Identification of Two Distinct Structural Motifs That, When Added to the C-Terminal Tail of the Rat LH Receptor, Redirect the Internalized Hormone-Receptor Complex from a Degradation to a Recycling Pathway
Mikiko Kishi,
Xuebo Liu,
Takashi Hirakawa,
David Reczek,
Anthony Bretscher and
Mario Ascoli
Department of Pharmacology (M.K., X.L., T.H., M.A.), The University
of Iowa College of Medicine, Iowa City, Iowa 52242-1109; and Department
of Molecular Biology and Genetics (D.R., A.B.), Cornell University,
Ithaca, New York 14853-2703
Address all correspondence and requests for reprints to: Dr. Mario Ascoli, Department of Pharmacology, 2319B BSB, 51 Newton Road, The University of Iowa, Iowa City, IA 52242-1109. E-mail:
mario-ascoli{at}uiowa.edu
 |
ABSTRACT
|
---|
We show that most of the internalized rat LH receptor is routed to
a lysosomal degradation pathway whereas a substantial portion of the
human LH receptor is routed to a recycling pathway. Chimeras of these
two receptors identified a linear amino acid sequence (GTALL) present
near the C terminus of the human LH receptor that, when grafted onto
the rat LH receptor, redirects most of the rat LH receptor to a
recycling pathway. Removal of the GTALL sequence from the human LH
receptor failed to affect its routing, however.
The GTALL sequence shows homology with the C-terminal tetrapeptide
(DSLL) of the ß2-adrenergic receptor, a motif that has
been reported to mediate the recycling of the internalized
ß2-adrenergic receptor by binding to
ezrin-radixin-moesin-binding phosphoprotein-50. Addition of the DSLL
tetrapeptide to the C terminus of the rat LH receptor also redirects
most of the internalized rat LH receptor to a recycling pathway but,
like the recycling of the human LH receptor, this rerouting is not
mediated by ezrin-radixin-moesin-binding phosphoprotein-50.
We conclude that most of the internalized rat LH receptor is
degraded because its C-terminal tail lacks motifs that promote
recycling and that two distinct, but homologous, motifs (DSLL at the C
terminus or GTALL near the C terminus) can reroute the internalized rat
LH receptor to a recycling pathway that is independent of
ezrin-radixin-moesin-binding phosphoprotein-50.
 |
INTRODUCTION
|
---|
INTERNALIZATION OF G protein-coupled
receptors (GPCRS) is a ubiquitous response that follows agonist-induced
activation (1, 2, 3). Most GPCRs are internalized via
clathrin-coated pits by a pathway that requires the formation of a
complex between the agonist-activated and phosphorylated GPCR and a
family of proteins known as the nonvisual or ßarrestins
(1, 2, 3). Once internalized, most GPCRs are recycled back to
the plasma membrane (1, 2, 3), but a few are routed to the
lysosomes and targeted for degradation (4, 5, 6, 7, 8).
The ß2-adrenergic receptor
(ß2AR) is a prototypical GPCR that is sorted to
the recycling pathway (9, 10, 11). The C-terminal tetrapeptide
of the wild-type ß2AR (DSLL) has been shown
(12, 13) to mediate the binding of this receptor to the
PDZ domains of ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50)
(see Refs. 14 and 15), also known as
Na+/H+-exchange regulatory factor (NHERF) (see Ref. 16).
EBP50 is an abundant phosphoprotein composed of two N-terminal PDZ
domains and a C-terminal domain that binds ezrin, a component of the
cortical cytoskeleton (17). Cao and co-workers
(11) recently showed that mutation of the DSLL motif of
the ß2-AR or overexpression of a C-terminally
truncated form of EBP50 that cannot bind ezrin results in the rerouting
of the internalized ß2-AR from the recycling
pathway to a lysosomal degradation pathway. Although these experiments
identified the molecular basis of the sorting of the internalized
ß2-AR, it is not yet known whether the
recycling or lysosomal targeting of other GPCRs is also mediated by
their interaction with, or lack of interaction with, EBP50,
respectively. This is an important issue because most internalized
GPCRs are routed to a recycling pathway (1, 2, 3), but there
is only one other GPCR (the P2Y1 purinergic receptor) that has a
C-terminal sequence (D-S/T-x-L) that promotes EBP50 binding
(13).
The rat (r), mouse (m), and porcine (p) LH receptors (LHRs) are among
the few GPCRs that recycle poorly after internalization. These
receptors are routed mostly to a lysosomal degradation pathway that has
been particularly well characterized using biochemical (4, 18, 19, 20) and microscopic approaches (5, 7).
Thus, it is now known that the complex formed by the r, m, or pLHR and
one of its agonists [human CG (hCG)] is internalized via
clathrin-coated pits (5) by a pathway that requires the
involvement of a nonvisual arrestin and dynamin (18, 19).
The r, m, or pLHR-hCG complex is resistant to dissociation by the mild
acidic pH that prevails in the endosomes (4), and a
substantial proportion of the internalized complex is routed to the
lysosomes where it dissociates before degradation (4, 5, 7, 20). By promoting the accumulation of the hCG-LHR complex in a
compartment where it can be degraded, this pathway is ultimately
responsible not only for the degradation of hCG (21) but
also for the net loss of cell surface LHR that ensues after exposure of
rodent or porcine target cells or cells expressing the recombinant
rodent or porcine LHR to agonists (22, 23, 24). Surprisingly,
however, the fate of the highly related human (h) LHR is different from
that of the rLHR. As shown herein, a substantial portion of the
hCG-hLHR complex is routed to a recycling pathway rather than to a
degradation pathway.
Since most of the internalized rLHR is routed to a lysosomal
degradation pathway, this GPCR provides an ideal model system to test
the hypothesis that the DSLL-mediated interaction of GPCRs with EBP50
is sufficient to target internalized GPCRs to the recycling pathway.
The experiments presented herein were initially designed as a classical
gain-of-function approach to test this hypothesis. Since the data
obtained supported a role for the DSLL motif, but excluded the
involvement of EPB50, we performed additional experiments that
ultimately resulted in the identification of a structural motif (GTALL)
present near the C terminus of the hLHR that, when grafted onto the
C-terminal tail of the rLHR, also redirects the internalized rLHR to a
recycling pathway. Like the DSLL-induced recycling, the GTALL-induced
recycling of the rLHR was also found to be independent of EBP50.
 |
RESULTS
|
---|
Addition of a DSLL Motif to the C-Terminal Tail of the rLHR
Promotes Binding to EBP50 and Reroutes the Internalized
Agonist-rLHR Complex from a Lysosomal Degradation to a Recycling
Pathway
The C-terminal tetrapeptide of the rLHR (ALTH, cf. Fig. 1
) does not conform to the consensus
sequence (D-S/T-x-L) necessary for binding EBP50. In view of the recent
results of Cao and co-workers (11), we initially
postulated that the well documented routing of the internalized
agonist-rLHR complex to the lysosomes (4, 5, 7) could be
due to the lack of an interaction of the rLHR with EBP50. This
hypothesis was tested by using a gain-of-function approach involving
the analysis of two mutants of the rLHR in which their C-terminal tails
were extended by similar sequences (DSLL or DSLA) that were predicted
to support or not support, respectively, the binding of the rLHR to
EBP50. Initial experiments (not presented) showed that extension of the
C-terminal tail of the rLHR by addition of the DSLL or DSLA sequences
had no effect on the expression of the receptor. Human kidney 293 cells
transiently transfected with epitope (N-terminal myc)-tagged versions
of wild-type rLHR (rLHR-wt), rLHR-DSLL, or rLHR-DSLA bound equivalent
amounts of 125I-hCG (
100 fmol/10
6 cells) and internalized the bound
125I-hCG at similar rates (t
1/2 of 100120 min).
