Department of Pharmacology The University of Iowa College of Medicine Iowa City, Iowa 52242-1109
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ABSTRACT |
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INTRODUCTION |
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In contrast to many other agonist-receptor complexes that dissociate in the acidic environment (pH 56) that prevails in endosomes (10), the agonist-LHR complex is rather insensitive to dissociation in this environment (1), and the agonist-LHR complex internalized into endosomes is delivered to the lysosomes in the intact form (i.e without dissociation of the agonist and receptor; see Refs. 1, 2). The more acidic environment that prevails in lysosomes as well as proteolysis promote the dissociation of the agonist-receptor complex, and both subunits of the agonist are eventually degraded to single amino acids (11). The lysosomal degradation of the LHR has not been formally documented, however. The net result of this pathway is to target the cell surface LHR for lysosomal degradation and as such, the endocytosis of the agonist-LHR complex is quantitatively responsible for the down-regulation of the cell surface LHR that occurs when mouse Leydig tumor cells are exposed to agonist (12).
Many of the newer experiments that have examined the agonist-induced internalization and subsequent down-regulation of the LHR have been performed with transfected 293 cells. While side-to-side comparisons using the same methodology have not been made, a perusal of the studies conducted using 293 cells expressing the recombinant LHR suggest that the extent of agonist-induced down-regulation is less in 293 cells expressing the recombinant LHR than in mouse Leydig tumor cells expressing the endogenous LHR (3, 6, 12, 13, 14, 15, 16). The experiments presented here were thus designed to determine the basis of this difference.
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RESULTS |
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Since the down-regulation of cell surface receptors that occurs upon agonist-induced activation is due to the balance of internalization/degradation of the agonist-occupied receptor, the recycling of the internalized receptor, and the synthesis/externalization of new receptors, we attempted to measure these parameters in MA-10 cells and in 293 cells stably transfected with the rLHR-wt or mutants thereof.
The results presented in the bottom panel of Fig. 1 show
that the t1/2 of internalization of the agonist-receptor
complex is approximately 50 min in Leydig tumor cells and about 100 min
in 293L(wt-12) cells. As expected (13), truncation of the C-terminal
tail of the rLHR at residue 653 increased the t1/2 of
internalization, and truncations at residues 631 or 628 reduced the
t1/2 of internalization in transfected 293 cells. Under our
standardized assay conditions the t1/2 of internalization
of the agonist-LHR complex in 293L(t6311) or 293L(t6281) cells was
comparable to that measured in MA-10 or R2C cells (i.e.
50 min).
Recycling of internalized receptors was next measured in MA-10,
293L(wt-12), and 293L(t6311) cells. In these experiments cells were
first allowed to bind and internalize a saturating concentration of hCG
during a 2-h incubation at 37 C. At this point the surface-bound hCG
was removed with a mild acid buffer, and the cells were reincubated at
37 C in hormone-free medium for up to 2 h (to allow for the
recycling of internalized receptors at the cell surface) before
measuring [125I]hCG binding at 4 C. The results
summarized in Fig. 2 show that MA-10,
293L(wt-12), and 293L(t6311) behave similarly in that there is little
or no replenishment of the cell surface receptor within 2 h of
hormone removal. Longer times were not examined because in other
systems recycling of internalized receptors occurs within minutes (see
Refs. 17, 18 for recent examples). Moreover, at longer times it is
difficult to distinguish recycling of internalized receptors from
externalization of newly synthesized receptors without the aid of
protein synthesis inhibitors. While protein synthesis inhibitors can be
readily used in 293 cells without adverse effects on cell viability,
they could not be used in MA-10 cells without compromising cell
viability.
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Taken together, these data suggest that the difference in the extent of down-regulation of the LHR detected in 293L(wt-12) and MA-10 cells is not due to differences in the recycling of the internalized receptor or the replenishment of new receptors at the cell surface. Since a decrease in the transcription of the endogenous LHR gene does not contribute to the hCG-induced down-regulation of the LHR in MA-10 cells (see Discussion and Ref. 12), the differences in the magnitude of down-regulation between MA-10 and 293L(wt-12) cells cannot be accounted for by the absence of a transcriptional effect on the rLHR cDNA that is stably incorporated into the 293 cell genome. Lastly, the enhanced agonist-induced down-regulation detected in 293L(t6311) cells, when compared with 293L(wt-12) cells, also cannot be explained by differences in the recycling of the internalized receptor or the replenishment of new receptors at the cell surface.
