Internalization and Homologous Desensitization of the GLP-1 Receptor Depend on Phosphorylation of the Receptor Carboxyl Tail at the Same Three Sites
Christian Widmann1,
Wanda Dolci and
Bernard Thorens
Institute of Pharmacology and Toxicology, University of
Lausanne CH-1005 Lausanne, Switzerland
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ABSTRACT
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Homologous desensitization and internalization of
the GLP-1 receptor correlate with phosphorylation of the receptor in a
33-amino acid segment of the cytoplasmic tail. Here, we identify the
sites of phosphorylation as being three serine doublets located at
positions 441/442, 444/445, and 451/452. The role of phosphorylation on
homologous desensitization was assessed after stable expression in
fibroblasts of the wild type or of mutant receptors in which
phosphorylation sites were changed in various combinations to alanines.
We showed that desensitization, as measured by a decrease in the
maximal production of cAMP after a first exposure of the cells to
GLP-1, was strictly dependent on phosphorylation. Furthermore, the
number of phosphorylation sites correlated with the extent of
desensitization with no, intermediate, or maximal desensitization
observed in the presence of one, two, or three phosphorylation sites,
respectively. Internalization of the receptor-ligand complex was
assessed by measuring the rate of internalization of bound
[125I]GLP-1 or the redistribution of the
receptor to an endosomal compartment after agonist binding. Our data
demonstrate that internalization was prevented in the absence of
receptor phosphorylation and that intermediate rates of endocytosis
were obtained with receptors containing one or two phosphorylation
sites. Thus, homologous desensitization and internalization require
phosphorylation of the receptor at the same three sites. However, the
differential quantitative impairment of these two processes in the
single and double mutants suggests different molec-ular
mechanisms controlling desensitization and internalization.
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INTRODUCTION
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Agonist binding to heterotrimeric G protein-coupled receptors
triggers several events that are thought to result from ligand-induced
changes in receptor conformation. First, dissociation of G
-subunits
from their ß
-partners leads to activation of the intracellular
signaling pathways. Usually, after this first event, there is a rapid
desensitization of the receptors. This is observed by a decrease in the
maximum, and/or an increase in the EC50, of the
dose-response curve for production of intracellular second messengers
in a second exposure of the cells to the agonist. Also occurring with a
rapid kinetics is the internalization of the receptor-ligand complex by
endocytosis through coated pits.
The molecular mechanisms underlying homologous and heterologous
desensitization of these receptors usually involve phosphorylation of
specific residues by different kinases. For instance, for the
ß2-adrenergic receptor, heterologous desensitization can be induced
by activation of protein kinase A (PKA) or protein kinase C (PKC), an
effect that requires the presence of a PKA/PKC consensus site in the
third intracellular loop of the receptor (1, 2, 3, 4). On the other hand,
homologous desensitization results from receptor phosphorylation by G
protein-coupled receptor-specific kinases (GRKs) (5), which form a
family of structurally related isoforms (6). This phosphorylation takes
place in the serine/threonine-rich C tail of the receptor (4) and
induces association of the receptor with ß-arrestins (7, 8, 9, 10). This
interaction prevents further activation of G proteins after agonist
binding to the receptor and is thought to form the basis for the
observed desensitization process.
Internalization of G protein-coupled receptors rapidly follows agonist
binding and has initially been proposed to be involved in receptor
desensitization (11, 12). More recently, studies have shown, on one
hand, that desensitization could be suppressed by removing the GRK
phosphorylation sites of the ß2-adrenergic receptor without impairing
receptor internalization (4, 13). On the other hand, mutants unable to
be internalized were still desensitized (14, 15). This indicated that
internalization of the receptor-ligand complex and homologous
desensitization were independent processes. This situation needs to be
reappraised, however, in light of work demonstrating that
internalization of the m2 muscarinic acetylcholine receptor can be
facilitated by overexpression of GRK2 and that expression of a dominant
negative mutant of this kinase decreases the rate of receptor
internalization (16). Also, endocytosis of an internalization-resistant
mutant of the ß2-adrenergic receptor could be induced by
overexpression of GRK2, which phosphorylated the receptor (17). These
two studies therefore suggest that phosphorylation by GRKs, if not
totally required, may nevertheless participate in the endocytosis
process. A role for ß-arrrestin in facilitating the internalization
of GRK-phosphorylated ß2-adrenergic receptor has further been
demonstrated (18), and ß-arrestin appears to function as a clathrin
adaptor in receptor endocytosis (19). Together, the above evidence
indicates that, although for some receptors desensitization and
internalization are two events that can proceed independently of each
other, phosphorylation of receptors by GRKs and consequent binding of
ß-arrestins may participate in both phenomena.
