loop (11, 12). The rFSHR is unusual, however, in that
agonist activation also promotes the phosphorylation of sites present
in the first intracellular loop (12).
The possible involvement of rFSHR phosphorylation in agonist-induced uncoupling and internalization has been studied using a number of phosphorylation-impaired rFSHR mutants. Our studies have shown that the mutation of S/T residues present in the third intracellular loop of the rFSHR impairs agonist-induced phosphorylation and uncoupling without affecting agonist-induced internalization, whereas the mutation of S/T residues present in the first intracellular loop of the rFSHR impairs agonist-induced phosphorylation, uncoupling, and internalization (12). Two signaling-impairing mutations of the rFSHR that preserve the phosphorylation sites have also been shown to completely prevent agonist-induced phosphorylation and to impair internalization (9). Overexpression of arrestin-3, however, rescues the internalization of both signaling-impaired mutants, even in the absence of receptor phosphorylation (9). Thus, it appears that the agonist-induced phosphorylation of the rFSHR is important, but not essential, for agonist-induced internalization.
In spite of our increased understanding of the location and function of the residues that are phosphorylated in the rFSHR, there is a paucity of information about the identity of the kinases that mediate the agonist-induced phosphorylation. The possible involvement of the cAMP-dependent protein kinases has been excluded by the following two findings: 1) the hFSH-induced phosphorylation of the rFSHR proceeds normally in a transfected cell line that does not respond with an increase in cAMP accumulation (because of overexpression of cAMP phosphodiesterase); and 2) the addition of cAMP analogs or the stimulation of endogenous cAMP synthesis with PGE2 does not result in the phosphorylation of the rFSHR expressed in transfected cells (10). In contrast, protein kinase C seems to be partially responsible for the agonist-induced phosphorylation. This conclusion is supported by the following findings. First, the binding of hFSH to the rFSHR expressed in transfected cells stimulates not only the cAMP pathway, but also the inositol phosphate/diacylglycerol-signaling pathway (10, 11). Second, the pharmacological activation of C kinase with phorbol 12-myristate-13-acetate (PMA) enhances the phosphorylation of the rFSHR (10, 11, 12). Third, the down-regulation of C kinase by chronic treatment of transfected cells with PMA inhibits the hFSH-induced phosphorylation by approximately 30% (10). Thus, while the agonist-induced phosphorylation of the rFSHR may indeed be partially mediated by protein kinase C, one needs to invoke the participation of at least one other kinase. This suggestion is also supported by the finding that two inactivating mutations of the rFSHR that prevent agonist-induced phosphorylation do not affect PMA-induced phosphorylation (9).
With these findings in mind, the experiments presented here were designed to identify the GRKs that are involved in the agonist-induced phosphorylation of the rFSHR and to assess their involvement in agonist-induced internalization.
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RESULTS |
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We did not attempt to detect GRK1 because the expression of this
kinase, also known as rhodopsin kinase, is restricted to the retina and
the pineal (3, 18). The expression of the other members of the GRK
family (GRK2-GRK6) was ascertained using Western blots with specific
GRK antibodies. The results presented in Fig. 1 show that we can readily detect GRK2
and GRK6 in 293 and MSC-1 cells. The data presented in Fig. 1
show that
MSC-1 cells express the
- and/or
-isoforms of GRK4 as well.
Although we do not have standards for the
- and
-isoforms of
GRK4, we conclude that one or both of these isoforms are present, as
opposed to the
- and ß-isoforms for the following reasons. First,
the antibody used in the Western blots shown in Fig. 1
(I-20 from
Santa Cruz Biotechnology, Inc., Santa Cruz, CA) recognizes
an epitope not present in the
- and ß-isoforms. Second, another
antibody that recognizes only the
- and
-isoforms of GRK4 (K-20
from Santa Cruz Biotechnology, Inc.) failed to give a
signal in Western blots of MSC-1 cells (data not shown). Third, the
apparent size of the GRK4 isoform detected in Western blots of MSC-1
cells (see Fig. 1
) is closer to the predicted sizes for the
- and
-isoforms of GRK4 (5761 kDa) than to the predicted sizes for the
- and ß-isoforms (6367 kDa) (19, 20). Antibodies directed
against GRK3 or GRK5 failed to give a signal in Western blots from 293
or MSC-1 cells but readily detected the recombinant proteins used as
controls (data not shown).