Mutations of the rLHR That Affect the Rate of Internalization of
the Agonist-Receptor Complex Affect the Extent of Agonist-Induced
Down-Regulation in Transfected 293 Cells
In the next series of experiments we tested the hypothesis
that the rate of internalization of the hCG-LHR complex is an important
determinant of the extent of down-regulation of the LHR. We considered
this hypothesis because 1) the experiments described above excluded
changes in recycling of the internalized receptors or in the
replenishment of new receptor at the cell surface as being important
determinants of down-regulation; and 2) the two truncations of the rLHR
that enhanced down-regulation in 293 cells also enhanced
internalization (cf. Fig. 1). It is also possible, however,
that truncations of the C-terminal tail affect down-regulation by other
mechanisms and that there is no cause-effect relationship between the
rate of internalization and the extent of down-regulation in 293
cells.
To more directly establish a causal effect between internalization and down-regulation, we took advantage of several previously established clonal lines of 293 cells that are stably transfected with point mutations of the rLHR that affect the rate of internalization of the agonist-rLHR complex. An aspartic-to-asparagine mutation in codon 383 in TM2 (D383N) and a tyrosine-to-phenylalanine mutation in codon 524 of TM5 (Y524F) of the rLHR have been previously shown to impair signal transduction as well as the internalization of hCG (7, 9), while an aspartic-to-tyrosine mutation in codon 556 of TM6 (D556Y) and a leucine-to-arginine mutation in codon 435 of TM3 (L435R) of the rLHR have been previously shown to induce constitutive activation and to enhance the internalization of hCG (7, 20, 21).
When compared with 293 cells expressing rLHR-wt, the extent of
agonist-induced down-regulation is higher in 293 cells expressing
rLHR-D556Y or rLHR-L435R, the two constitutively active mutants that
internalize hCG at a faster rate than rLHR-wt (Fig. 4). Note that although the
t1/2 of internalization of hCG is shorter in
293Lmyc-(L435R-2) cells than in 293L(D556Y-6) (
7 and
37
min, respectively) the magnitude of down-regulation is greater in
293L(D556Y-6) cells than in 293L(mycL435R-2) cells. hCG induces
approximately 80% down-regulation in 293L(D556Y-6) cells compared with
about 30% in 293L(wt-12) cells, and about 55% down-regulation in
293Lmyc(L:435R-2) cells compared with approximately 10% in
293Lmyc(wt-11) cells. This difference may very well be explained by the
finding that new rLHR-L435R is replenished at the cell surface at a
faster rate than rLHR-D556Y or rLHR-wt (7). This faster rate of
replenishment will, of course, balance the increased rate of
internalization of the hCG-receptor complex and ultimately reduce the
extent of down-regulation observed at steady state.
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Thus, four mutations that enhance the rate of agonist-induced internalization of the recombinant rLHR (i.e. rLHR-t631, rLHR-t628, rLHR-D556Y, and rLHR-L435R) also enhance the extent of agonist-induced down-regulation of the rLHR expressed in 293 cells, while only one (i.e. rLHR-D383N) of the three mutations that reduce the rate of agonist-induced internalization (i.e. rLHR-D383N, rLHR-Y524F, and rLHR-t653) reduce the extent of agonist-induced down-regulation. It should also be noted that only two (rLHR-D556Y and rLHR-L435R) mutations that enhance internalization induce constitutive activation, and that only two (rLHR-D383N and rLHR-Y524F) mutations that impair internalization impair signal transduction (7, 9, 14).
Cotransfections of 293 Cells with the rLHR-wt and Nonvisual
Arrestins or G Protein-Coupled Receptor Kinase 2 (GRK2) Enhance the
Internalization of the hCG-LHR Complex and the hCG-Induced
Down-Regulation of the Cell Surface rLHR
While the experiments summarized above suggest a relationship
between the rate of internalization of hCG and the extent of
down-regulation of the cell surface rLHR, this suggestion is based
entirely on the study of receptor mutants. Thus we sought to also
examine this issue using manipulations that affect the endocytosis of
the rLHR-wt. We chose to use cotransfections of 293 cells with the
rLHR-wt and GRK2 or arrestins because the GRK-catalyzed phosphorylation
of the rLHR and the interaction of the rLHR with the nonvisual
arrestins are expected to enhance the internalization of the hCG-LHR
complex (4, 5, 22), while the interaction of the rLHR with visual
arrestin is not expected to enhance internalization (5).