Internalization of single transmembrane receptors such as those for
transferrin, low-density lipoproteins, insulin, and epidermal growth
factor require a tyrosine residue present in a tight-turn-forming motif
of the sequence NPXY (20, 21). Internalization of other membrane
proteins, such as the T lymphocyte CD3 antigen (22), the IgGFc receptor
(23), or the glucose transporter GLUT4 (24), depends in great part on
the presence of a dileucine internalization motif. For G-coupled
receptors, however, no specific internalization motif has been
described, although mutations of single amino acids may prevent
internalization in some instances. This is the case for tyrosine 326 of
the ß2-AR receptor (14), for three threonine residues of the
cytoplasmic tail of the m3 muscarinic acetylcholine receptor (25), and
for specific lysine residues of the yeast
-pheromone receptor
(26).
The glucagon-like peptide-1 receptor is a G-coupled receptor expressed
by pancreatic ß-cells (27). Binding of GLP-1 activates the adenylyl
cyclase pathway, which ultimately results in the strong potentiation of
glucose-induced insulin secretion (28, 29). We previously described
that homologous and heterologous (PKC-induced) desensitization of the
receptor strictly correlated with receptor phosphorylation in the last
33-amino acid segment of the receptor C tail (30). Furthermore, we
showed that the PKC phosphorylation sites were four serine doublets
present in this segment of the receptor and that serine doublet at
position 431/432 was the major phosphorylation site (31). In this study
we characterize the sites phosphorylated after agonist binding and
demonstrate that these phosphorylation sites are required not only for
homologous desensitization but also for internalization of the
receptor-ligand complex.
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RESULTS
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Binding of GLP-1 to its receptor expressed in insulinomas and
fibroblasts (30) or in COS cells (see below) induces phosphorylation of
the receptor by a PKA- and PKC-independent mechanism. Previous
experiments demonstrated that, in fibroblasts, phosphorylation took
place in the carboxyl-terminal 33-amino acid segment of the receptor
and that this phosphorylation strictly correlated with desensitization
of the cAMP response (30).
Here, to identify the phosphorylated amino acids, we first performed a
phosphoamino acid analysis of the phosphate-labeled receptor expressed
in fibroblasts or COS cells. Figure 1
shows that
phosphorylation of the GLP-1 receptor expressed in both cell types
occurs only on serine residues. To determine which serines were
phosphorylated in the carboxyl-terminal segment of the receptor, we
constructed several deletion and point mutants of the receptor. Figure 2
shows the sequence of the receptor cytoplasmic tail
and of the different mutants tested. Ten serines are present in the
region of the receptor that contains the phosphorylation sites and that
extends from serine 431 to the carboxyl end at position 463. Eight of
these serines are present as four doublets, and two individual serines
are at positions 461 and 463. In a first set of experiments,
carboxyl-tail deletion mutants were transiently expressed in COS cells.
Binding affinity and coupling to production of cAMP were identical for
the truncated mutants and the wild type receptor (not shown). After
radiolabeling with radioactive orthophosphate, the cells were exposed
to GLP-1 for 15 min and lysed, and the receptor was immunoprecipitated
and analyzed by gel electrophoresis. Figure 3A
shows
phosphorylation of the wild type receptor and of deletion mutants
CT451 and
CT444. No phosphorylation of the receptor, however,
could be detected in the
CT441 and
CT431 mutants. This suggests
that phosphorylation takes place at least on the last three serine
doublets but not on the doublet at position 431/2. Identical results
were obtained with the same truncation mutants stably transfected in
fibroblasts (Ref. 30 and not shown), therefore indicating no cell type
differences in receptor phosphorylation.

