The C-Terminal Tail of the Rat Lutropin/Choriogonadotropin (CG) Receptor Independently Modulates Human (h)CG-Induced Internalization of the Cell Surface Receptor and the Lysosomal Targeting of the Internalized hCG-Receptor Complex
Mikiko Kishi and
Mario Ascoli
Department of Pharmacology The University of Iowa College of
Medicine Iowa City, Iowa, 52242-1109
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ABSTRACT
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The analysis of 21 progressive
truncations of the C-terminal tail of the rat LH/CG receptor (rLHR)
revealed the presence of a region delineated by residues 628649 that,
when removed, enhanced the degradation of the internalized human (h)CG.
The analysis of these truncations also revealed the presence of a
region delineated by residues 624631 that, when removed, enhanced the
rate of internalization of hCG. Since there is little overlap between
these two regions, we conclude that the structural features of the rLHR
that mediate internalization and degradation of the internalized
hormone are different. Detailed analyses of cells expressing a
truncation at Y637 (designated rLHR-t637)
showed that the enhanced degradation of hCG observed in the these cells
is due to an increase in the rate of transfer of the internalized
hCG-rLHR complex from the endosomes to the lysosomes rather than to the
enhanced dissociation of the hCG-rLHR complex in the lysosomes.
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INTRODUCTION
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The LH/CG receptor (LHR) is a member of the
rhodopsin-like subfamily of G protein-coupled receptors (GPCRs) (1, 2).
Like other members of this family, the LHR is internalized after
agonist binding by a pathway that requires receptor activation (3, 4, 5, 6)
and phosphorylation (7, 8). This pathway has been extensively
characterized using biochemical approaches (8, 9, 10, 11, 12) as well as by
electron (13) and confocal (14) microscopy. The results from all of
these approaches are consistent and show that the internalization of
the agonist-LHR complex proceeds via clathrin-coated pits (13) and is
dependent on the participation of the nonvisual arrestins and dynamin
(8, 10, 11, 12). Whereas most internalized GPCRs recycle back to the plasma
membrane from the endosomal compartment, the agonist-LHR complex is
instead routed to the lysosomes (9, 13, 14). Once in the lysosomes the
hCG-LHR complex dissociates, and both subunits of the hormone are
degraded (15). Although the lysosomal degradation of the LHR has not
been directly measured, its degradation has been inferred from the lack
of recycling of the internalized receptor, and the net loss of cell
surface and total LHR (i.e. down-regulation) caused by
agonist-induced activation (9, 13, 16, 17).
Since there is virtually no information available about the
structural features of the LHR that target the internalized agonist-LHR
complex to the lysosomes, the studies presented here were designed to
address this question. We focused our attention on the C-terminal
cytoplasmic tail of the rat (r) LHR because previous experiments from
this and other laboratories have shown that mutation or removal of
certain residues present in the C-terminal tail of the LHR can have
pronounced effects on the targeting of the LHR precursor to the plasma
membrane, and on the agonist-induced cAMP accumulation, uncoupling,
internalization, and down-regulation of the cell surface LHR (11, 17, 18, 19, 20, 21).
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RESULTS
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Effect of Truncations of the Cytoplasmic Tail of the rLHR on
Agonist-Induced Internalization and on the Degradation of the
Internalized hCG
To study the effects of the C-terminal region of the rLHR on the
trafficking of the rLHR, we prepared 21 progressive C-terminal
truncations. Each deletion mutant was transiently transfected in 293
cells, and internalization was measured during a 30-min incubation at
37 C with a concentration of 125I-hCG equivalent
to the Kd. The results are expressed as the
internalization index, which is defined as the ratio of
internalized/surface-bound ligand (22). This index can be readily used
to approximate the rate of internalization because a plot of the
internalization index vs. time is linear for approximately
60 min, and the slope of this plot gives the rate of internalization
(10, 11, 22). The results presented (Fig. 1
) show that two truncations (at residues
663 and 654) induced a slight (<2-fold) enhancement in
internalization, but truncations of their flanking residues
(i.e. residues 664, 655, or 653) did not. Truncations
starting at residue 631 up to residue 624 enhance the internalization
of hCG approximately 2-fold, however. Additional truncations, starting
at residue 622, were poorly expressed (if at all) at the cell surface
and could not be analyzed. The cell surface expression of all the
C-terminal constructs shown in Fig. 1
was similar to that of rLHR-wt,
however.

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Figure 1. Effect of Truncations of the C-Terminal Tail of the
rLHR on the Receptor-Mediated Internalization of 125I-hCG
293 Cells were transiently transfected with the indicated constructs,
and the internalization index was measured at the end of a 30-min
incubation with 125I-hCG as described in Materials
and Methods. The total (i.e. surface +
internalized) amount of radioactivity associated with the cells at this
time point was 10,00020,000 cpm/well. The mature rLHR-wt is
674 residues long (34 ), and the different C-terminal truncations are
designated as txxx(X) where xxx denotes the position and (X) denotes
the identity of the C terminus. Each value represents the average
± SEM of 350 independent transfections. The absence of
an error bar indicates that the SEM is too small to be
shown. The dashed line across the figure highlights the
internalization index displayed by cells expressing rLHR-wt. The two
arrows highlight the two mutants that were later chosen
for further analysis. The asterisks denote statistically
significant differences (P < 0.05 from rLHR-wt).