We (23) and others (24) have previously shown the presence of GRK2 and
nonvisual arrestins in mock-transfected 293 cells. The results
presented in Fig. 5A document again the
presence of endogenous GRK2 in 293 cells and show the increased
expression of this kinase after transfection with a GRK2 expression
vector.2 The results presented in Fig. 5B
document the enhanced expression of visual arrestin, arrestin 3, and
ß-arrestin in 293 cells transfected with the appropriate expression
vectors.
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DISCUSSION |
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Using standardized assay conditions we clearly showed that the extent
of agonist-induced down-regulation of the LHR is indeed much lower in
293 cells expressing rLHR-wt than in rat or Leydig tumor cells (Fig. 1). This decreased down-regulation does not appear to be due to
differences in the fate of the internalized agonist-receptor complex
because 1) this complex has been shown to accumulate in the lysosomes
in target and transfected cells (1, 2); and 2) target or transfected
cells degrade more than 90% of the internalized agonist during each
round of endocytosis (3, 9, 11). Here we show that these differences
cannot be accounted for by differences in the extent of recycling of
the internalized receptor because this is minimal in Leydig tumor cells
and in 293 cells expressing rLHR-wt (Fig. 2
).
The possibility that differences in the extent of hCG-induced
down-regulation of the LHR in target and 293 cells are partially due to
an hCG-induced reduction in the transcription of the endogenous LHR
gene expressed in MA-10 or R2C cells has already been excluded. While
it is clear that hCG, acting through cAMP as a second messenger,
decreases the transcription of the endogenous LHR gene and the levels
of LHR mRNA in MA-10 cells (12, 27, 28), this effect is quantitatively
unimportant to down-regulation. Thus, the magnitude of hCG-induced
down-regulation of the LHR is unaffected in a subclone of MA-10 cells
that express a cAMP-resistant phenotype and do not respond to hCG with
a decrease in LHR mRNA (12). The effects of hCG on the levels of mRNA
transcribed from the LHR cDNA incorporated into the 293 cell genome
have not been examined, but we have shown that cAMP tends to increase
LHR mRNA without affecting the levels of cell surface LHR in
transfected 293 cells (28). While a cAMP-induced increase in LHR mRNA
in transfected cells could theoretically attenuate the extent of the
hCG-induced down-regulation of the LHR in these cells, this does not
appear to be the case. If a cAMP-induced increase in LHR mRNA was an
important attenuator of down-regulation in transfected 293 cells, one
would expect an enhancement of down-regulation in 293 cells expressing
signaling-impairing mutations of the LHR and perhaps even an impairment
of down-regulation in 293 cells expressing constitutively active
mutants of the rLHR. In fact, the data presented here document the
opposite effect in that signaling-impairing mutations attenuate
down-regulation while mutations that induced constitutive activation
enhance it (Fig. 4 and Refs. 7, 9). The three truncations of the
cytoplasmic tail of the rLHR that impair or enhance internalization and
down-regulation (Fig. 1
) have little or no effect on hCG-induced cAMP
accumulation (14, 29). Overexpression of arrestin-3, a manipulation
that enhances internalization and down-regulation of the rLHR-wt (Table 1
), also has no effect on hCG-induced cAMP accumulation (4). Lastly,
the rate of replenishment of new receptors at the cell surface, a rate
that is affected by the transport of the LHR precursor to the cell
surface and by the synthesis of new LHR (19), is also not responsible
for differences in the extent of down-regulation between target and
transfected cells because this rate is very similar in 293 cells
expressing rLHR-wt and in MA-10 cells (Fig. 3
and Refs. 6, 7).
The data presented here argue that the difference in the extent of
agonist-induced down-regulation of the LHR observed in rodent Leydig
tumor cells and 293 cells expressing the rLHR-wt is due mostly to
differences in the rate at which these cells internalize the
agonist-receptor complex. Thus, there is a positive correlation between
the rate of internalization of the agonist-LHR complex and the extent
of down-regulation in three different cell lines (MA-10, R2C and
transfected 293 cells) that express the mouse or rat LHR-wt (Fig. 1).
