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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, 2–319B BSB, 51 Newton Road, The University of Iowa, Iowa City, IA 52242-1109. E-mail: mario-ascoli{at}uiowa.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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) 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 100–120 min).
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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 242–358, see
Ref. 28) is not capable of forming a complex with the DSLL
mutant of the rLHR.
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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.
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). 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
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). 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).
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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.
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). 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.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 298–358) 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 70–80%
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 (0.5–2 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 (0.3–90 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 128–249 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.
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ACKNOWLEDGMENTS |
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. 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 |
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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 128–249) 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.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-carbon
template for the transmembrane helices in the rhodopsin family of
G-protein-coupled receptors. J Mol Biol 272:144–164[CrossRef][Medline]
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 J. D. Paasche, T. Attramadal, K. Kristiansen, M. P. Oksvold, H. K. Johansen, H. S. Huitfeldt, S. G. Dahl, and H. Attramadal Subtype-Specific Sorting of the ETA Endothelin Receptor by a Novel Endocytic Recycling Signal for G Protein-Coupled Receptors Mol. Pharmacol., May 1, 2005; 67(5): 1581 - 1590. [Abstract] [Full Text] [PDF]
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 R S Bhaskaran and M Ascoli The post-endocytotic fate of the gonadotropin receptors is an important determinant of the desensitization of gonadotropin responses J. Mol. Endocrinol., April 1, 2005; 34(2): 447 - 457. [Abstract] [Full Text] [PDF]
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U. M. Munshi, C. L. Clouser, H. Peegel, and K. M. J. Menon Evidence that Palmitoylation of Carboxyl Terminus Cysteine Residues of the Human Luteinizing Hormone Receptor Regulates Postendocytic Processing Mol. Endocrinol., March 1, 2005; 19(3): 749 - 758. [Abstract] [Full Text] [PDF]
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 S. Shenolikar, J. W. Voltz, R. Cunningham, and E. J. Weinman Regulation of Ion Transport by the NHERF Family of PDZ Proteins Physiology, December 1, 2004; 19(6): 362 - 369. [Abstract] [Full Text] [PDF]
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G. A. Vargas and M. von Zastrow Identification of a Novel Endocytic Recycling Signal in the D1 Dopamine Receptor J. Biol. Chem., September 3, 2004; 279(36): 37461 - 37469. [Abstract] [Full Text] [PDF]
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P. M. Haggie, B. A. Stanton, and A. S. Verkman Increased Diffusional Mobility of CFTR at the Plasma Membrane after Deletion of Its C-terminal PDZ Binding Motif J. Biol. Chem., February 13, 2004; 279(7): 5494 - 5500. [Abstract] [Full Text] [PDF]
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C. Galet, T. Hirakawa, and M. Ascoli The Postendocytotic Trafficking of the Human Lutropin Receptor Is Mediated by a Transferable Motif Consisting of the C-Terminal Cysteine and an Upstream Leucine Mol. Endocrinol., February 1, 2004; 18(2): 434 - 446. [Abstract] [Full Text] [PDF]
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T. Hirakawa, C. Galet, M. Kishi, and M. Ascoli GIPC Binds to the Human Lutropin Receptor (hLHR) through an Unusual PDZ Domain Binding Motif, and It Regulates the Sorting of the Internalized Human Choriogonadotropin and the Density of Cell Surface hLHR J. Biol. Chem., December 5, 2003; 278(49): 49348 - 49357. [Abstract] [Full Text] [PDF]
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M. Tanowitz and M. von Zastrow A Novel Endocytic Recycling Signal That Distinguishes the Membrane Trafficking of Naturally Occurring Opioid Receptors J. Biol. Chem., November 14, 2003; 278(46): 45978 - 45986. [Abstract] [Full Text] [PDF]
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H. Krishnamurthy, H. Kishi, M. Shi, C. Galet, R. S. Bhaskaran, T. Hirakawa, and M. Ascoli Postendocytotic Trafficking of the Follicle-Stimulating Hormone (FSH)-FSH Receptor Complex Mol. Endocrinol., November 1, 2003; 17(11): 2162 - 2176. [Abstract] [Full Text] [PDF]
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Y. Qiu, P.-Y. Law, and H. H. Loh {micro}-Opioid Receptor Desensitization: ROLE OF RECEPTOR PHOSPHORYLATION, INTERNALIZATION, AND RESENSITIZATION J. Biol. Chem., September 19, 2003; 278(38): 36733 - 36739. [Abstract] [Full Text] [PDF]
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C. Galet, L. Min, R. Narayanan, M. Kishi, N. L. Weigel, and M. Ascoli Identification of a Transferable Two-Amino-Acid Motif (GT) Present in the C-Terminal Tail of the Human Lutropin Receptor that Redirects Internalized G Protein-Coupled Receptors from a Degradation to a Recycling Pathway Mol. Endocrinol., March 1, 2003; 17(3): 411 - 422. [Abstract] [Full Text] [PDF]
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S. Chauvin, M. Bencsik, T. Bambino, and R. A. Nissenson Parathyroid Hormone Receptor Recycling: Role of Receptor Dephosphorylation and {beta}-Arrestin Mol. Endocrinol., December 1, 2002; 16(12): 2720 - 2732. [Abstract] [Full Text] [PDF]
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 Y. Sun and M. C. Gershengorn Correlation between Basal Signaling and Internalization of Thyrotropin-Releasing Hormone Receptors: Evidence for Involvement of Similar Receptor Conformations Endocrinology, August 1, 2002; 143(8): 2886 - 2892. [Abstract] [Full Text] [PDF]
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J.-G. Li, C. Chen, and L.-Y. Liu-Chen Ezrin-Radixin-Moesin-binding Phosphoprotein-50/Na+/H+ Exchanger Regulatory Factor (EBP50/NHERF) Blocks U50,488H-induced Down-regulation of the Human kappa Opioid Receptor by Enhancing Its Recycling Rate J. Biol. Chem., July 19, 2002; 277(30): 27545 - 27552. [Abstract] [Full Text] [PDF]
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 M. Ascoli, F. Fanelli, and D. L. Segaloff The Lutropin/Choriogonadotropin Receptor, A 2002 Perspective Endocr. Rev., April 1, 2002; 23(2): 141 - 174. [Abstract] [Full Text] [PDF]
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T. Hirakawa, C. Galet, and M. Ascoli MA-10 Cells Transfected with the Human Lutropin/Choriogonadotropin Receptor (hLHR): A Novel Experimental Paradigm to Study the Functional Properties of the hLHR Endocrinology, March 1, 2002; 143(3): 1026 - 1035. [Abstract] [Full Text] [PDF]
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 S. Martin, J.-P. Vincent, and J. Mazella Recycling ability of the mouse and the human neurotensin type 2 receptors depends on a single tyrosine residue J. Cell Sci., January 1, 2002; 115(1): 165 - 173. [Abstract] [Full Text] [PDF]
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M. Cong, S. J. Perry, L. A. Hu, P. I. Hanson, A. Claing, and R. J. Lefkowitz Binding of the beta 2 Adrenergic Receptor to N-Ethylmaleimide-sensitive Factor Regulates Receptor Recycling J. Biol. Chem., November 21, 2001; 276(48): 45145 - 45152. [Abstract] [Full Text] [PDF]
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