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The internalization of hCG mediated by three of these truncated mutants
(rLHR-t653, rLHR-t631 and rLHR-t628) has been previously measured (17, 18). As in previous reports, the data presented in Fig. 1
show that
rLHR-t631 and rLHR-t628 internalize hCG at a faster rate than rLHR-wt.
In contrast to previous publications that reported a slower rate of
internalization for rLHR-t653 (17, 18), however, Fig. 1
now shows that
this mutant internalizes hCG at about the same rate as rLHR-wt. The
reason for this discrepancy is simple: we have found that the rLHR-t653
construct used in previous experiments had an additional mutation
(V497A in the second extracellular loop) that went unnoticed until now,
and this additional mutation is actually responsible for the decreased
rate of internalization previously reported for rLHR-t653. The
rLHR-t653 plasmid used for the present experiments does not have the
additional mutation that went previously unnoticed. Additional
experiments with rLHR-V497A are now underway to determine how a
mutation of an extracellular amino acid affects the rate of
internalization.
Because the internalized rLHR-receptor complex is delivered to the
lysosomes in the intact form (9, 13), measurements of the degradation
of the internalized hCG can be used to indirectly (but conveniently)
measure the targeting of the internalized hCG-receptor complex to the
lysosomes using a protocol that allows for the measurement of hormone
degradation in a manner that is independent of the rate of
internalization (3, 23, 24). Therefore, we transfected 293 cells with
each of the truncated receptors and subjected the transiently
transfected cells to a two-incubation procedure. During the first
incubation the cells were allowed to internalize
125I-hCG for 2 h at 37 C. The cells were
then washed with a neutral buffer to remove the free
125I-hCG and with an acidic buffer to remove the
surface-bound 125I-hCG (t = 0). Due to the
acid release, more than 90% of the cell-associated radioactivity is
located intracellularly at t = 0 regardless of the plasmid used to
transfect the cells at this point (data not shown). A second incubation
at 37 C was performed to allow the cells to process the internalized
agonist; at the end of this incubation (t = 2 h) the medium
was assayed for degraded and undegraded hormone released, and the
cells were used to determine the amount of
125I-hCG that remained cell-associated.
Since internalization was stopped from occurring at t = 0, all the
degraded and undegraded 125I-hCG found at t
= 2 h is derived from the 125I-hCG that was
internalized during the first incubation. Under these conditions, cells
expressing rLHR-wt release approximately 40% of the radioactivity that
was initially internalized back to the medium as degraded hormone (Fig. 2
). The data presented in Fig. 2
also
show that most truncations of the C-terminal tail have no effect on
degradation, whereas others enhance degradation. We did not, however,
find any truncations that inhibited degradation. A truncation at
residue 652 enhances degradation but truncations at residue 653 or 651
do not. Truncations of residues 649628 also serve to delineate a
broader region of the C-terminal tail that enhances degradation. The
maximal stimulatory effect on degradation was noted upon removal of the
628649 region in particular when the C-terminal tail of the rLHR was
truncated at position 637. In cells expressing rLHR-t637, the amount of
degraded hormone reached a maximum of approximately 60%.

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Figure 2. Effect of Truncations of the C-Terminal Tail of the
rLHR on the Degradation of the Internalized 125I-hCG
Cells were transiently transfected with the indicated constructs and
incubated with 125I-hCG for 2 h at 37 C. At this time
the cells were washed with a neutral buffer (to remove the free
hormone) and briefly treated with an isotonic pH 3 buffer (to remove
the surface-bound hormone). They were then incubated for another 2
h at 37 C. At the end of this incubation the medium was collected and
used to measure the undegraded and degraded hormone that were released
back into the medium, and the cells were used to determine the amount
of radioactivity that remained cell-associated. The degraded hormone
released is expressed as a percent of the sum of the radioactivity
present in each of these three compartments. At the end of the second
2-h incubation the amount of undegraded hormone released back into the
medium accounted for at most 5% of the radioactivity present in all
compartments, and this low percentage was not affected by any of the
truncations tested (data not shown). The internalized radioactivity
present at the beginning of the 2-h incubation was 1020,000 cpm/well.
The mature rLHR-wt is 674 residues long (34 ), and the different
C-terminal truncations are designated as txxx(X) where xxx denotes the
position and (X) denotes the identity of the C terminus. Each value
represents the mean ± SEM of 310 independent
transfections. The absence of an error bar indicates that the
SEM is too small to be shown. The dashed
line across the figure highlights the degradation of
125I-hCG measured in cells expressing rLHR-wt. The two
arrows highlight the two mutants that were later chosen
for further analysis. The asterisks denote statistically
significant differences (P < 0.05 from rLHR-wt).