Figure 2A
shows that the rLHR-wt or
rLHR-DSLA mutants do not bind to EBP50, but the rLHR-DSLL mutant does.
The three bands of rLHR detected in the pull-down assays (
68 kDa,
85 kDa, and
165 kDa) correspond to an immature intracellular
precursor, the mature cell surface receptor, and an aggregate/oligomer
of the immature intracellular precursor, respectively
(25, 26, 27). The data presented in Fig. 2B
document that the
interaction of rLHR-DSLL with EBP50 occurs through the PDZ domain(s)
rather than the ezrin-radixin-moesin (ERM) binding domain of EBP50.
Thus, a glutathione-S-transferase (GST) fusion protein of
the ERM binding domain of EBP50 (i.e. residues 242358, see
Ref. 28) is not capable of forming a complex with the DSLL
mutant of the rLHR.

View larger version (53K):
[in this window]
[in a new window]
|
Figure 2. Association of rLHR-DSLL with EBP50
A, Detergent lysates of 293 cells transiently transfected with rLHR-wt,
rLHR-DSLL, or rLHR-DSLA were prepared and partially purified on a
lectin column as described in Materials and Methods.
Aliquots of the partially purified lysates containing the same amount
of solubilized protein were allowed to bind to GST or GST-EBP50 that
had been previously bound to glutathione agarose. The bound proteins
were eluted, and visualized on Western blots developed using a
monoclonal antibody to the myc epitope (9E10) and the ECL visualization
system. B, Detergent lysates of 293 cells transiently transfected with
rLHR-DSLL were prepared and partially purified on a lectin column as
described in Materials and Methods. Aliquots of the
partially purified lysates containing the same amount of solubilized
protein were allowed to bind to GST, GST-EBP50, or GST-EBP50(242358)
that had been previously bound to glutathione agarose. After the bound
proteins were eluted, they were visualized on Western blots developed
using a monoclonal antibody to the myc epitope (9E10) and the ECL
visualization system. The results of a representative experiment are
shown in each panel.
|
|
Since most of the internalized 125I-hCG
remains bound to the rLHR in endosomes and lysosomes (4, 5, 7, 20), measurements of the fate of the internalized
125I-hCG can be used to indirectly (but
conveniently) assess the targeting of the internalized hCG-receptor
complex. Thus, to study the fate of the internalized receptor cells
expressing rLHR-wt, rLHR-DSLL or rLHR-DSLA were allowed to bind and
internalize 125I-hCG for 2 h at 37 C. After
removal of the free and bound 125I-hCG, the fate
of the intracellular 125I-hCG was followed by
reincubating the cells at 37 C in medium without any added hormone. The
data summarized in Fig. 3
show that cells
expressing rLHR-wt quickly recycle approximately 15% of the
internalized 125I-hCG to the cell surface and
eventually degrade about 70% of the internalized hormone by the end of
a 4-h incubation. In contrast, cells expressing rLHR-DSLL quickly
recycle about 45% of the internalized 125I-hCG
to the cell surface and degrade approximately 40% of the internalized
125I-hCG during a 4-h incubation. The behavior of
rLHR-DSLA was intermediate between that of rLHR-wt and rLHR-DSLL. These
cells recycled about 25% and degraded approximately 55% of the
internalized hormone.

View larger version (15K):
[in this window]
[in a new window]
|
Figure 3. Fate of the Internalized 125I-hCG in
Cells Transiently Transfected with rLHR-wt, rLHR-DSLL, or rLHR-DSLA
Transiently transfected 293 cells were incubated with a
saturating concentration of 125I-hCG for 2 h at 37 C.
After washing to remove the free hormone, the surface-bound hormone was
released by a brief exposure of the cells to an isotonic pH 3 buffer,
and the cells were placed in hormone free medium at 37 C (t = 0 in
the figure) as described in Materials and Methods. At
the times indicated the medium was removed and saved. The cells were
washed with cold medium and they were briefly exposed again to the
isotonic pH 3 buffer, thus releasing any internalized hormone that had
recycled back to the surface. The acid-stripped cells were solubilized
with NaOH. The radioactivity that remained associated with the cells
after the acid elution (top panel) and the radioactivity
released by the acid treatment (middle panel) were
subsequently quantitated in a -counter. The saved medium was used to
determine the amount of degraded and undegraded 125I-hCG
released as described in Materials and Methods. Only the
degraded 125I-hCG is shown (lower panel)
because the amount of undegraded 125I-hCG released into the
medium was low (<5% of the initial counts per min) in cells
expressing any of these receptors. Three 35-mm wells were used for each
time point. Two of them contained 125I-hCG only, and the
third also contained an excess of nonlabeled hCG. The radioactivity
associated with the third well was used to correct for nonspecific
binding. Each point represents the mean ± SE of three
independent transfections. The absence of an error bar indicates that
the SEM is too small to be shown. Results are expressed as
% of the total radioactivity present at t = 0 (10,00020,000
cpm/well in individual experiments).
|
|
Dominant-Negative Mutants of EBP50 Do Not Interfere with the
Trafficking of the rLHR-DSLL Mutant
A dominant-negative approach was next used to determine whether
the functional effects described above are due to an interaction
between the rLHR-DSLL and endogenous EBP50. The presence of EBP50 in
293 cells could be readily documented by Western blots (Fig. 4A
). Overexpression of the hemagglutinin
(HA)-tagged forms of the wild-type EBP50 or a modified form of EPB50
(designated EBP50
) lacking the ERM-binding domain (11)
could also be readily demonstrated using antibodies to the HA epitope
(Fig. 4B
) or to an N-terminal epitope of EBP50 (Fig. 4C
). Expression of
a FLAG-tagged form of the ERM binding domain of EBP50 (designated
ERM-BD) was also readily demonstrated (Fig. 4C
).1

View larger version (22K):
[in this window]
[in a new window]
|
Figure 4. Detection of Endogenous EBP50 and of Several
Transfected Constructs
A, Endogenous EBP50 was detected in lysates of untransfected 293 cells
using a monoclonal antibody to residues 128249 of human EBP50. B and
C, 293 cells were transiently transfected with an empty vector,
HA-EBP50, or HA-EBP50 as indicated. The expression of the
transfected constructs was ascertained using the 12CA5 monoclonal
antibody to the HA-epitope (panel B) or a monoclonal antibody to
residues 128249 of human EBP50 (panel C). In panel C the endogenous
EBP50 is not visible in the cells transfected with the empty vector
simply because of the short exposure used. D, 293 cells were
transiently transfected with an empty vector or a construct encoding
for the FLAG-tagged version of the ERM binding domain of EBP50
(ERM-BD). The expression of the transfected constructs was ascertained
using the M2 monoclonal antibody to the FLAG-epitope. The
results of a representative experiment are shown.