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Figure 1. Phosphoamino Acid Analysis of the GLP-1 Receptor
Phosphoamino acid analysis was performed on immunoprecipitated and
gel-purified receptors prepared from fibroblasts (leftpanel) or COS cells (right panel) prelabeled for 2 h
with [32P]orthophosphate and exposed for 15 min to 10
nM GLP-1. The left part of each panel shows the
receptor immunoprecipitated from cells exposed (+) or not (-) to
GLP-1 and purified by SDS-gel electrophoresis. The phosphoaminoacid
analysis is on the right part of each panel. The position of
phosphoserine (S), phosphothreonine (T), and phosphotyrosine (Y) is
indicated.
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Figure 2. Sequence of the GLP-1 Receptor C-Tail Mutants
The top line shows the sequence of the wild type
receptor carboxy-terminal cytoplasmic tail (position 412 to 463).
Sequence of the truncated mutants and of the point mutants is indicated
below the wild type sequence; only the mutated amino
acids are shown.
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Figure 3. Phosphorylation of the Receptor Mutants
A, The wild type or truncated mutants of the GLP-1 receptor were
transiently transfected in COS cells. After labeling with
[32P]orthophosphate, the cells were exposed (+) or not
(-) for 15 min to 10 nM GLP-1, and the receptors were
immunoprecipitated and separated on SDS-polyacrylamide gels.
Phosphorylation is observed in the wild type and the CT451 and
CT444 receptor mutant but not in the other two truncation mutants.
B, The wild type receptor or point mutants having a single serine
doublet left intact and the others mutated to alanines were transiently
transfected in COS cells, and phosphorylation after agonist stimulation
was assessed as in panel A). The three distal serine doublets were
phosphorylated but not that present at position 431/432.
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To identify the phosphorylation sites, we next evaluated the
phosphorylation of four mutants in which a single serine doublet was
left intact while the other three were mutated to alanines. These
mutants were then transiently transfected in COS cells, and their
phosphorylation after exposure to GLP-1 was assessed as described
above. Figure 3B
shows that the mutants 451SS, 444SS, and 441SS were
phosphorylated after GLP-1 binding while mutant 431SS was not. These
data thus indicate that the last three serine doublets are
phosphorylated after agonist binding to its receptor. This is in
agreement with the results obtained with the deletion mutants.
Furthermore, because no phosphorylation can be detected in the 431SS
mutant, we conclude that serines at positions 461 and 463, which are
retained in this mutant, are not phosphorylated.
To determine whether phosphorylation was correlated with receptor
homologous desensitization, we established fibroblast cell lines
expressing the wild type or the different mutant receptors under the
control of the metallothionein promoter, as described (31). Figure 4
(left panel) shows that desensitization of
the wild type receptor could be measured by a decrease in the
Vmax for cAMP production by about 50%. In these cells only
a small shift in the EC50 for cAMP production was observed
(from 2 to 5.8 nM). Mutation of all three serine doublets
to alanine generated a receptor in which desensitization was almost
completely suppressed (Vmax =
90% of nondesensitized
receptor) (Fig. 4
, right panel). We similarly tested cell
lines expressing mutant receptors with only a single doublet or two
doublets mutated to alanine and evaluated the Vmax for cAMP
production after desensitization. Figures 5
and 7
show a
summary of the data obtained for all the clones tested. For the wild
type receptor, Vmax was reduced to 58% of the
nondesensitized receptor. With a single doublet mutated to alanine,
Vmax was reduced to 7580% of nondesensitized receptor.
Double and triple mutants showed the same reduction of Vmax
after desensitization to
90% of the nondesensitized receptor. These
data indicate a correlation between the extent of phosphorylation and
the decrease in Vmax after desensitization. Phosphorylation
on at least two serine doublets is required to observe intermediate
desensitization, and maximal desensitization is only obtained when
phosphorylation occurs at all three identified sites.