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Lastly, it is worth emphasizing that a comparison of the data presented
in Figs. 1
and 2
readily demonstrates that the effects of truncations
of the C-terminal tail on internalization and degradation are not co-
linear, even with severe truncations of the C-terminal tail that
affect both of these processes. For example, a truncation at residue
637 enhanced the degradation of the internalized hCG but clearly had no
effect on internalization of the surface-bound hormone, whereas
truncations at residues 624 and 626 enhanced the internalization of hCG
but had no effect on the degradation of the internalized hormone.
Since the aim of this study was to delineate structural features of the
rLHR that affect degradation of the internalized hormone, we performed
a more complete time course of degradation in cells expressing rLHR-wt,
rLHR-t655, a truncation mutant that does not affect degradation, and
rLHR-t637, the mutant with the more pronounced effect on degradation.
These data, presented in Fig. 3
, clearly
document the enhanced degradation of the internalized
125I-hCG mediated by rLHR-t637. In the rest of
the experiments presented here, we further characterized the
trafficking of the internalized hCG mediated by rLHR-t637.

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Figure 3. Time Course of Degradation of the Internalized
125I-hCG by 293 Cells Transiently Transfected with rLRH-wt,
rLHR-t655, and rLHR-t637
Cells were transiently transfected with the indicated constructs and
incubated with 125I-hCG for 2 h at 37 C. At this time
(time = 0) the cells were washed with a neutral buffer (to remove
the free hormone) and briefly treated with an isotonic pH 3 buffer (to
remove the surface-bound hormone). They were then placed back in warm
medium and incubated at 37 C. At the indicated times the medium was
collected and used to measure the undegraded and degraded hormone that
were released back into the medium, and the cells were used to
determine the amount of radioactivity that remained cell-associated.
The radioactivity released as degraded hormone and the radioactivity
that remained cell associated are expressed as a percent of the sum of
the radioactivity present in each of these three compartments. The
radioactivity released as undegraded hormone accounted for at most 5%
of the sum of these three compartments and is not shown. The
internalized radioactivity present at t = 0 was
10,000-20,000 cpm/well. Each value represents the mean ±
SEM of four independent transfections. The absence of an
error bar indicates that the SEM is too small to be shown.
The asterisks denote statistically significant
differences (P < 0.05 from rLHR-wt).
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Trafficking of the Internalized 125I-hCG
Receptor Complex between the Endosomes and Lysosomes
The enhanced degradation of the internalized
125I-hCG mediated by rLHR-t637 could be due to
the increased delivery of 125I-hCG to the
lysosomes and/or to the enhanced degradation of the
125I-hCG that accumulates in the lysosomes. These
possibilities were tested by measuring the trafficking of the
internalized 125I-hCG-rLHR complex between the
endosomes and lysosomes. To facilitate analysis of the
125I-hCG-rLHR complex (see below), the
experiments described below were done using modified forms of the
rLHR-wt and rLHR-t637 (designated myc-rLHR-wt and myc-rLHR-t637)
containing the myc-epitope at the N terminus (25). The addition of the
myc-epitope to the rLHR was previously shown to have no effect on the
rate of internalization of the hCG-receptor complex (25), and
additional experiments (not presented) showed that the intracellular
trafficking of the complex formed by hCG and the myc-tagged rLHR-wt is
similar to that of the complex formed with the nontagged rLHR-wt.
Three independent but complementary experimental approaches,
subcellular fractionation (9), electron microscopy (13), and confocal
microscopy (14), have been used to track the fate of the hormone and
the receptor during the receptor-mediated endocytosis of hCG. The
results obtained with these three approaches are internally consistent,
and they all show that the internalized hCG-rLHR complex is delivered
to the lysosomes in the intact form. Whereas the microscopic approaches
provide formal and unambiguous data about the subcellular location of
the complex, the subcellular fraction approach is much more amenable to
quantitation, and this analysis was chosen for the studies described
below. Cells were homogenized and the postnuclear supernatant was
fractionated on a Percoll gradient (9, 26). Figure 4
shows that biochemical markers for
plasma membranes (i.e. 125I-hCG
bound to the surface of 293 cells transfected with the rLHR) and
endosomes (i.e. internalized
125I-transferrin) migrate toward the top of the
gradient, whereas a biochemical marker for lysosomes
(ß-hexosaminidase activity) migrates to the bottom of the gradient.
Thus the gradient chosen can separate endosomes from lysosomes but
cannot separate plasma membranes from endosomes. The lack of separation
of plasma membranes and endosomes is not a problem for our experiments
because the surface-bound 125I-hCG can be
released from the cells by a brief exposure to pH 3 (see
Materials and Methods) at the beginning of the experiment
(see below). Thus, any 125I-hCG migrating in the
position of plasma membranes/endosomes would have to be located in the
endosomal compartment rather than the plasma membrane.