|
|
The functional effects of these constructs on the trafficking of the
internalized 125I-hCG were measured in cells
cotransfected with rLHR-wt or rLHR-DSLL. Whereas we did not expect
EBP50 to have an effect on the trafficking of the hCG internalized by
cells expressing rLHR-wt, we were surprised to find that
overexpression of EBP50 did not further increase the recycling or
further prevent the degradation of the 125I-hCG
internalized by cells expressing rLHR-DSLL (Fig. 5
). We were also surprised by the finding
that a construct of EBP50 that lacks the ERM binding domain
(i.e. EBP50
) did not prevent the recycling or promote the
degradation of the 125I-hCG internalized by cells
expressing rLHR-DSLL (Fig. 6
) because
this same construct has been previously shown to prevent the recycling
and to promote the degradation of the internalized ß 2-AR
in 293 cells (11).2 An
additional EBP50 construct (designated ERM-BD) that lacks the two PDZ
domains and is also expected to interfere with the functions of the
endogenous EBP50 failed to prevent the recycling or promote the
degradation of the 125I-hCG internalized by cells
expressing rLHR-DSLL (Fig. 5
).

View larger version (22K):
[in this window]
[in a new window]
|
Figure 5. Effect of Several Constructs on the Fate of the
125I-hCG Internalized by Cells Cotransfected with rLHR-wt
or rLHR-DSLL
Cells were cotransfected with combinations of rLHR-wt or rLHR-DSLL and
HA-EBP50, HA-EBP50 , or the FLAG-tagged version of the ERM binding
domain of EBP50 (ERM-EBP50) as indicated. The fate of the internalized
125I-hCG was measured as described in the legend to Fig. 3 , except that only the 2 h time point was analyzed. The top
panel shows the proportion of internalized hormone that
remained cell associated, the middle panel shows the
proportion of the internalized hormone that recycled to the membrane,
and the lower panel shows the proportion of internalized
hormone that was degraded and released into the medium. Results
represent the mean ± SE of three independent
transfections and are expressed as % of the total radioactivity
present at t = 0 (10,00020,000 cpm/well in individual
experiments). The absence of an error bar indicates that the
error is too small to be shown.
|
|

View larger version (13K):
[in this window]
[in a new window]
|
Figure 6. Fate of the Internalized 125I-hCG in
Cells Transiently Transfected with rLHR-wt or hLHR-wt
The fate of the internalized 125I-hCG was determined in
transiently transfected cells exactly as described in the legend to
Fig. 3 . Results are expressed as percent of the total radioactivity
present at t = 0 (10,00020,000 cpm/well in individual
experiments) and they represent the mean ± SE of
three independent transfections. The absence of an error bar indicates
that the SEM is too small to be shown.
|
|
When Grafted onto the C-Terminal Tail of the rLHR, a Motif Present
in the C-Terminal Tail of the hLHR Reroutes the Internalized
Agonist-rLHR Complex from a Lysosomal Degradation to a Recycling
Pathway
There are two hypotheses that are consistent with the results
presented above. First it is possible that the DSLL motif added to the
C terminus of the rLHR may promote the interaction of this receptor
with a protein distinct from EBP50 that also has some affinity for the
DSLA motif and can, therefore, route the internalized hCG-receptor
complex to the recycling pathway. Alternatively, it is also possible
that the addition of the DSLL or DSLA motifs to the C-terminus of the
rLHR disrupts the interaction of the rLHR with a protein(s) that
normally route the internalized hormone-rLHR complex to a lysosomal
degradation pathway.
Since the amino acid sequence of the human (h) LHR and the rLHR are
highly homologous (
87% identity, see Ref. 29), but
they internalized hCG at vastly different rates (19), we
speculated that the intracellular routing of the hLHR and rLHR may also
be different. If this were the case we could then take advantage of the
availability of several hLHR/rLHR chimeras (19) to
differentiate between the two hypotheses proposed above. As shown in
Fig. 6
, we found that the fate of internalized agonist-hLHR complex is
indeed different from that of the agonist-rLHR complex. When compared
with cells expressing rLHR-wt, cells expressing hLHR-wt recycle more
and degrade less of the internalized hormone. The extent of recycling
of the internalized agonist-hLHR complex detected in cells expressing
hLHR-wt is in fact very similar to that detected with rLHR-DSLL
(cf. Fig. 3
).
The results presented in Table 1
show that replacing the C-terminal tail of the rLHR with that of the
hLHR (i.e. the rrh chimera) or replacing the serpentine
domain and the C-terminal tail of the rLHR with those of the hLHR
(i.e. the rhh chimera) increased recycling and decreased
degradation in such a way that the routing of these two chimeras is
closer to that of hLHR-wt than to that of rLHR-wt. Although the routing
of the rrh chimera is not identical to that of the rhh chimera, it is
still reasonable to conclude that the C-terminal tail of the hLHR
contains sufficient structural information to reroute the rLHR from a
degradation to a recycling pathway. Surprisingly, however, the
complementary manipulation had little or no effect on the routing of
the hLHR. As shown in Table 1
, the recycling and degradation of hCG
mediated by the hhr and the hrr chimeras more closely resembles the
hLHR-wt than the rLHR-wt.
View this table:
[in this window]
[in a new window]
|
Table 1. Fate of the Internalized 125I-hCG
in Cells Transiently Transfected with the rLHR, hLHR, and Mutants
Thereof
|
|
In the next series of experiments we concentrated on identifying the
structural features present in the C-terminal tail of the hLHR that
promote the recycling of the rLHR. The alignment of the amino acid
sequences of the C-terminal tail of three different species of the LHR
that are routed mostly to the lysosomes (rat, mouse, and porcine, see
Refs. 4, 5, 7 , and 18, 19, 20) and one that
recycles more (human LHR) show that there are only 10 amino acid
residues that are different between the hLHR and the r, m, and pLHR
group (these are shown in bold in Fig. 1
). Since most of
these divergent residues are present near the C terminus, additional
mutants focused on this region. We prepared and analyzed three mutants
of the rLHR (designated rLHR-mt1, -mt2, and -mt3) in which short amino
acid sequences near the C terminus of the rLHR were substituted with
the corresponding sequences of the hLHR as shown in Fig. 1
.