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GRK2, GRK4, and GRK6 Can Phosphorylate the rFSHR
Since MSC-1 cells express GRK2, GRK4/
, and GRK6, we next
attempted to determine whether these three kinases can phosphorylate
the rFSHR. These experiments were done by measuring basal and
agonist-stimulated phosphorylation of the rFSHR in 293 cells
cotransfected with the rFSHR and the different GRKS.
The results presented in Fig. 2A display
representative experiments documenting that transient transfection of
293 cells with optimal amounts of the appropriate plasmids results in
increased expression of GRK2,
GRK4
2, or GRK6. Note that
Western blots of cells transfected with the GRK2 or GRK4
expression
vectors not only reveal proteins of the appropriate size
(i.e. 80 and 67 kDa, respectively), but they also reveal
proteins of slightly higher molecular weights. Although the identity of
these bands is not known, a similar phenomenon has been described by
others upon transfection of GRK2 (23). These investigators suggested
that the high molecular weight product detected in GRK2-transfected
cells may represent improperly processed GRK2 (23).
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Kinase-Deficient Mutants of GRK2 or GRK6 Inhibit the
hFSH-Stimulated Phosphorylation of the rFSHR by Kinases Endogenous to
293 Cells
While the experiments presented above show that GRK2, GRK4, and
GRK6 can phosphorylate the agonist-stimulated rFSHR, overexpression
studies cannot be used to discern the identity of the endogenous
kinases that phosphorylate the agonist-occupied rFSHR (see
Discussion). In an attempt to address this question we
assessed agonist-induced receptor phosphorylation after transfection of
293 cells with the rFSHR and kinase-deficient mutants of GRK2 or GRK6.
We did not attempt to use a kinase-deficient mutant of GRK4 because
this kinase is not detectable in 293 cells (cf. Fig. 1).
Kinase-deficient mutants of many protein kinases have been prepared by mutation of a lysine residue that is universally conserved in their catalytic domain and is directly involved in the phosphotransfer reaction (25, 26). Such constructs can be readily used as dominant-negative mutants to inhibit endogenous kinases (23, 25, 26).
C20-GRK2-K220M, a previously described isoprenylated
version of a kinase-deficient mutant of GRK2, has been shown to inhibit
the agonist-induced phosphorylation of the ß2-adrenergic receptor
expressed in 293 cells. The K220M mutation of this construct impairs
its kinase activity, and the isoprenylation signal targets the
kinase-deficient enzyme to the plasma membrane. The constitutive
association of the C20-GRK2-K220M mutant with the plasma
membrane is believed to enhance the ability of this construct to
inhibit endogenous GRK2, which is a cytosolic enzyme that is
translocated to the membrane by Gß/ as part of the
agonist-induced activation of GPCRs (3, 18). GRK6-(K215M,K216M) is
a new construct harboring a mutation in the catalytic
domain3 of GRK6 that is expected to
impair its kinase activity (25, 26). Like the wild-type GRK6, however,
the GRK6-(K215M,K216M) construct should be constitutively associated
with the plasma membrane by a covalently attached palmitate group (3, 18).