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Figure 4. Distribution of Biochemical Markers on Percoll
Gradients
Postnuclear supernatants of 293 cells were prepared and fractionated on
Percoll gradients as described in Materials and Methods.
The top panel shows the distribution of the
125I-hCG bound to the cell surface of 293 cells transiently
transfected with rLHR-wt. The middle panel shows the
distribution of 125I-transferrin internalized by the
endogenous transferrin receptor of 293 cells. The bottom
panel shows the distribution of endogenous
ß-hexosaminadase activity present in 293 cells. The results of a
representative experiment are shown.
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To measure the transit of the internalized
125I-hCG from endosomes to lysosomes, we first
incubated 293 cells expressing myc-rLHR-wt or myc-rLHR-t637 with
leupeptin and 125I-hCG for 15 min at 37 C. The
presence of leupeptin, a lysosomal enzyme inhibitor, does not affect
the rate of internalization of the hCG-receptor complex or the
trafficking of the internalized complex to the lysosomes, but it
prevents lysosomal degradation and allows the undegraded hormone to
accumulate in the lysosomes (9, 15). At this point (t = 0) the
cells were washed to remove the surface-bound hormone, treated with a
pH 3 buffer to release the surface-bound hormone, and then reincubated
at 37 C (in the presence of leupeptin) to allow processing of the
internalized hormone while preventing degradation. At t = 0 and at
two subsequent time points thereafter, the cells were homogenized and
the postnuclear supernatants were analyzed on Percoll gradients to
track the movement of the internalized 125I-hCG
between the endosomes and lysosomes. The representative experiment
shown in Fig. 5
and the summary shown in
Fig. 6
show that at t = 0 most of
the radioactivity derived from internalized
125I-hCG is present in endosomes, and that the
radioactivity present in this compartment decreases with time, at the
expense of an increase in the radioactivity present in the lysosomes.
These results also show that the transit of the internalized
125I-hCG from endosomes to lysosomes is faster in
cells expressing myc-rLHR-t637 than in cells expressing myc-rLHR-wt.
Thus, whereas at t = 0 cells expressing myc-rLHR-wt or
myc-rLHR-t637 displayed similar amounts of
125I-hCG in the endosomes and lysosomes, the
transfer of internalized 125I-hCG from endosomes
to lysosomes at the 90-min time point in cells expressing myc-rLHR-t637
was equivalent to that detected in cells expressing rLHR-wt at the
270-min time point (Fig. 6
).

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Figure 5. Analysis of the Distribution of the Internalized
125I-hCG Present in 293 Cells Expressing myc-rLHR-wt or
myc-rLHR-t637
Cells were transiently transfected with the indicated constructs and
incubated with 125I-hCG and leupeptin for 20 min at 37 C.
At this time (time = 0) the cells were washed with a neutral
buffer (to remove the free hormone) and briefly treated with an
isotonic pH 3 buffer (to remove the surface-bound hormone). They were
then placed back in medium containing leupeptin and incubated at 37 C.
At the indicated times the cells were collected, homogenized, and
analyzed on Percoll gradients as described in Materials and
Methods. The results of a representative experiment are shown
in which the radioactivity present in each fraction is expressed as %
of the total radioactivity present in the gradient. The total amount of
radioactivity present in each gradient was 10,00020,000 cpm.
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Figure 6. Quantitative Analysis of the Transfer of the
Internalized 125I-hCG between Endosomes and Lysosomes in
293 Cells Expressing myc-rLHR-wt or myc-rLHR-t637
Cells were transiently transfected with the indicated constructs and
incubated with 125I-hCG and leupeptin for 20 min at 37 C.
At this time (time = 0) the cells were washed with a neutral
buffer (to remove the free hormone) and briefly treated with an
isotonic pH 3 buffer (to remove the surface-bound hormone). They were
then placed back in medium containing leupeptin and incubated at 37 C.
At the indicated times the cells were collected, homogenized, and
analyzed on Percoll gradients as described in Materials and
Methods and shown in Fig. 5 . The radioactivity associated with
endosomes (fractions 315) and lysosomes (fractions 2836) was
calculated as a percent of the total radioactivity present in the
gradient and plotted as a function of the length of time of the second
incubation. Each value shows the mean ± SEM of six
independent transfections. The absence of an error bar indicates that
the SEM is too small to be shown. The
asterisks denote statistically significant differences
(P < 0.05 from rLHR-wt).
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The nature of the 125I-hCG radioactivity
associated with the endosomes and lysosomes was next determined by
immunoprecipitation with a monoclonal antibody (9E10) to the myc
epitope. Since the rLHR used for these experiments is tagged with the
myc epitope, any 125I-hCG radioactivity
immunoprecipitated by the 9E10 antibody could be safely considered to
be receptor-bound rather than free. As shown in Fig. 7
, approximately 75% and 50% of the
125I-hCG present in the endosomes and lysosomes,
respectively, of 293 cells transfected with the myc-rLHR-wt or
myc-rLHR-t637 can be immunoprecipitated with an antibody to the myc
epitope. We conclude that most of the 125I-hCG
present in the endosomes and lysosomes of cells expressing myc-rLHR-wt
or myc-rLHR-t637 is receptor-bound. This conclusion is in agreement
with previous biochemical and morphological data obtained with the
endogenous or transfected LHR (9, 13, 14).