These results are summarized in Table 1
and show that rLHR-mt1 and
rLHR-mt2 enhance the recycling and decrease the degradation of the
internalized hCG-rLHR complex whereas rLHR-mt3 has no effect on
recycling or degradation. We conclude from these experiments that, when
grafted onto the rLHR, the GTALL sequence present near the C terminus
of the hLHR (see Fig. 1
) is responsible for rerouting the rLHR from a
degradation to a recycling pathway.
To confirm that the rerouting of the rLHR was caused by the addition of
the GTALL sequence of the hLHR rather than by the removal of the QPIPP
sequence of the rLHR (the sequences exchanged in rLHR-mt2, see Fig. 1
)
we analyzed two additional mutants in which the relevant portions of
the C-terminal tail of the rLHR and the hLHR were removed by truncation
of their C-terminal tails at residues 664 and 686, respectively (see
Fig. 1
). As shown in Table 1
, the removal of the QPIPP sequence of the
rLHR caused by truncation at residue 664 had no effect on the fate of
the internalized hCG-rLHR complex. Lastly, the removal of the GTALL
motif by truncation of the hLHR at residue 686 also had little or no
effect on the fate of the internalized hLHR (Table 1
).
The hLHR-wt and rLHR-mt2 Recycle by an EBP50-Independent
Pathway
Because of the involvement of EBP50 in the recycling of the
internalized ß 2-AR (11), it was
important to test whether this protein is also involved in the
recycling of the hLHR-wt and rLHR-mt2.
The pull-down assays shown in Fig. 7
show
that, in contrast to rLHR-DSLL, the binding of hLHR-wt and rLHR-mt2 to
EBP50 is minimal or undetectable. The functional assays summarized in
Fig. 8
also show that overexpression of
EBP50 or two distinct dominant negative mutants of EBP50 has no effect
on the trafficking of the hCG internalized by the hLHR-wt or by
rLHR-mt2.

View larger version (51K):
[in this window]
[in a new window]
|
Figure 7. Lack of Association of hLHR-wt and rLHR-mt2 with
EBP50
Detergent lysates of 293 cells transiently transfected with hLHR-wt,
rLHR-mt2, or rLHR-DSLL were prepared and partially purified on a lectin
column as described in Materials and Methods. Aliquots
of the partially purified lysates containing the same amount of
solubilized protein were allowed to bind to GST or GST-EBP50 that had
been previously bound to glutathione agarose. After the bound proteins
were eluted, they were visualized on Western blots developed using a
monoclonal antibody to the myc epitope (9E10) and the ECL visualization
system. The results of a representative experiment are shown.
|
|

View larger version (27K):
[in this window]
[in a new window]
|
Figure 8. Effect of Several Constructs on the Fate of the
125I-hCG Internalized by Cells Cotransfected with hLHR-wt
or rLHR-mt2
Cells were cotransfected with combinations of hLHR-wt or rLHR-mt2 and
HA-EBP50, HA-EBP50 , or the FLAG-tagged version of the ERM binding
domain of EBP50 (ERM-EBP50) as indicated. The fate of the internalized
125I-hCG was measured as described in the legend to Fig. 3 , except that only the 2 h time point was analyzed. The top
panel shows the proportion of internalized hormone that
remained cell associated; the middle panel shows the
proportion of the internalized hormone that recycled to the membrane,
and the lower panel shows the proportion of internalized
hormone that was degraded and released into the medium. Results
represent the mean ± SE of three independent
transfections and are expressed as % of the total radioactivity
present at t = 0 (10,00020,000 cpm/well in individual
experiments). The absence of an error bar indicates that the error is
too small to be shown.
|
|
 |
DISCUSSION
|
---|
The biochemical data presented here on the fate of the hCG
internalized by the rLHR are in agreement with previous results
obtained using electron microscopy (5), confocal
microscopy (7), and biochemical approaches (4, 20, 21) in target or transfected cells that express the rodent or
porcine LHR. When considered together, they show that most of the
125I-hCG-rLHR complex internalized by 293 cells
transfected with rLHR-wt is routed to the lysosomal degradation pathway
and that there is little recycling of the internalized hormone or
receptor. Surprisingly, the new data presented here also show that the
complex formed by hCG and the highly homologous hLHR is routed
differently. In this case a substantial portion of the internalized
complex is recycled to the membrane rather than routed to the lysosomes
for degradation.
More importantly, the results presented here define two discrete but
distinct experimental manipulations that reroute a substantial portion
of the internalized hCG-rLHR complex from a lysosomal degradation
pathway to a recycling pathway. These are 1) the addition of a DSLL
sequence to the C terminus of the rLHR; and 2) substitution of a QPIPP
sequence present near the C terminus of the rLHR with the corresponding
sequence (GTALL) of the hLHR.
The data presented here with rLHR-DSLL are interesting in view of the
recent results of Cao and co-workers (11) with the
ß2-AR. They used a loss-of-function approach to
show that the interaction of the C-terminal DSLL sequence of the
ß 2-AR with the PDZ domains of EBP50 is
necessary for the recycling of the internalized
ß2-AR. The data presented here utilize a
classical gain-of-function approach to test whether the DSLL sequence
is sufficient to promote the recycling of a GPCR (the rLHR) that is
normally routed mostly to a lysosomal degradation pathway. We show that
the DSLL sequence promotes the association of the rLHR with EBP50 and
is indeed sufficient to reroute a substantial portion of the
internalized rLHR to a recycling pathway. Importantly, however, our
data also show that the forced interaction of the rLHR-DSLL with EBP50
is not responsible for the rerouting of the internalized hCG-rLHR
complex. This conclusion is supported by three different findings.
First, modification of the rLHR with a C-terminal sequence (DLSA) that
does not support EBP50 binding (12, 13) does not promote
the association of the rLHR with EBP50 but it has a weak effect on
rerouting. Second, over expression of EBP50 does not further enhance
the recycling of the hCG internalized by the rLHR-DSLL. Third, and
perhaps more importantly, overexpression of two dominant-negative
mutants of EBP50 does not override the rerouting of the internalized
hCG-rLHR complex induced by addition of the DSLL motif to the rLHR. The
discrepant effects on the involvement of EBP50 in the recycling of the
rLHR-DSLL mutant (this paper) and the ß2-AR
(11) do not appear to be due to differences in the cell
types used, as both sets of experiments were done using 293 cells.