The ability of C20-GRK2-K220M and GRK6-(K215M,K216M) to act
as specific dominant-negative mutants of GRK2 and GRK6, respectively,
was tested in 293 cells cotransfected with the rFSHR, a suboptimal
amount of the wild-type kinases (1 µg/100-mm dish), and an excess (9
µg/100-mm dish) of the dominant- negative mutants. As shown in Fig. 4, these transfection conditions result
in increased expression of both the wild-type and the kinase-deficient
mutants. As expected from the amounts of plasmid used, the expression
of the transfected GRK6-(K215M,K216M) was higher than that of the
transfected GRK6. Surprisingly, however, the expression of the
transfected GRK2 was higher than that of the transfected
C20-GRK2-K220M, in spite of the fact that we used 9 times
more C20-GRK2-K220M than GRK2 in the transfections. This is
perhaps due to the nature of the protein encoded by
C20-GRK2-K220M, which, in addition to harboring a
single-point K-to-M mutation in the catalytic site, has a C-terminal
mutation that allows for isoprenylation of the mutant, whereas the
wild-type GRK2 is not isoprenylated (see above).
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Since the rFSHR also becomes phosphorylated when transfected 293 cells
are stimulated with PMA (9, 10, 11, 12), we conducted an additional test of the
specificity of the kinase-deficient GRKs by testing their effects on
the PMA-induced phosphorylation of the rFSHR. The results presented
in Fig. 6 show that transfection of 293
cells with C20-GRK2-K220M or GRK6-(K215M,K216M) (see
expression data shown in Fig. 4
) did not inhibit the PMA-induced
phosphorylation of the cotransfected rFSHR.
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These results indicate that the two GRKs that are endogenously expressed in 293 cells participate in the hFSH-induced phosphorylation of the transfected rFSHR.
Functional Correlates of GRK-Mediated Phosphorylation of the
rFSHR
Since a binary complex formed by some phosphorylated GPCRs and
arrestins serve as common molecular intermediates in the functional
uncoupling of the receptors from their effector systems and the
internalization of the agonist-receptor complex (see
Introduction), we tested the effects of different GRKs, as
well as arrestin-3 and dominant-negative mutants, on the
receptor-mediated internalization of FSH.
The data presented in Fig. 8 show that
cotransfection of the rFSHR with GRK2, GRK4
, or GRK6 result in a 2-
to 3-fold increase in the receptor-mediated internalization of hFSH.
These data also show that cotransfection with arrestin-3 enhanced
internalization 2- to 3-fold and that combinations of the GRKs and
arrestin-3 enhanced internalization 3- to 4-fold. Thus, the effects of
coexpression of arrestin-3 with GRK2, GRK4
, or GRK6 were more
pronounced than those of arrestin-3 alone or GRKs alone, but were
certainly not additive.
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Since the inhibitors of GRK2 and GRK6 have a similar effect on phosphorylation but display a differential effect on internalization, these results suggest that the GRK2-catalyzed phosphorylation of the rFSHR is more important for internalization than the GRK6-catalyzed phosphorylation.
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DISCUSSION |
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We concentrated our efforts on characterizing the involvement of GRK2
and GRK6 on the phosphorylation and internalization of the rFSHR
because these two kinases are present in MSC-1 cells (a Sertoli-like
cell line) and in 293 cells, the cells used for transfections (Fig. 1).
Moreover, experiments in which GRK2 or GRK6 were cotransfected with the
rFSHR in 293 cells indicate that both of these GRKs are capable of
phosphorylating the agonist-occupied rFSHR (Figs. 2
and 3
). The
majority of cotransfection experiments have revealed no specificity in
the ability of GRKs to phosphorylate a given GPCR (reviewed in Refs. 3, 18). On the other hand, there is one report showing that
cotransfection of the
2C10-adrenergic receptor with GRK5 or GRK6
does not result in increased agonist-induced receptor phosphorylation,
while cotransfection with GRK1 or GRK2 does. This report is
particularly relevant because, like the rFSHR (9, 12), the
2C10-adrenergic receptor is phosphorylated in an acidic S/T cluster
present in the third intracellular loop (13). In spite of the
similarity in the phosphorylation sites of the
2C10-adrenergic and
the rFSHR receptors, however, our overexpression studies with GRK2 and
GRK6 (Figs. 2
and 3
) did not reveal the kind of GRK specificity
documented with the
2C10-adrenergic receptor.