Second, all mutations of the rLHR that enhance the rate of
internalization of the agonist-rLHR complex in transfected 293 cells
also enhance the extent of agonist-induced down-regulation of the rLHR
in these cells (Figs. 1
and 4
). Third, three manipulations that enhance
the rate of internalization of the agonist-rLHR complex in transfected
293 cells (i.e. cotransfections with GRK2, arrestin-3, or
ß-arrestin) also enhance the extent of down-regulation of the rLHR-wt
in these cells. Lastly, cotransfection with a related construct that
has little or no effect on internalization (visual arrestin) does not
enhance down-regulation (Table 1
).
If the rate of internalization is important to the extent of
down-regulation, one would predict that manipulations that slow down
internalization should also impair down-regulation. This has been found
to be the case only in some instances, however. For example, the slow
rate of internalization of a weak partial agonist in MA-10 cells (8) is
accompanied by a reduction in the extent of down-regulation (12). In
contrast, when using transfected 293 cells, where the rate of
internalization of the agonist-receptor complex is already slow
compared with that of target cells (cf. Fig. 1), mutations
of the rLHR that increase the t1/2 of internalization by
less than about 2-fold (i.e rLHR-t653 and rLHR-Y524F) are
not accompanied by a reduction in the extent of down-regulation (Figs. 1
and 4
). One mutation that increased the t1/2 of
internalization by 3- to 4-fold (rLHR-D383N, see Fig. 4
) did abolish
down-regulation, however. Taken together with the results discussed
above, these data suggest that the extent of down-regulation in 293
cells stays fairly constant when the t1/2 of
internalization is between 100 and 200 min (cells expressing rLHR-wt,
rLHR-t653, or rLHR-Y524F), but it is enhanced when the t1/2
of internalization is shorter than about 100 min (cells expressing
rLHR-t631, rLHR-t628, rLHR-D556Y, and rLHR-L435R or cells expressing
rLHR-wt but overexpressing GRK2, ß-arrestin, or arrestin-3), and is
impaired when the t1/2 of internalization is longer than
about 200 min (cells expressing rLHR-D383N).
Results of experiments presented above and elsewhere (3, 4, 7, 13) have
shown that the activation and phosphorylation of the rLHR facilitate
the internalization of the agonist-LHR complex and have identified some
cellular proteins involved in this process. The importance of receptor
activation is readily demonstrated by the slower rate of
internalization of a complex formed between the mLHR-wt and weak
partial agonist (8) and by the findings that rLHR mutations that impair
signal transduction internalize agonist at a slow rate while mutations
of the rLHR that induce constitutive activation internalize agonist at
a fast rate (7, 9). The importance of receptor phosphorylation is also
readily demonstrated by the findings that a phosphorylation-deficient
mutant of the rLHR internalizes agonist at a slow rate (3, 4) while
overexpression of GRKs, a manipulation that enhances LHR
phosphorylation, also stimulate the internalization of the agonist-LHR
complex (Table 1). Thus, GRKs are among the cellular proteins that may
affect internalization. Dynamin and nonvisual arrestins also
participate in the internalization of the agonist-LHR complex as judged
by the finding that their specific dominant-negative mutants (4)
inhibit the internalization of this complex and by the finding that
overexpression of nonvisual arrestins stimulate the endocytosis of the
agonist-LHR complex (Table 1
and Ref. 4). It is therefore possible that
the differences in the rate of internalization of the agonist-LHR
complex reported here between MA-10/R2C cells and 293 cells expressing
the recombinant rLHR could be due to differences in the expression of
GRKs, nonvisual arrestins, dynamin (and perhaps other as yet
unidentified proteins that participate in internalization) between
these cells. While antibodies to all these proteins are available, we
could not use them to accurately measure their relative levels in
MA-10, R2C, and 293 cells because these cell lines are from different
species (mouse, rat, and human, respectively), and the interspecies
cross-reactivity of all these antibodies is not known.
When using a single cell line (i.e. 293 cells), the rate of
internalization of the agonist-rLHR complex can be readily manipulated
by mutations that affect receptor activation (Fig. 4 and Refs. 7, 9), by mutations that affect receptor phosphorylation (3, 4), and by
overexpression of GRKs or nonvisual arrestins or their
dominant-negative mutants (Table 1
and Ref. 4). Other structural
features of the LHR must be involved in internalization, however, as
illustrated by the findings that some truncations of the C-terminal
tail of the rLHR (such as rLHR-t653) impair endocytosis, while others
(such as rLHR-t631 and rLHR-t628) enhance endocytosis (Fig. 4
and Refs.