Also, the constructs used by us to overexpress the wt EBP50 or one of
its dominant negative mutants (EBP50D) are identical to those used in
the ß2-AR studies (11). One reason
for this discrepancy includes the possibility that the binding affinity
of the DSLL motif to EBP50 (or other PDZ domain-containing proteins)
could be dependent on other structural features of the protein
containing the DSLL motif. Since it is now known that there is at least
one other PDZ domain-containing protein (designated CAP70) that can
recognize the consensus sequence (D-S/T-x-L) necessary for binding
EBP50 (30), it is possible that the DSLL-induced recycling
of the rLHR is mediated by CAP70 or by other as-yet-unidentified
proteins that recognize the DSLL motif.
When considered together the results presented here with rLHR-DSLL,
rLHR-DSLL, and the rLHR/hLHR mutants exclude the hypothesis that the
hCG-rLHR complex is actively routed to the lysosomes because the rLHR
has structural motifs that target it to the lysosomes. Our results
instead are consistent with the conclusion that the internalized
hCG-rLHR complex is routed mostly to the lysosomes because the rLHR
lacks a sorting motif that is necessary for recycling. Thus, if the
C-terminal tail of the rLHR contained sorting motifs that routed the
internalized hCG-rLHR complex to the lysosomes, one would expect that
truncations of the C-terminal tail of the rLHR would reroute it to a
recycling pathway. As shown here and elsewhere (20),
however, progressive truncations of the C-terminal tail of the rLHR do
not reroute the internalized hCG-rLHR complex to a recycling pathway.
If anything, severe truncations tend to enhance transfer of the complex
to the lysosomes (20). Second, and perhaps more
importantly, a substantial portion of the internalized hCG-rLHR complex
can be rerouted from a degradation to a recycling pathway by the
addition of a DSLL motif at the C terminus (as discussed above) or by
substituting a QPIPP sequence present near the C terminus of the rLHR
with the corresponding sequence (GTALL) of the hLHR. This latter effect
is due to the addition of the GTALL sequence rather than to the removal
of the QPIPP sequence, because as noted above, progressive truncations
of the C-terminal tail of the rLHR do not reroute the internalized
hCG-rLHR complex from a lysosomal degradation to a recycling
pathway.
The behavior of the hhr chimera and hLHR-t686 clearly show that
exchanging the GTALL motif of the hLHR for the QPIPP motif of the rLHR
(as it occurred in the hhr chimera) or removing the GTALL motif from
the C-terminal tail of the hLHR (as it occurred in hLHR-t686) do not
redirect the internalized hCG-hLHR complex from a recycling to a
degradation pathway. We can thus conclude that although the GTALL motif
is sufficient to redirect a substantial portion of the rLHR from a
degradation to a recycling pathway (see above), this motif is not
necessary to promote the default sorting of the hLHR to a recycling
pathway. This finding is consistent with two distinct hypotheses.
First, it is possible that the sorting of the internalized hLHR to a
recycling pathway is completely independent of the GTALL motif.
Alternatively, the sorting of the internalized hLHR may occur by a
pathway that involves redundant motifs, one of which is the GTALL
motif. If redundant sorting motifs do exist in the hLHR, the GTALL
motif would be neither necessary nor sufficient to promote the
recycling of this receptor. Conversely, if the rLHR does not have any
motifs that promote recycling, then grafting the GTALL motif on its
C-terminal tail would be sufficient to reroute most of the internalized
rLHR to a recycling pathway as shown herein.
One way to differentiate between these two hypotheses would be to
search for additional structural motifs of the hLHR that participate in
recycling. A comparison of the trafficking of the hLHR-wt with the hrr
and hhr chimeras suggest that, if the hLHR has redundant structural
features that promote recycling, these features must be located in the
extracellular domain. Since the extracellular domain of the LHR is the
main determinant of ligand binding affinity (31), we
propose that the additional feature of the LHR that may participate in
recycling is the binding affinity of this receptor for hCG. We
specifically propose that the routing of the rLHR to a degradation
pathway may require two features, a high binding affinity for hCG and
the absence of a GTALL motif in the C-terminal tail. We further propose
that the routing of the hLHR to a recycling pathway may require only
one of two features, a low binding affinity for hCG or the presence of
a GTALL motif in its C-terminal tail. This proposal is best illustrated
by the data shown in Table 2
where we
summarize the hCG binding affinity, the presence/absence of the GTALL
motif, and the fate of the internalized hCG-receptor complexes in cells
expressing the most informative LHR mutants characterized here. These
data show that the only form of the LHR that is routed mostly to a
degradation pathway (i.e. the rLHR-wt) binds hCG with a high
affinity and does not have a GTALL motif in its C-terminal tail. The
presence of a GTALL motif in mutants that have the extracellular domain
of the rLHR and bind hCG with a high affinity (i.e. rrh,
rLHR-mt1, and rLHR-mt2) results in rerouting of a substantial amount of
the internalized receptors to a recycling pathway. The deletion of the
GTALL motif (as it occurred in hLHR-t686) or the substitution of the
GTALL motif in the hLHR (as it occurred in the hhr and hrr chimeras)
does not affect routing, and the only common feature among these forms
of the hLHR is the low hCG binding affinity caused by the presence of
the extracellular domain of the hLHR. This is illustrated in Table 2
with the hLHR-wt, as well as the hhr and hrr chimeras. The second
hypothesis proposed above is, therefore, consistent with all the
available data. The proposal that hCG binding affinity may contribute
to the fate of the internalized hCG-LHR complex is also consistent with
previous reports showing that all three members of the family of the
LHR that display minimal recycling (i.e. rat, porcine, and
mouse) bind hCG with high affinity (
0.2 nM;
this paper and Refs. 19, 32 , and 33).
Moreover, ligand binding affinity has already been shown to be an
important determinant of the fate of internalized growth
factor-receptor complexes (34, 35).
Although the precise locations of the two sorting motifs (DSLL and
GTALL) that reroute the internalized hCG-rLHR complex to the recycling
pathway are not identical, they both are located in the C-terminal
tail, a finding that agrees with the perceived importance of this
region in the intracellular routing of other GPCRs (8, 10, 11, 36). We do not yet know whether the DSLL or GTALL motifs
redirect the internalized hCG-rLHR complex to a recycling pathway by
the same or by different mechanisms, but it is clear that this
rerouting is not mediated by EBP50. Although addition of the DSLL
tetrapeptide to the C-terminal tail of the rLHR promotes the binding of
this receptor to EBP50, the presence of the GTALL motif in the
C-terminal tail of the rLHR does not promote EBP50 binding. More
importantly, dominant negative constructs of EBP50 do not interfere
with the DSLL- or GTALL-induced rerouting of the internalized hCG-rLHR
complex to a recycling pathway. These are important observations
because of the paucity of information about the structural features of
GPCRs that contribute to intracellular sorting and the identity of the
cellular proteins that mediate the sorting of internalized receptors.
In this respect it is interesting to note that the DSLL and GTALL
motifs share some structural features. They both have a phosphate
acceptor (DSLL and GTALL) and a downstream
dileucine motif (DSLL and GTALL). Given the
involvement of phosphorylation (10, 11, 37) and dileucine
motifs (38) in protein trafficking, it is possible that
both of these features are needed to sort the internalized rLHR to a
recycling pathway. Future studies will examine this possibility.