Since there is no difference in the proportion of
125I-hCG that is associated with the endosomes or
lysosomes of cells expressing myc-rLHR-wt or myc-rLHR-t637, yet the
lysosomal accumulation of the 125I-hCG-receptor
complex and the degradation of 125I-hCG is faster
in the latter, we conclude that the increased degradation of the
internalized 125I-hCG mediated by rLHR-t637 is
due to an increase in the rate of transfer of the internalized
125I-hCG-rLHR complex from endosomes to
lysosomes.
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DISCUSSION
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Whereas much has been recently learned about the molecular and
cellular basis of the agonist-induced internalization of GPCRs in
general (reviewed in Refs. 27, 28, 29) and the LHR in particular (5, 8, 10, 11, 12), little is known about the structural features of these
receptors that modulate other aspects of their cellular trafficking.
Since the internalized LHR is one of the few GPCRs that is targeted to
the lysosomal degradation pathway rather than to the recycling pathway
(9, 13), it provides a good opportunity to define the structural
features of GPCRs that are involved in lysosomal targeting. With this
in mind we analyzed a series of truncations of the C-terminal tail of
the rLHR for agonist-induced internalization, for the transit of the
internalized hCG- receptor complex to lysosomes, and for the
lysosomal degradation of hCG.
C-Terminal truncations at C663 and
A654 enhanced agonist-induced internalization,
but truncations at Q664 or S652 or additional
truncations between these two residues did not affect internalization
(Fig. 1
). Thus, the enhanced internalization mediated by truncations at
C663 and A654 appear to be
due to the creation of new C-terminal sequences rather than to the
removal of a given region. The nature of the C terminus itself does not
appear to be important at least for the alanine residue since two other
truncations (at residues 636 and 626) also create C-terminal alanines,
but only one of them, the truncation at residue 626, enhances
internalization (Fig. 1
). Progressive truncations starting at
Arg631 and continuing to
Arg624 enhanced agonist-induced internalization
about 2-fold, and they serve to define a region of approximately 8
residues that enhances internalization when removed (shown by the
top horizontal bar in Fig. 8
).

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Figure 8. Amino Acid Sequence of the C-Terminal Tail of the
rLHR
The amino acid sequence of the relevant portion of the C-terminal tail
(starting at residue 621) of the rLHR (accession number P16235,
SWISS-PROT databank) is shown. The asterisks at the top and the
bottom of the amino acid sequence show the truncations that
enhanced the internalization of hCG and the degradation of the
internalized hCG, respectively. Likewise, the top and the bottom
dark horizontal bars denote the regions of the rLHR that, when
removed, enhance the internalization of hCG and the degradation of the
internalized hCG, respectively (see Figs. 1 and 2 ). The
arrow indicates the C terminus of rLHR-t637, the
truncated rLHR that was extensively characterized in the experiments
presented herein. The four serine residues that become phosphorylated
upon agonist stimulation (7 8 30 41 ) are enclosed in
rectangles.
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Two of the truncations examined here (rLHR-t631 and rLHR-t628) have
been previously shown to enhance the internalization of hCG (17, 18).
The internalization indexes reported here (Fig. 1
) can be readily
translated into t1/2s of about 100 min for
rLHR-wt and about 50 min for rLHR-t631 and rLHR-t628. These values are
comparable to those previously measured in stably transfected cells
lines (17) and are consistent with other reports from this laboratory
showing that the rate of internalization of hCG mediated by the rLHR-wt
or mutants thereof is the same in stably and transiently transfected
293 cells (5, 10, 11, 12, 17).
Previous results from this laboratory have shown that the
agonist-induced phosphorylation of the rLHR maps to
S635, S639,
S649, and S652 (enclosed in
rectangles in Fig. 8
) in the C-terminal of the rLHR (7, 8, 30). We have also shown that the agonist-induced internalization of the
rLHR is impaired by the simultaneous mutation of these four residues
(7) or by the individual mutation of S635,
S639, or S649, but not by
the individual mutation of S652 (8). Thus, while
truncations of the C-terminal tail up to S649
were not expected to remove phosphorylation sites that influence
internalization, all truncations upstream of S649
would remove at least one of the phosphate acceptors that influences
internalization and would thus be expected to impair internalization.