It is generally accepted that while GRK overexpression studies provide
useful information (see above), they cannot be used to discern the role
of endogenous GRKs on the phosphorylation of a given GPCR because
overexpression of GRKs may force a reaction that does not normally
occur at the ratio of GRK/receptor that prevails in the cell. A better
way to address this question is to use overexpression of
dominant-negative mutants as specific inhibitors of a given GRK. Thus,
the possible involvement of endogenous GRK2 or GRK6 on the
agonist-induced phosphorylation of the rFSHR expressed in 293 cells was
probed by overexpression of Gt, as well as kinase-negative mutants
of GRK2 and GRK6. Overexpression of G
t, a Gß/
scavenger (30),
is expected to specifically inhibit GRK2 by preventing the
Gß/
-promoted translocation of this kinase to the membrane (3, 18, 29). It should not affect the activity of GRK6, however, because this
kinase is constitutively associated with the plasma membrane via a
covalently attached palmitate (3, 18). Specificity studies performed
against the transfected wild-type kinases show that overexpression of a
C20-GRK2-K220M inhibited the cotransfected GRK2 but not the
cotransfected GRK6, and overexpression of GRK6-(K215M,K216M) inhibited
the cotransfected GRK6 but not the cotransfected GRK2 (Fig. 5
). In
addition, overexpression of C20-GRK2-K220M or
GRK6-(K215M,K216M) had no effect on the PMA-induced phosphorylation
of the transfected rFSHR (Fig. 6
).
Together with the studies discussed above, the ability of
C20-GRK2-K220M, Gt, and GRK6-(K215M,K216M) to inhibit
the agonist-induced phosphorylation of the rFSHR catalyzed by kinases
endogenous to 293 cells (Fig. 7
) show that GRK2 and GRK6 participate in
the phosphorylation of the rFSHR. When considered together with
previous data from this laboratory, these results allow us to conclude
that three different kinases, protein kinase C, GRK2, and GRK6,
participate in the agonist-induced phosphorylation of the rFSHR.
Although it is difficult to ascertain the relative importance of each
of these three kinases, the most effective inhibitor of phosphorylation
was G
t, an inhibitor of GRK2 (cf. Fig. 6
), and the least
effective inhibitor of agonist-induced phosphorylation of the rFSHR
was the down-regulation of protein kinase C (10). The dominant-negative
mutants of GRK2 and GRK6 were intermediate, but equally effective
(cf. Fig. 7
). it is also important to note that while
C20-GRK2-K220M and G
t are both inhibitors of GRK2, G
t was more
effective than C20-GRK2-K220M in inhibiting the
agonist-induced phosphorylation of the rFHSR catalyzed by endogenous
kinases (Fig. 7
). The reasons for this difference were not explored,
but since these plasmids encode for different proteins, it is possible
that G
t is expressed at higher levels than
C20-GRK2-K220M leading to a more efficient inhibition of
GRK2. By virtue of its ability to scavenge Gß/
, G
t should also
be an inhibitor of any kinase that depends on Gß/
for activity or
for translocation to the plasma membrane. There is only one other
kinase, GRK3, that is known to be dependent on Gß/
for membrane
translocation, however, and this kinase was not detectable in 293 cells
by Western blots (see Results). Cotransfection studies with
mixtures of the dominant-negative mutants have not yet been performed,
but addition of a protein kinase C inhibitor (Bisindolylmaleimide) to
cells overexpressing C20-GRK2-K220M failed to enhance the
effects of the dominant-negative kinase on rFSHR phosphorylation (data
not shown).