13, 14). The structural features of the rLHR removed by these
truncations appear to be more important than receptor activation,
because receptor activation (as measured by second messenger
generation) is not impaired by any of these three truncations (14).
They are also more important than phosphorylation because the sites
phosphorylated in response to agonist stimulation are four serine
residues (serine635, serine639,
serine649, and serine652) located between
residues 631 and 653 (3, 4, 14). Thus, agonist-induced phosphorylation
of rLHR-t653 is normal or only slightly reduced when compared with
rLHR-wt while the agonist-induced phosphorylation of rLHR-t631 and
rLHR-t628 are undetectable (14).
Further experiments on the effects of truncations or mutations of the C-terminal tail of the LHR on internalization and on its interaction with nonvisual arrestins may provide important information about how this region of the LHR affects internalization.
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MATERIALS AND METHODS |
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Expression vectors encoding for visual arrestin (in pBC12B1), ß-arrestin, and arrestin-3 (both in pcDNA3.1) have been described previously (31) and were generously provided by J. L. Benovic (Thomas Jefferson University, Philadelphia, PA). A full-length GRK2 (32) was subcloned into pcDNA1.1/Amp for expression studies.
Transient transfections of human embryonic kidney (293) cells were done using calcium phosphate as described by Chen and Okayama (33). After an overnight incubation the cells were washed, placed back in culture medium, and used 24 h later (4, 34).
The establishment and properties of clonal cell lines of 293 cells stably transfected with rLHR-wt, designated 293L(wt-12); rLHR-t631, designated 293L(t6311); rLHR-t653, designated 293L(t6536); rLHR-t628, designated 293L(t6281); rLHR-D383N, designated 293L(D383N-9); rLHR-Y524F, designated 293L(Y524F-22); and rLHRD556Y, designated 293L(D556Y-6), have been described previously (7, 14, 29). Clonal lines of 293 cells stably transfected with a myc-tagged rLHR-wt, designated 293Lmyc(wt-11); and with a myc-tagged rLHR-L435R mutant, designated 293Lmyc(L435R-2), have also been described (7, 19). All of these cell lines express between 100,000 and 200,000 cell surface receptors per cell (7, 14, 19, 29).
The origin and handling of MA-10 cells, a clonal strain of mouse Leydig tumor cells that were adapted to culture in this laboratory and express the LHR endogenously, have been described (35). R2C cells are a clonal strain of rat Leydig tumor cells (36) available from the American Type Culture Collection (Manassas, VA). These cells were maintained using the same culture conditions previously described for MA-10 cells (35).
Internalization Assays
The endocytosis of [125I]hCG was measured as
follows (6, 26). Cell monolayers (in 35-mm wells), were washed twice
with assay medium (Waymouths MB752/1 containing 1 mg/ml BSA and 20
mM HEPES, pH 7.4) and then preincubated in 1 ml of assay
medium for 60 min at 37 C. Each well then received 40 ng/ml
[125I]hCG with or without 25 IU/ml of crude hCG (to
correct for nonspecific binding), and the incubation was continued at
37 C. At 5- to 10-min intervals, groups of cells were placed on ice and
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 (11). 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
endocytotic rate constant (ke) was calculated from the slope of the
line obtained by plotting the internalized radioactivity against the
integral of the surface-bound radioactivity (7, 26, 37, 38). The
half-life of internalization (t1/2) is defined as
0.693/ke.
Assay of Receptor Down-Regulation
These assays were done with minor modifications of previously
described methods (1). Cell monolayers (in 35-mm wells) were incubated
at 37 C with a saturating concentration of hCG (100 ng/ml) for 24
h. The cells were then cooled to 4 C for 30 min, washed twice with 2-ml
aliquots of cold HBSS containing 1 mg/ml BSA (to remove the free
hormone), treated with 1 ml of cold 50 mM glycine, 150
mM NaCl, pH 3, for 24 min to remove the residual
surface-bound hormone, and then washed twice with 2-ml aliquots of
assay medium. Each well then received 100 ng/ml 125I-hCG
with or without 25 IU/ml of crude hCG (to correct for nonspecific
binding), and the cells were incubated for 4 h at 4 C. The free
hormone was then removed by washing three times with 2-ml aliquots of
cold HBSS containing 1 mg/ml BSA, 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 bound hormone. All binding data were
then expressed as percent of the radioactivity detected in cells
incubated without hCG, but otherwise treated under identical
conditions. For the stably transfected 293 cell lines, 100% binding
varied between 100,000 and 200,000 cpm/well. For MA-10 and R2C cells
and for the transiently transfected 293 cells, 100% binding varied
between 20,000 and 40,000 cpm/well. Since a saturating concentration of
[125I]hCG was used, only a small fraction (<10%) of the
added [125I]hCG was specifically bound to the cells.