Furthermore, the availability of different constructs of the LHR that
are routed to the lysosomes or to a recycling pathway should also now
allow us to identify proteins that may be involved in the sorting of
the internalized LHR and to determine whether the fate of the
internalized LHR has an effect on its biological functions.
 |
MATERIALS AND METHODS
|
---|
Plasmids and Cells
Full-length cDNAs encoding for the hLHR and rLHR (39, 40) were subcloned into pcDNAI/Neo (rLHR) or pcDNA 3.1(hLHR) for
expression. The preparation and characterization of myc-rLHR-wt and
myc-hLHR-wt, modified forms of the LHR containing the myc epitope at
the N terminus, have also been described (26, 41). The
different mutants of the rLHR and hLHR used here were constructed by
standard PCR strategies using the myc-rLHR-wt or myc-hLHR-wt as
templates. The wild-type and mutant rLHR cDNAs were subcloned into the
eukaryotic expression vector pcDNAI/Neo (Invitrogen, San
Diego, CA) for transfection. The preparation of the rLHR/hLHR chimeras
and the preparation of C-terminally truncated mutants of the rLHR
(rLHR-t664) and the hLHR (hLHR-t686) have also been described
(19, 20).
Bacterial expression vectors encoding for GST fusion proteins of the
full-length EBP50 or derivatives thereof were prepared as described
(15, 28). GST fusion proteins were also prepared as
described elsewhere (15, 28). Expression vectors (pcDNA3,
Invitrogen) encoding the C-terminally HA-tagged EBP50-wt
and EBP50
(an EBP50 mutant lacking the last 61 amino acids) are
described in Ref. 11 and were kindly provided by Tracy Cao
and Mark von Zastrow (University of California at San Francisco). An
additional expression vector (pcDNA3.1, Invitrogen)
encoding for EBP50
without an epitope tag was also constructed from
the full-length expression vector encoding for the EBP50 GST fusion
protein using standard PCR strategies. An expression vector for an
N-terminally FLAG-tagged construct encoding for the ERM binding domain
of EBP50 (designated ERM-BD) was prepared by subcloning a portion
of EBP50 (coding for residues 298358) into the pFLAG-CMV2 vector
(Sigma, St. Louis, MO).
Human embryonic kidney (293) cells were obtained from the
American Type Culture Collection (Manassas, VA; CRL 1573)
and maintained in DMEM containing 10 mM HEPES, 10% newborn
calf-serum, and 50 µg/ml gentamicin, pH 7.4. Cells were plated in
35-mm wells or 100-mm dishes and transfected with not more than 2 or 10
µg of plasmid DNA, respectively (42), when 7080%
confluent. After an overnight incubation with the transfection mixture,
the cells were washed and used 24 h later. Clonal lines of 293
cells stably expressing the HA-tagged version of EBP50
were obtained
by selection of the transfected cells with 700 µg/ml of G418
(43). Resistant colonies were then tested for the
expression of EBP50
using an antibody to the HA epitope as described
below.
Fate of the Internalized Hormone
Transiently transfected cells were incubated with
125I-hCG (0.52 nM) for 2 h at
37 C. After washing to remove the free hormone, the surface-bound
hormone was released by a brief exposure of the cells to an isotonic pH
3 buffer (21). Warm, hormone-free medium was then added
back (t = 0), and the cells were returned to the incubator. At the
times indicated the medium was removed and saved. The cells were washed
with cold medium, and they were briefly exposed again to the isotonic
pH 3 buffer, thus releasing any internalized hormone that had recycled
back to the surface. The acid-stripped cells were solubilized with
NaOH. The radioactivity that remained associated with the cells after
the acid elution and the radioactivity released by the acid treatment
were subsequently quantitated in a
counter. The saved medium was
precipitated with 10% trichloroacetic acid to determine the amount of
degraded and undegraded 125I-hCG released
(21).
hCG Binding Assays
The equilibrium binding parameters for
125I-hCG were measured using intact cells that
had been cotransfected with dynamin-K44A to prevent internalization
(see Ref. 19). Binding parameters were measured during a
1-h incubation of intact cells (plated in 35-mm wells) with seven
different concentrations (0.390 nM) of
125I-hCG at room temperature. All binding assays
were corrected for nonspecific binding (measured in the presence of an
excess of partially purified hCG). The binding data were simply fitted
to a sigmoidal equation (44) using DeltaGraph software
(Delta Point, Monterey, CA), and this equation was used to calculate
the maximal amount of cell-associated hormone and the apparent
dissociation constant (Kd).
Other Methods
The interaction between the myc-tagged rLHR-wt, rLHR-DSLL, or
rLHR-DSLA with different EBP50 derivatives was determined by measuring
the ability of detergent lysates prepared from transiently transfected
cells to bind to the indicated GST fusion proteins. Lysates of cells
expressing the myc-tagged rLHR constructs were prepared and partially
purified on a wheat germ agglutinin agarose column as described
elsewhere (25, 26), except that the lysis buffer contained
1% NP-40 and 60 mM octylglucoside. Equal amounts of
partially purified lysate protein were incubated with 25 µg of the
appropriate GST fusion proteins bound to glutathione agarose, washed,
and eluted exactly as described in Ref. 15 . The eluted
samples were resolved on SDS gels and electrophoretically blotted as
described elsewhere (25). Blots were visualized using a
monoclonal antibody to the myc epitope (9E10) and the enhanced
chemiluminescence (ECL) detection system.
The expression of the endogenous EBP50 or the different EBP50
constructs transfected was ascertained on Western blots of cell lysates
prepared as described above but without lectin purification. Depending
on the construct transfected, blots were visualized using a monoclonal
antibody to residues 128249 of human EBP50 (Transduction Laboratories, Inc., Lexington, KY), a monoclonal antibody to the
HA epitope (12CA5 from Roche Molecular Biochemicals,
Indianapolis, IN), or a monoclonal antibody to the FLAG epitope (M2
from Sigma, St. Louis, MO) and the ECL detection
system.
Hormones and Supplies
Purified hCG (CR-127,
13,000 U/mg) was kindly provided by Dr.
A. Parlow and 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, and
it was used only to correct for nonspecific binding.
125I-hCG was prepared as previously described
(45). Cell culture supplies and reagents were obtained
from Corning, Inc. (Corning, NY) and Life Technologies, Inc., Gaithersburg, MD), respectively. All other
chemicals were obtained from commonly used suppliers.
 |
ACKNOWLEDGMENTS
|
---|
We wish to thank Tracy Cao and Mark von Zastrow
(University of California at San Francisco, San Francisco, CA) for the
expression vectors encoding for the HA-tagged forms of EBP50 and
EBP50
. We also thank Deborah Segaloff (University of Iowa, Iowa
City, IA) for her comments on this manuscript and Professor Masatomo
Mori (First Department of Internal Medicine, Gunma University, Gunma,
Japan) for his support. The initial hLHR plasmid used in these
experiments was kindly provided to us by Ares Serono (Randolph,
MA).
 |
FOOTNOTES
|
---|
This work was supported by grants from the NIH: CA-40629 to MA and
GM-36652 to AB. The services and facilities provided by the Diabetes
and Endocrinology Research Center of the University of Iowa (supported
by NIH grant DK-2529 5) are also gratefully acknowledged.