To our knowledge this is the first report on the involvement of GRKs in the phosphorylation of the glycoprotein hormone receptors. While previous studies from other laboratories have examined the functional impact of GRK overexpression or ablation on some functions of the glycoprotein hormone receptors, none of them have examined receptor phosphorylation. Thus, it has been reported that GRK2 and GRK5 may be responsible for the agonist-induced desensitization of the TSH receptor in a rat thyroid cell line (35, 36). Likewise, cotransfection of GRK2 or the four isoforms of GRK4 have been previously reported to enhance the agonist-induced uncoupling of the lutropin/choriogonadotropin receptor (19).
In the studies presented here we also attempted to correlate rFSHR
phosphorylation with the rFSHR-mediated internalization of FSH
because the agonist-induced internalization of some GPCRs is
facilitated by GRK-catalyzed phosphorylation (see
Introduction). Overexpression of GRK2, GRK4, or GRK6 is
similarly effective in enhancing hFSH-stimulated rFSHR phosphorylation
(Figs. 2
and 3
) and the internalization of [125I]hFSH
(Fig. 8
). Moreover, arrestin-3 alone is capable of enhancing the
internalization of [125I]hFSH, and the effects of GRKs on
internalization are enhanced by arrestin-3 (Fig. 8
). Thus, it is clear
that the enhanced phosphorylation elicited by GRK overexpression or the
overexpression of arrestin-3 result in an increase in receptor-mediated
agonist internalization (also see Ref. 9).
While the overexpression studies establish a link between
phosphorylation and internalization, the results obtained with the
kinase-deficient mutants and with Gt indicate that the GRK2-mediated
rFSHR phosphorylation is important for internalization, but the
GRK6-mediated rFSHR phosphorylation is not (Table 1
). The idea that the
rFSHR phosphorylation mediated by different GRKs may have a different
functional impact merits further consideration in view of the finding
that the agonist-induced phosphorylation of the rFSHR occurs in two
distinct domains, the first and third intracellular loops. Moreover,
the phosphorylation of these two domains may not be functionally
equivalent, as documented by the finding that mutation of the
phosphorylation sites present in the third intracellular loop of the
rFSHR inhibits uncoupling but not internalization, while mutation of
the phosphorylation sites present in the first intracellular loop of
the rFSHR inhibits uncoupling as well as internalization (12). Thus,
there are now two lines of evidence, mutation of individual
phosphorylation domains of the rFSHR (12) and inhibition of different
GRKs (this paper) that suggest different roles for different
phosphorylation events in the rFSHR. It will now be of interest to
determine whether the two domains phosphorylated upon agonist
activation of the rFSHR are preferentially phosphorylated by GRK2 or
GRK6. If we can also establish an experimental paradigm to examine
agonist-induced uncoupling in transiently transfected cells, we should
also be able to determine whether the dominant-negative mutants of GRK2
or GRK6 have a differential effect on the agonist-induced uncoupling of
the rFSHR.
The idea that phosphorylation of different domains, or even different residues, has a different impact on the functional properties of GPCRs is also supported by recent studies from our laboratory and others. Thus, individual mutations of the four serine residues that become phosphorylated in the LH/CG receptor in response to agonist stimulation show that two of these residues are involved in uncoupling and internalization, the third residue is involved in internalization but not uncoupling, and the fourth residue is not involved in either (37). Similar conclusions can be made from individual mutation of the two phosphorylation clusters present in the third intracellular loop of the m2 muscarinic receptor. In this receptor the phosphorylation of only one of the two clusters is necessary for uncoupling, but the phosphorylation of both clusters is necessary for internalization (15).
Finally, the data presented here also show that a dominant-negative
mutant of the nonvisual arrestins is a more effective inhibitor of
internalization than the two inhibitors of GRK2 (Table 1). The
dominant-negative mutant of the nonvisual arrestins is, in fact, as
effective in inhibiting internalization as a dominant-negative mutant
of dynamin (Table 1
). This finding suggests that the binding of
nonvisual arrestin to the rFSHR is more important than phosphorylation
in the internalization of the agonist-rFSHR complex. This conclusion is
in agreement with previous results from this laboratory showing that
the loss of agonist-induced phosphorylation of two signaling-deficient
mutants of the rFSHR can be rescued by overexpression of GRK2, but this
enhanced phosphorylation rescues the slow rate of internalization of
the agonist-rFSHR complex in only one of the two mutants.