As shown here, however, truncations that remove only
S649 (i.e. rLHR-t643)
S649 and S639
(i.e. rLHR-t637 and rLHR-t636), or
S649, S639, and
S635 (i.e. rLHR-t632) have no effect
on internalization (Fig. 1
). Moreover, as already noted above, more
severe truncation enhances internalization (Fig. 1
). These findings
suggest that, in addition to the phosphorylation sites, the C-terminal
tail of the rLHR may contain other structural features that (directly
or indirectly) influence endocytosis independently of receptor
phosphorylation. Other experiments have already shown that the
internalization of phosphorylation-deficient mutants of the rLHR can be
rescued by cotransfection of cells with one of the nonvisual arrestins
(8). Thus, while phosphorylation may be necessary for the
agonist-induced internalization of the rLHR, it is certainly not
sufficient. In the absence of rLHR phosphorylation agonist- induced
internalization can occur normally (or even at enhanced rates) by
overexpression of one of the nonvisual arrestins (8) or by truncation
of the C-terminal tail as shown herein.
The effect of C-terminal truncations on the degradation of the
internalized hCG revealed that a truncation at
Ser652 enhanced degradation, but truncations at
flanking residues, A653 and
P651, did not (Figs. 2
and 8
). Thus, the enhanced
degradation mediated by rLHR-t652 appears to be due to the creation of
a new C-terminal sequence rather than to the removal of a given region.
Although another truncation that enhances degradation (i.e.
rLHR-t649) also terminates in a serine residue, there are additional
truncations that enhance degradation but do not terminate in a serine
residue (Figs. 2
and 8
). Thus, the nature of the C terminus itself does
not appear to be an important determinant of degradation. Progressive
truncations starting at Ser649 and
continuing to Leu628 enhanced the degradation of
the internalized hormone, and they serve to define a region of
approximately 22 residues that enhances degradation when removed (Figs. 2
and 8
). The most dramatic effects on degradation were observed using
truncations that did not affect internalization (such as rLHR-t643 and
rLHR-t637), whereas some truncations that enhanced internalization had
no effect on degradation (such as rLHR-t624 and rLHR-t626). Thus, the
regions of the C-terminal tail that modulate these two aspects of the
trafficking of the rLHR overlap to some extent, but are clearly not
co-linear (Fig. 8
).
To our knowledge this is the first report showing that the C-terminal
tail of GPCRs influences the rate of transit of internalized receptors
among different compartments of the endocytic pathway. We have shown
here that the removal of residues 628649 (Figs. 2
and 8
) enhances the
degradation of the internalized hCG by promoting the transfer of the
hormone receptor complex from the endosomes to the lysosomes. Other
investigators have already shown that the C-terminal tail of some GPCRs
such as the thrombin, substance P,
ß2-adrenergic, arginine-vasopressin type 2, and
TSH and LH receptors (14, 31, 32, 33) have a great influence on the sorting
of these internalized GPCRs to the degradation or recycling pathways.
Thus, whereas the internalized substance P receptor recycles from
endosomes to the plasma membrane, the internalized thrombin receptor is
targeted to the lysosomes. However, the fate of chimeras of these two
receptors is dictated by the origin of the C-terminal tail (31).
Likewise, whereas the internalized TSH receptor recycles from endosomes
to the plasma membrane, the internalized LH receptor is targeted to the
lysosomes. In this case too, the fate of chimeras of these two
receptors is dictated by the origin of the C-terminal tail (14). The
628649 region of the rLHR identified here as influencing degradation
of the internalized hormone is enriched in basic and polar amino acids,
but it does not contain known protein-targeting motifs. It is also not
known if the 628649 region directly affects trafficking of the
internalized receptor or if it does so indirectly by interacting with
another intracellular region(s) of the rLHR.
Previous results from this (9) and other laboratories (13, 14) have
shown that once internalized, the hCG-LHR complex traverses the
endosomal compartment in an intact form and is delivered to the
lysosomes without dissociation. Once in the lysosomes, the complex
dissociates and the free hormone (and presumably the receptor) are
degraded (9). While such a pathway is best documented using microscopy
(13, 14), these methods are not readily amenable to quantitation. We
carefully quantitated this process in MA-10 cells a number of years ago
by assessing the state of the internalized hormone (i.e.
free vs. receptor-bound and intact vs. degraded)
in endosomes and lysosomes separated by Percoll gradient centrifugation
(9). We have now used similar protocols to determine the fate of the
internalized hCG in transfected 293 cells and to begin to understand
the reasons why some C-terminal truncations of the rLHR result in
enhanced degradation of the internalized hCG (Figs. 2
and 3
). The
results obtained here with 293 cells expressing the rLHR-wt document
the trafficking of the hCG-LHR complex from endosomes to lysosomes
(
Figs. 46

) and recapitulate the results previously reported by us and
others in target or transfected cells (9, 13, 14). We have previously
shown that approximately 99% and 60% of the
125I-hCG present in the endosomes and lysosomes,
respectively, of MA-10 cells treated with leupeptin is receptor bound
(9). Here we show that about 75% and 50% of the
125I-hCG present in the endosomes and lysosomes,
respectively, of transfected 293 cells treated with leupeptin is
receptor bound (Fig. 6
). Whereas the quantitative results are somewhat
different in these two cell lines, the data clearly show that a
substantial portion of the hCG present in these two fractions is
receptor bound. These small differences may also be due to the
methodology used. In previous experiments we used polyethylene glycol
to precipitate the receptor-bound 125I-hCG
whereas in the current experiments we used immunoprecipitation with an
antibody to the epitope-tagged rLHR to quantitate the receptor-bound
125I-hCG. Our quantitative biochemical studies,
together with the microscopic approaches used by Milgrom and co-workers
(13, 14), clearly show that most of the internalized
125I-hCG-LHR complex is routed to the lysosomes
rather than recycled back to the plasma membrane in MA-10 cells,
porcine Leydig cells, transfected 293 cells, or transfected mouse L
cells. More importantly, the biochemical approaches described here
allowed us to conclude that the enhanced degradation of
125I-hCG detected in 293 cells expressing
rLHR-t637 (Fig. 3
) is due to an increase in the rate of transfer of the
internalized 125I-hCG-LHR complex from the
endosomes to the lysosomes (
Figs. 46

) rather than to the increased
dissociation of the internalized 125I-hCG-LHR
complex.