Abbreviations: ß2AR, ß2-adrenergic
receptor; ECL, enhanced chemiluminescence; ERM, ezrin-radixin-moesin;
GPCR, G protein-coupled receptor; GST,
glutathione-S-transferase; HA, hemagglutinin; LHR, LH
receptor; mLHR, mouse LHR; pLHR, porcine LH; rLHR, rat LHR.
1 This construct could not be detected with the
commercially available antibody to EBP50 because this antibody
recognizes an epitope (residues 128249) that was removed from
ERM-BD. 
2 The same results were also obtained using
transient co-transfections of 293 cells with myc-rLHR-DSLL and a
different construct of EBP50
that was not tagged with the HA-epitope
or by transient expression of myc-rLHR-DSLL in 293 cells stably
expressing the HA-tagged version of EBP50
(data not shown). 
Received for publication January 23, 2001.
Accepted for publication June 5, 2001.
 |
REFERENCES
|
---|
-
Carman CV, Benovic JL 1998 G-protein-coupled receptors:
turn-ons and turn-offs. Curr Opin Neurobiol 8:335344[CrossRef][Medline]
-
Lefkowitz RJ 1998 G protein-coupled receptors. III. New roles
for receptor kinases and ß-arrestins in receptor signaling and
desensitization. J Biol Chem 273:1867718680[Free Full Text]
-
Ferguson SS, Caron MG 1998 G protein-coupled receptor
adaptation mechanisms. Semin Cell Dev Biol 9:119127[CrossRef][Medline]
-
Ascoli M 1984 Lysosomal accumulation of the hormone-receptor
complex during receptor-mediated endocytosis of human
choriogonadotropin. J Cell Biol 99:12421250[Abstract]
-
Ghinea N, Vuhai MT, Groyer-Picard M-T, Houllier A,
Schoëvaërt D, Milgrom E 1992 Pathways of internalization of
the hCG/LH receptor: immunoelectron microscopic studies in Leydig cells
and transfected L cells. J Cell Biol 118:13471358[Abstract]
-
Hein L, Ishii K, Coughlin SR, Kobilka BK 1994 Intracellular
targeting and trafficking of thrombin receptors. J Biol Chem 269:2771927726[Abstract/Free Full Text]
-
Baratti-Elbaz C, Chinea N, Lahuna O, Loosfelt H, Pichon C,
Milgrom E 1999 Internalization and recycling pathways of the
thyrotropin receptor. Mol Endocrinol 13:17511765[Abstract/Free Full Text]
-
Trejo J, Coughlin SR 1999 The cytoplasmic tails of
protease-activated receptor-1 and substance P receptor specify sorting
to lysosomes versus recycling. J Biol Chem 274:22162224[Abstract/Free Full Text]
-
Zhang J, Barak LS, Winkler KE, Caron MG, Ferguson SSG 1997 A
central role for ß-arrestins and clathrin-coated vesicle-mediated
endocytosis in ß2-adrenergic receptor
resensitization. J Biol Chem 272:2700527014[Abstract/Free Full Text]
-
Oakley RH, Laporte SA, Holt JA, Barak LS, Caron MG 1999 Association of ß-arrestin with G protein-coupled receptors during
clathrin-mediated endocytosis dictate the profile of receptor
resensitization. J Biol Chem 274:3224832257[Abstract/Free Full Text]
-
Cao TT, Deacon HW, Reczek D, Bretscher A, von Zastrow M 1999 A
kinase-regulated PDZ-domain interaction controls endocytic sorting of
the ß 2-adrenergic receptor. Nature 401:286290[CrossRef][Medline]
-
Hall RA, Premont RT, Chow CW, et al. 1998 The
ß2-adrenergic receptor interacts with the
Na+/H+-exchanger regulatory factor to control Na+/H+ exchange. Nature 392:626630[CrossRef][Medline]
-
Hall RA, Ostedgaard LS, Premont RT, et al. 1998 A C-terminal
motif found in the ß2-adrenergic receptor, P2Y1 receptor and cystic
fibrosis transmembrane conductance regulator determines binding to the
Na+/H+ exchanger regulatory factor family of PDZ proteins. Proc Natl
Acad Sci USA 95:84968501[Abstract/Free Full Text]
-
Reczek D, Berryman M, Bretscher A 1997 Identification of
EBP50: a PDZ-containing phosphoprotein that associates with members of
the Ezrin-Radixin-Moesin family. J Cell Biol 139:169179[Abstract/Free Full Text]
-
Short DB, Trotter KW, Reczek D, Kreda sM, Bretscher A, Boucher
RC, Stutts MJ, Milgram SL 1998 An apical PDZ protein anchors the cystic
fibrosis transmembrane conductance regulator to the cystoskeleton.
J Biol Chem 273:1979719801[Abstract/Free Full Text]
-
Weinman EJ, Steplock D, Wang Y, Shenolikar S 1995 Characterization of a protein cofactor that mediates protein kinase A
regulation of the renal brush border membrane Na+/H+ exchanger. J
Clin Invest 95:21432149[Medline]
-
Bretscher A 1999 Regulation of cortical structure by the
ezrin-radixin-moesin protein family. Curr Opin Cell Biol 11:109116[CrossRef][Medline]
-
Nakamura K, Ascoli M 1999 A dileucine-based motif in the
C-terminal tail of the lutropin/choriogonadotropin receptor inhibits
endocytosis of the agonist-receptor complex. Mol Pharmacol 56:728736[Abstract/Free Full Text]
-
Nakamura K, Liu X, Ascoli M 2000 Seven non-contiguous
intracellular residues of the lutropin/choriogonadotropin receptor
dictate the rate of agonist-induced internalization and its sensitivity
to non-visual arrestins. J Biol Chem 275:241247[Abstract/Free Full Text]
-
Kishi M, Ascoli M 2000 The C-terminal tail of the rat
lutropin/choriogonadotropin receptor independently modulates
hCG-induced internalization of the cell surface receptor and the
lysosomal targeting of the internalized hCG-receptor complex. Mol
Endocrinol 14:926936[Abstract/Free Full Text]
-
Ascoli M 1982 Internalization and degradation of
receptor-bound human choriogonadotropin in Leydig tumor cells. Fate of
the hormone subunits. J Biol Chem 257:1330613311[Abstract/Free Full Text]
-
Lloyd CE, Ascoli M 1983 On the mechanisms involved in the
regulation of the cell surface receptors for human choriogonadotropin
and mouse epidermal growth factor in cultured Leydig tumor cells.