Overexpression of arrestin-3, however, rescues the internalization of
both mutants (12).
In summary, the results presented here show that GRK2 and GRK6 participate in the agonist-induced phosphorylation of the rFSHR and suggest that only the GRK2-induced phosphorylation facilitates the internalization of the agonist-rFSHR complex. When considered with previous data from this laboratory (12), our results also suggest that even when the phosphorylation of the rFSHR is impaired by inhibiting GRK2 (this paper) or by preventing agonist-induced activation (12), the agonist-rFSHR complex can still be internalized by a pathway that depends on a nonvisual arrestin.
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MATERIALS AND METHODS |
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Inserts encoding the full-length bovine GRK2 (39) and human GRK6 (28)
were subcloned into pcDNA1.1/Amp for expression studies. A mutant form
of GRK6 predicted to be deficient in kinase activity (25, 26) was
prepared using PCR strategies to mutate two adjacent lysine residues
present in GRK6 to methionine. This mutant, designated
GRK6-(K215M,K216M), was also subcloned into pcDNA1.1/Amp for expression
studies. A full-length human GRK4 subcloned into pcDNA 1.1 (19) was
generously donated by Dr. Robert Lefkowitz (Duke University, Durham,
NC). An expression vector (pcDNA1.1) encoding for a prenylated,
kinase-deficient mutant of GRK2 (designated
C20-GRK2-K220M4, see Ref.
23) was generously donated by Dr. Marc Caron (Duke University). A
plasmid encoding for a human influenza hemagglutinin-tagged
dominant-negative mutant form of dynamin (i.e. dynamin K44A,
see Ref. 32) was obtained from Dr. Sandra Schmid (Scripps Research
Institute, La Jolla, CA) and subcloned into pcDNA3.1
(Invitrogen) for transfection. The expression vectors
(both in pcDNA3.1) encoding for arrestin-3 and ß-arrestin(319418)
have been described (31). An expression vector for bovine transducin
(designated G
t) in pcDNA1 was purchased from the American Type Culture Collection (Manassas, VA).
Transient cotransfections of human embryonic kidney (293) cells were done using calcium phosphate as described by Chen and Okayama (40). Cells plated in 100-mm dishes were transfected when 7080% confluent using a maximum of 20 µg of plasmid DNA (the total amount of plasmid transfected was kept constant by including the appropriate amounts of empty expression vector). After an overnight incubation the cells were washed, trypsinized, and replated in 100- mm dishes (12 x 107 cells per dish) for phosphorylation experiments or in 35-mm wells (510 x 105 cells per well) for binding and internalization experiments. All cells were used 24 h later.
Intact Cell Phosphorylation Assays
Transiently transfected cells (in 100-mm dishes) were
metabolically labeled with 200300 µCi/ml of
32Pi for 3 h at 37 C. The methods used to
lyse the cells, to immunoprecipitate the rFSHR, and to resolve the
immunoprecipitates on SDS gels have been described previously (10, 11, 41). Receptor phosphorylation was determined after incubation of the
32Pi-prelabeled cells at 37 C with buffer only
for 1560 min, 100 ng/ml hFSH for 60 min, or 200 nM PMA
for 15 min (11). Although there were some variations on receptor
expression among the different conditions tested, there were no
consistent effects of arrestin or GRK overexpression (or their
dominant-negative mutants) on FSHR expression. Regardless of the
plasmids transfected, the amount of receptor used in all
immunoprecipitation experiments was equalized based on measurements of
[125I]hFSH binding performed in the same batch of cells
used for the phosphorylation assays (see above). In 70 independent
transfections, the binding of [125I]hFSH to transiently
transfected cells (determined during a 1-h incubation of intact cells
with 100 ng/ml [125I]hFSH) was found to be 3.2 ±
0.3 ng/106 cells.