Measurement of the Replenishment of the Cell Surface rLHR
Since proteolysis of intact cells under mild conditions has been
shown to destroy the cell surface rLHR, the rate of replenishment of
the rLHR at the cell surface can be measured by following the time
course of recovery of [125I]hCG binding to intact cells
at 4 C as a function of time after removal of the protease (7, 19).
Cells (plated in 100-mm dishes) were placed on ice and washed twice
with 4-ml portions of cold HBSS. The cell surface rLHR was then
proteolyzed by incubating the cells on ice for 3045 min in cold HBSS
supplemented with 250 µg/ml of Protease type XIV (19). Protease
activity was quenched by the addition of 4 ml of Waymouths MB752/1
medium supplemented with 20 mM HEPES, 15% horse serum, 1
mM phenylmethyl sulfonylfluoride, 2 mM EDTA,
and 5 mM N-ethylmaleimide. The cells were then
scraped from the plate and collected by centrifugation. The pellet was
resuspended in the same medium, and the cells were collected by
centrifugation again and resuspended in DMEM supplemented with 10%
newborn calf serum, 20 mM HEPES, 50 µg/ml gentamicin, pH
7.4. The cells were then distributed into 35-mm wells and placed in a
CO2 incubator at 37 C to allow the cell surface rLHR to
recover. At predetermined times the cells were used for
[125I]hCG binding assays as described above, and the
amount of hormone bound was expressed relative to the last time point
used (i.e. 24 h). The amount of [125I]hCG
bound at this time point varied between 100,000 and 200,000 cpm/well
for the two 293 cell lines used.
Immunoblots
Cells 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. (Hercules,
CA). The lysates were resolved on SDS gels and electrophoretically
transferred to polyvinylidenefluoride membranes as described
elsewhere (39). After blocking (39), 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 was detected using a mouse monoclonal antibody (3A10, at a final dilution of 1:100) (40). Visual arrestin, ß-arrestin, and arrestin-3 were detected with a mouse monoclonal antibody (F4C1; final dilution, 1:2,000) directed against an epitope common to all known arrestins (41).
Other Methods
Statistical analysis (t test with two-sided
P values) was performed using Instat (GraphPad Software, Inc., San Diego, CA).
Hormones and Supplies
Purified hCG (CR-127, 12,000 IU/mg) was obtained from the
National Hormone and Pituitary Agency of the NIDDK (Baltimore, MD).
This material was used for iodinations and in the experiments designed
to measure down-regulation. [125I]hCG was prepared using
the purified hCG as described previously (42), to give a specific
radioactivity of 25,00030,000 cpm/ng. Crude hCG (
3,000 IU/mg) was
obtained from Sigma Chemical Co. (St. Louis, MO) and was
used exclusively for the correction of nonspecific binding (see above).
Cell culture supplies and reagents were obtained from Corning, Inc. (Corning, NY) and Life Technologies, Inc.
(Gaithersburg, MD), respectively. Human kidney 293 cells and the rat
Leydig tumor cell line (R2C) were purchased from the American Type Culture Collection. 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 CA-40629 (to M.A.) and DK-25295, which supports The Diabetes and Endocrinology Research Center of The University of Iowa. K.N. was partially supported by a fellowship from the Lalor Foundation. M. de F.M.L. was supported by a fellowship from the Fudaçao de Amparo A Pesquisa Do Estado de Sao Paulo, Brazil (FAPESP, 96/14548).
1 Present address: Laboratorio de Farmacologia, Instituto Butantan,
Avenue Dr. Vital, Brazil 1500, 05503900, Sao Paulo, Brazil.
2 The presence of a high molecular weight form of
GRK2 in transfected cells has been previously noted by us (23 ) and
others (25 ). Although the identity of this band has not been
established, it has been suggested that it represents an incompletely
processed form of GRK2 (25 ).
Received for publication March 2, 1999. Revision received May 11, 1999. Accepted for publication May 14, 1999.
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REFERENCES |
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