J Cell Biol 96:521526[Abstract]
-
Wang H, Segaloff DL, Ascoli M 1991 Lutropin/choriogonadotropin
down-regulates its receptor by both receptor mediated endocytosis and a
cAMP-dependent reduction in receptor mRNA. J Biol Chem 266:780785[Abstract/Free Full Text]
-
Nakamura K, Lazari MFM, Li S, Korgaonkar C, Ascoli M 1999 Role
of the rate of internalization of the agonist-receptor complex on the
agonist-induced down-regulation of the lutropin/choriogonadotropin
receptor. Mol Endocrinol 13:12951304[Abstract/Free Full Text]
-
Hipkin RW, Sánchez-Yagüe J, Ascoli M 1992 Identification and characterization of a luteinizing hormone/chorionic
gonadotropin (LH/CG) receptor precursor in a human kidney cell line
stably transfected with the rat luteal LH/CG receptor complementary
DNA. Mol Endocrinol 6:22102218[Abstract]
-
Fabritz J, Ryan S, Ascoli M 1998 Transfected cells express
mostly the intracellular precursor of the lutropin/choriogonadotropin
receptor but this precursor binds choriogonadotropin with high
affinity. Biochemistry 37:664672[CrossRef][Medline]
-
VuHai-LuuThi MT, Misrahi M, Houillier A, Jolivet A, Milgrom E 1992 Variant forms of the pig lutropin/choriogonadotropin receptor.
Biochemistry 31:83778383[Medline]
-
Reczek D, Bretscher A 1998 The carboxyl-terminal region of
EBP50 binds to the a site in the amino-terminal domain of Ezrin that is
masked in the dormant molecule. J Biol Chem 273:1845218458[Abstract/Free Full Text]
-
Segaloff DL, Ascoli M 1993 The lutropin/choriogonadotropin
(LH/CG) receptor ... 4 years later. Endocr Rev 14:324347[Abstract]
-
Wang S, Yue H, Derin RB, Guggino WB, Li M 2000 Accessory
protein facilitated CFTR-CFTR interaction, a molecular mechanism to
potentiate the chloride channel activity. Cell 103:169179[Medline]
-
Xie Y-B, Wang H, Segaloff DL 1990 Extracellular domain of
lutropin/choriogonadotropin receptor expressed in transfected cells
binds choriogonadotropin with high affinity. J Biol Chem 265:2141121414[Abstract/Free Full Text]
-
Buettner K, Ascoli M 1984 Na+ modulates
the affinity of the lutropin/choriogonadotropin (LH/CG) receptor.
J Biol Chem 259:1507815084[Abstract/Free Full Text]
-
Loosfelt H, Misrahi M, Atger M, Salesse R, Vu Hai-Luu Thi MT,
Jolivet A, Guiochon-Mantel A, Sar S, Jallal B, Garnier J, Milgrom E 1989 Cloning and sequencing of porcine LH-hCG receptor cDNA: variants
lacking transmembrane domain. Science 245:525528[Medline]
-
French AR, Sudlow GP, Wiley HS, Lauffenburger DA 1994 Postendocytic trafficking of epidermal growth factor-receptor complexes
is mediated through saturable and specific endosomal interactions.
J Biol Chem 269:1574915755[Abstract/Free Full Text]
-
Ceresa BP, Schmid SL 2000 Regulation of signal transduction by
endocytosis. Curr Opin Cell Biol 12:204210[CrossRef][Medline]
-
Bremnes T, Paasche JD, Mehlum A, Sandberg C, Bremnes B,
Attramadal H 2000 Regulation and intracellular trafficking pathways of
the endothelin receptors. J Biol Chem 275:1759617604[Abstract/Free Full Text]
-
Innamorati G, Sadeghi HM, Tran NT, Birnbaumer M 1998 A serine
cluster prevents recycling of the V2 vasopressin receptor. Biochemistry 95:22222226
-
Kirchhausen T 1999 Adaptors for clathrin-mediated traffic.
Annu Rev Cell Dev Biol 15:705732[CrossRef][Medline]
-
Minegishi T, Nakamura K, Takakura Y, et al. 1990 Cloning and
sequencing of human LH/hCG receptor cDNA. Biochem Biophys Res Commun 172:10491054[Medline]
-
McFarland KC, Sprengel R, Phillips HS, et al. 1989 Lutropin-choriogonadotropin receptor: an unusual member of the G
protein-coupled receptor family. Science 245:494499[Medline]
-
Min L, Ascoli M 2000 Effect of activating and inactivating
mutations on the phosphorylation and trafficking of the human
lutropin/choriogonadotropin receptor. Mol Endocrinol 14:17971810[Abstract/Free Full Text]
-
Chen C, Okayama H 1987 High-efficiency transformation of
mammalian cells by plasmid DNA. Mol Cell Biol 7:27452752[Medline]
-
Hipkin RW, Wang Z, Ascoli M 1995 Human chorionic gonadotropin-
and phorbol ester stimulated phosphorylation of the LH/CG receptor maps
to serines 635, 639, 645 and 652 in the C-terminal cytoplasmic tail.
Mol Endocrinol 9:151158[Abstract]
-
De Lean A, Munson PJ, Rodbard D 1978 Simultaneous analysis of
families of sigmoidal curves: application to bioassay, radioligand
assay and physiological doseresponse curves. Am J Physiol
235:E97E102
-
Ascoli M, Puett D 1978 Gonadotropin binding and stimulation of
steroidogenesis in Leydig tumor cells. Proc Natl Acad Sci USA 75:99102[Abstract]
-
Gudermann T, Birnbaumer M, Birnbaumer L 1992 Evidence for dual
coupling of the murine luteinizing hormone receptor to adenylyl cyclase
and phosphoinositide breakdown and Ca+2
mobilization. J Biol Chem 267:44794488[Abstract/Free Full Text]
-
Baldwin JM, Schertler GFX, Unger VM 1997 An
-carbon
template for the transmembrane helices in the rhodopsin family of
G-protein-coupled receptors. J Mol Biol 272:144164[CrossRef][Medline]
-
Wang Z, Liu X, Ascoli M 1997 Phosphorylation of the
lutropin/choriogonadotropin receptor facilitates uncoupling of the
receptor from adenylyl cyclase and endocytosis of the bound hormone.
Mol Endocrinol 11:183192[Abstract/Free Full Text]
-
Lazari MFM, Bertrand JE, Nakamura K, et al. 1998 Mutation of
individual serine residues in the C-terminal tail of the
lutropin/choriogonadotropin (LH/CG) receptor reveal distinct structural
requirements for agonist-induced uncoupling and agonist-induced
internalization. J Biol Chem 273:1831618324[Abstract/Free Full Text]