After immunoprecipitation and electrophoresis, autoradiograms of the dried gels were obtained using Kodak BioMax MS film and intensifying screens (Eastman Kodak Co., Rochester, NY). The autoradiograms were scanned and quantitated using a molecular imaging system (Bio-Rad Laboratories, Inc., Hercules, CA). All images were captured in a digital format for presentation. Basal receptor phosphorylation (i.e. that detected in 32Pi-labeled cells incubated with buffer only, see above) varied by at most 10% among all the conditions tested, and thus all data presented are expressed as fold over basal.
Internalization Assays
Transiently transfected cells (in 35-mm wells) were preincubated
in 1 ml of Waymouths MB752/1 containing 1 mg/ml BSA and 20
mM HEPES, pH 7.4 for 60 min at 37 C. Each well then
received 40 ng/ml [125I]hFSH, and the incubation was
continued for 12 min at 37 C. The cells were then washed twice with
2-ml aliquots of cold HBSS containing 1 mg/ml BSA. The surface-bound
hormone was then released by incubating the cells in 1 ml of cold 50
mM glycine, 150 mM NaCl, pH 3, for 24 min
(42, 43). The acidic buffer was removed, and the cells were washed once
more with another aliquot of the same buffer. The acid buffer washes
were combined and counted, and the cells were solubilized with 100 µl
of 0.5 N NaOH, collected with a cotton swab, and counted to
determine the amount of internalized hormone. The results of these
experiments are expressed as an internalization index (which is defined
as the ratio of internalized [125I]hFSH/surface-bound
[125I]hFSH, see Ref. 44) because plots of internalization
index vs. time are linear for at least 30 min. Under the
experimental conditions used here, such plots can be readily used to
calculate rates of internalization.
Immunoblots
Cell were lysed in a solution containing 1% Triton X-100, 200
mM NaCl, 20 mM HEPES, 1 mM EDTA, pH
7.4, during a 30-min incubation at 4 C. The lysates were clarified by
centrifugation (100,000 x g for 30 min), and the
amount of protein present in the supernatants was measured using the DC
protein assay from Bio-Rad Laboratories, Inc.. The lysates
were resolved on SDS gels and electrophoretically transferred to
polyvinylidenefluoride (PVDF) membranes as described elsewhere
(41). After blocking (41), expression of the different proteins was
determined by incubating the blots overnight with the indicated
concentrations of the primary antibodies listed below. Horseradish
peroxidase-labeled secondary antibodies were then used during a 1-h
incubation at a final dilution of 1:5,000, and the proteins were
finally visualized using the enhanced chemiluminescence (ECL) system of
detection from Amersham Pharmacia Biotech (Arlington
Heights, IL).
GRK2 and C20-GRK2-K220M were detected using a mouse
monoclonal antibody (3A10, at a final dilution of 1:100) (45) a rabbit
polyclonal antibody (AB9, at a final dilution of 1:3,000) (46), or a
commercially available rabbit polyclonal antibody (C-15 from
Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at a final
concentration of 0.5 µm/ml. GRK3 was detected using a rabbit
polyclonal antibody (C-14, final concentration = 0.5 µg/ml) from
Santa Cruz Biotechnology, Inc.. GRK4 was detected using
rabbit polyclonal antibodies specific for the - and ß-isoforms
(K-20) or the
- and
-isoforms (I-20) from Santa Cruz Biotechnology, Inc. at final concentrations of 0.5 µg/ml. GRK5
was detected using 0.5 µg/ml of a rabbit polyclonal antibody
designated GRK5(C-20) from Santa Cruz Biotechnology, Inc..