 |
MATERIALS AND METHODS
|
---|
Plasmids and Cells
A full-length cDNA encoding the rLHR in pcDNAI/Neo has been
described (34). Truncations of the C-terminal tail of the rLHR were
constructed using PCR strategies to introduce a stop codon in the
position immediately following the new desired C terminus. The identity
of all mutants was verified by automated DNA sequencing (performed by
the DNA core of The Diabetes and Endocrinology Research Center of the
University of Iowa). All transient transfections in 293 cells were done
using the rLHR-wt and mutants thereof subcloned in the pcDNAI/Neo. Some
experiments (
Figs. 57

) were done using rLHR-wt and rLHR-t637 that
were tagged with the myc-epitope. These constructs (designated
myc-rLHR-wt and myc-rLHR-t637) have the myc-epitope at the N terminus,
and the preparation and characterization of the parent construct have
been described (25).
Human embryonic kidney (293) cells (CRL 1573) were obtained from the
American Type Culture Collection (Manassas, VA) and
maintained in DMEM containing 10 mM HEPES, 10% newborn
calf serum, and 50 µg/ml gentamicin, pH 7.4. Transient transfections
were done using the calcium phosphate method of Chen and Okayama (35).
Cells were plated in 35-mm wells and transfected with not more than 2
µg of plasmid DNA when 7080% confluent. After an overnight
incubation, the cells were washed and used 24 h later. Plasmids
were prepared using QIAGEN kits.
Internalization Assays
The endocytosis of 125I-hCG was measured
using intact cells incubated with 40 ng/ml
125I-hCG at 37 C for 30 min as described
previously (10, 11, 12, 22, 23, 24). All internalization data are expressed as
an internalization index, which is defined as the ratio of the
internalized to surface-bound hormone (22). This index is used because
under the assay conditions used here, plots of the internalization
index against time are linear for up to about 60 min and can be used to
calculate a rate constant and a half-life for internalization (10, 11, 22).
Degradation of the Internalized Hormone
This was measured using previously established protocols (3, 11, 23, 24). Briefly, transfected cells were washed and then allowed to
bind and internalize 40 ng/ml 125I-hCG for 2
h at 37 C. At this point the cells were placed on ice, washed to remove
the free hormone, and treated with an acidic buffer to remove the
hormone bound to the cell surface. The cells were then placed back in
warm assay medium, and a second incubation (at 37 C) was performed to
allow the cells to process and degrade the hormone that had been
internalized during the first incubation. At the end of the second
incubation (which lasted from 1 to 4 h as described in the figure
legends) the dishes were placed on ice, the medium was saved, and the
undegraded and degraded hormone were measured by precipitation with
trichloracetic acid (15). The cells were solubilized with NaOH to
determine the amount of hormone that remained cell-associated.
Fate of the Internalized Hormone
Transfected cells were washed and then allowed to bind and
internalize 125I-hCG for 20 min at 37 C in
Waymouths MB752/1 containing 1 mg/ml BSA and 20 mM HEPES,
pH 7.4 (assay medium) and 200 µM leupeptin. At this point
(t = 0 in the figures) the cells were placed on ice and washed two
to three times with 2-ml portions of cold HBSS containing 1 mg/ml BSA
(wash medium). 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 (15). This buffer was removed
and the cells were washed once more with the same acid buffer and then
once with cold assay medium. The cells were then placed back in 1 ml of
warm assay medium containing 200 µM leupeptin, and a
second incubation (lasting 90 or 270 min) at 37 C was conducted to
allow the cells to process the hormone that had been internalized
during the first incubation. In the presence of leupeptin, however, the
internalized hormone is not degraded and it accumulates in the
lysosomes (9, 15). At the indicated times the dishes were placed on
ice, the cells were washed twice with cold homogenization buffer (0.25
M sucrose, 10 mM HEPES, 1 mM EDTA,
pH 7.4), scraped into a small volume of the same buffer, and collected
by centrifugation at 4 C. The cells were then resuspended in cold
homogenization buffer, and they were lysed by forcing them through a
21-gauge needle 10 times. Postnuclear supernatants were prepared by
centrifuging the homogenates at 800 x g for 10 min at
4 C. The supernatants were saved, and the pellets were rehomogenized
and centrifuged again. The two supernatants were combined and a 2-ml
aliquot was thoroughly mixed with 8 ml of a Percoll solution (with a
density of 1.046 g/ml) prepared in homogenization buffer (9, 26). These
mixtures were then centrifuged at 33,000 x g for 20
min (70.1 Ti rotor in a L780 ultracentrifuge, Beckman Coulter, Inc., Palo Alto, CA) at 4 C. The contents of the gradients were
then collected from the top (250 µl/fraction), and aliquots of each
fraction were assayed for radioactivity with the aid of a
-counter.