GRK6 and GRK6-(K215M,K216M) were detected using 0.5 µg/ml of a rabbit
polyclonal antibody designated GRK6(C-20) from Santa Cruz Biotechnology, Inc.. Arrestin-3 was detected with a mouse
monoclonal antibody (F4C1, final dilution = 1:2,000) directed
against an epitope common to all known arrestins (47). ß-Arrestin
(319418) was detected using a polyclonal antibody (KEE, final
dilution = 1:2,000) raised against a C-terminal 16 residue peptide
(48). The hemagglutinin-tagged dynamin was detected using the 12CA5
monoclonal antibody at a final concentration of 5 µg/ml
(Boehringer Mannheim, Indianapolis, IN).
Other Methods
Statistical analysis (t test with two-sided
P values) was performed using Instat (Graph Pad Software,
San Diego, CA).
Hormones and Supplies
The rabbit antibody to the rFSHR (Anti-F) has been described.
The National Hormone and Pituitary Agency of the National Institute of
Diabetes and Digestive and Kidney Diseases kindly provided purified
hFSH (AFP-5720D) and PMSG. [125I]hFSH was prepared as
previously described (49). [32P]orthophosphate was
obtained from DuPont-New England Nuclear (Boston, MA). Phosphate-free
DMEM was purchased from ICN Biomedicals, Inc. (Irvine,
CA). Nonidet P-40, Triton X-100, protease inhibitors,
N,N',N''-triacetylchitotriose, protein A-agarose,
and BSA were from Sigma Chemical Co. (St. Louis, MO).
Okadaic acid and cypermethrin were purchased from Alexis Biochemicals
(Woburn, MA). Wheat germ agglutinin agarose was from Vector Laboratories, Inc. (Burlingame, CA). Cell culture supplies and
reagents were obtained from Corning, Inc. (Corning, NY)
and Gibco BRL (Grand Island, NY), respectively. The enhanced
chemiluminescence reagents were from Amersham Pharmacia Biotech (Arlington Heights, IL), and the horseradish
peroxidase-labeled secondary antibodies were from Bio-Rad Laboratories, Inc.). Human embryonic kidney cells (293) were
obtained from the American Type Culture Collection
(CRL-1573) and MSC-1 cells (16, 17) were generously donated by Dr.
Michael Griswold (Washington State University, Pullman, WA). All other
materials were obtained from commonly used suppliers.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by NIH Grants HD-28962 to M.A and GM-47417 and GM-44944 to J.L.B. The services and facilities provided by the Diabetes and Endocrinology Research Center of the University of Iowa were supported by NIH Grant DK-25295. J.L.B. is an Established Investigator of the American Heart Association. M. de F.M.L. was supported by a fellowship from the Fudaçao de Amparo A Pesquisa Do Estado de São Paulo, Brazil (FAPESP, 96/14548). K.N. was partially supported by a fellowship from the Lalor Foundation.
1 Present address: Laboratorio de Farmacologia, Instituto Butantan, Av.
Dr. Vital, Brazil 1500, 05503900, Sao Paulo, Brazil.
2 There are four splice variants of GRK4
(cf. Refs. 19 and 20) and only two of them, and
,
were identified in MSC-1 cells (cf. Fig. 1
). However,
all expression experiments were done using GRK4
(the longest splice
variant) because there appear to be no differences in the ability of
these four variants to enhance the agonist-induced desensitization of
the gonadotropin receptors (19 ).
3 Since GRK6 has two adjacent lysines (codons 215
and 216) in the conserved kinase catalytic domain (28 ), both of them
were mutated to methionine.
4 This construct was initially named
C20ßARK1-K220M (23 ). In order to be consistent with the
more current nomenclature used in this paper, we renamed it
C20-GRK2-K220M, because ßARK1 is now known as GRK2.
Received for publication December 8, 1998. Revision received March 10, 1999. Accepted for publication March 16, 1999.
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REFERENCES |
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