These Percoll gradients were calibrated by analyzing the migration of
biochemical markers for plasma membranes, endosomes, and lysosomes.
Since a 4 C incubation of cells with 125I-hCG
prevents internalization (9, 13, 15), we used the
125I-hCG radioactivity associated with 293 cells
transiently transfected with rLHR-wt and allowed to bind
125I-hCG at 4 C as a marker for plasma membranes.
Since internalized transferrin localizes only to endosomes (36, 37, 38), we
used the 125I-transferrin radioactivity
internalized by 293 cells during a 20-min incubation at 37 C as a
marker for endosomes. Lastly, ß-hexosaminidase activity (measured as
described in Ref. 39) was used as a marker for lysosomes.
Analysis of Receptor-Bound 125I-hCG
After separation by Percoll gradient centrifugation (cf. Figs. 4
and 5
) the fractions containing the resolved endosomes and lysosomes
(fractions 315 and 2836, respectively) from cells expressing
rLHR-wt or rLHR-t637 were pooled. Each pool received 200 µl of
protease inhibitors (Complete protease inhibitor cocktail from
Roche Molecular Biochemicals, Indianapolis, IN) and 500
µl of 10% NP-40. The volume was adjusted to 5 ml with homogenization
buffer, and the samples were incubated for 45 min at 4 C. The
solubilized samples were then centrifuged at 100,000 x
g for 60 min at 4 C to remove the Percoll and insoluble
material.
Duplicate aliquots of the supernatants were used to determine the total
amount of 125I-hCG present, and duplicate
aliquots (500 µl) were mixed with an antibody to the myc-epitope
(9E10) that was previously bound to protein G-Sepharose (see below).
This mixture was rotated overnight at 4 C, and the beads were recovered
by centrifugation (4 C) and washed two times with 0.1% NP-40, 20
mM HEPES, 0.15 M NaCl, pH 7.4). The beads were
then counted in a
counter to determine the amount of
125I-hCG that was bound to the receptor. Control
samples in which equivalent amounts of free
125I-hCG were immunoprecipitated as described
above revealed that only about 10% of the free
125I-hCG was immunoprecipitated. In contrast,
when cells transiently transfected with myc-rLHR-wt were incubated with
125I-hCG at 4 C followed by detergent
solubilization, approximately 70% of the solubilized radioactivity
could be immunoprecipitated using the method described above.
Prebinding of the 9E10 antibody to the protein G-Sepharose was
accomplished by mixing 50 µl of a 20-fold dilution of 9E10 antibody
(i.e. a concentrated cell culture supernatant of the 9E10
hybridoma cell line) with 25 µl of protein G-agarose (50% slurry in
0.1% NP-40, 20 mM HEPES, 0.15
M NaCl, and 1 mM EDTA, pH
7.4) for at least 3 h at 4 C. The bound antibody was recovered by
centrifugation at 4 C, and the agarose beads were washed twice with the
same solution before mixing them with the lysate (see above).
Hormones and Supplies
Purified hCG (CR-127,
13,000 U/mg) was kindly provided by the
National Hormone and Pituitary Agency of the National Institute of
Diabetes and Digestive and Kidney Diseases. Partially purified hCG
(
3,000 U/mg) was purchased from Sigma (St. Louis, MO),
and it was used only to correct for nonspecific binding.
125I-hCG was prepared as previously described
(40). Percoll and 125I-transferrin were purchased
from Amersham Pharmacia Biotech. Cell culture supplies and
reagents were obtained from Corning, Inc. (Corning, NY)
and Life Technologies, Inc., respectively. All other
chemicals were obtained from commonly used suppliers.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Deborah L. Segaloff for critically reading this
manuscript and Professor Masatomo Mori (First Department of Internal
Medicine, Gunma University) for his support.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Mario Ascoli, Department of Pharmacology, 2319A BSB 51 Newton Road, The University of Iowa, Iowa City, Iowa 52242-1109.
This work was supported by NIH Grant CA-40629 to M.A. The services and
facilities provided by the Diabetes and Endocrinology Research Center
of the University of Iowa (supported by NIH Grant DK-25295) are also
gratefully acknowledged.
Received for publication November 30, 1999.
Revision received February 1, 2000.
Accepted for publication March 2, 2000.
 |
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