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Figure 4. Desensitization of the GLP-1 Receptor and of the
Phosphorylation Negative Mutant
The wild type receptor or the 431SS mutant in which the three distal
serine doublets were mutated to alanines were stably transfected in
Chinese hamster lung fibroblasts. The dose-dependent production of cAMP
in response to increases in GLP-1 concentrations was measured in
untreated cells (control) or cells preexposed for 15 min to 10
nM GLP-1. Wild type receptor-expressing cells
(left panel) show a marked reduction in the maximal
production of cAMP after agonist-induced desensitization whereas this
desensitization was considerably reduced in cells expressing the
phosphorylation mutant (right panel).
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Figure 7. Functional Modifications in Receptor
Phosphorylation Site Mutants
The figure shows the sequence of the cytoplasmic tail of the receptor.
The sites phosphorylated in response to GLP-1 binding (homologous
phosphorylation, this study) or in response to PKC activation
[heterologous desensitization (31 )] are indicated by
arrows. The mutated sequences are presented
below the wild type sequence, and the corresponding
modifications of desensitization and internalization are indicated on
the right. Desensitization is expressed as the percent
of maximal cAMP production by desensitized compared with
nondesensitized receptors. Mutations of single serine doublets lead to
intermediate levels of desensitization whereas mutations of two or
three serine doublets almost completely suppress desensitization.
Internalization is expressed as the percent of bound
[125I]GLP-1, which becomes internalized after 5 min of
initiating endocytosis. Mutations of single serine doublets lead to
reduction of internalization, but the effect of the 451AA mutation is
less marked than that of the other mutations. Removal of two serine
doublets leads to a more extensive reduction of internalization.
Complete suppression of internalization, however, requires mutations of
the three doublets. The differential effect of double and triple
mutations on receptor desensitization and internalization suggests that
both mechanisms, although dependent on receptor phosphorylation, may be
regulated by different molecular mechanisms.
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We previously demonstrated that agonist binding induces a fast
(t1/2 = 23 min) internalization of the ligand-receptor
complex (32). We further showed that truncation of the cytoplasmic tail
at position 444 generated a receptor that was no longer internalized
(30). This receptor could, however, still be desensitized after
activation of PKC which phosphorylates the receptor mostly on serines
431/432. These preliminary experiments thus indicated that the sequence
containing the homologous desensitization phosphorylation sites was
required for agonist-induced internalization. As no known
internalization motifs are present in this region, we evaluated the
role of the identified phosphorylation sites in receptor
internalization by measuring the extent of endocytosis of
[125I]GLP-1 bound by the different receptor mutants at 0
and 5 min after the cells were warmed. Preliminary measurement of
internalization kinetics with the wild type (32) and various mutant
receptors indicated that internalization proceeded linearly over this
period of time. Figure 6A
shows the acid wash-resistant
[125I]GLP-1 measured at 0 and 5 min. A nondissociable
component representing about 25% of initial binding is present at time
0 in all clones except in that expressing the 444/451AA mutant for
which this value, for an unknown reason, is 40%. The inset
in Fig. 6A
shows the differences between the acid wash-resistant
[125I]GLP-1 at time 0 and 5 min, which represents the
peptide internalized over this period of time. Internalization is
maximal for the wild type receptor; it is reduced when any single
phosphorylation site is mutated to alanines; it is further decreased
when two sites are mutated and is suppressed in the absence of
phosphorylation sites (see Fig. 7
). It appears, however,
that mutation of position 451/452 has a smaller effect in reducing
internalization than mutation of the two other sites. Also, mutation of
the three sites has a distinctly more pronounced effect on blocking
internalization as compared with the double mutant. These two features
indicate different correlation between phosphorylation and
internalization and phosphorylation and desensitization. Figure 6B
shows, by western blot analysis, the redistribution of the receptors to
light density vesicles (endosome-enriched) after exposure of the cells
to GLP-1. Whereas the wild type receptor content of light density
vesicles is increased and that of the heavy density vesicles (plasma
membrane-containing) is decreased upon exposure of the cells to GLP-1,
no such redistribution can be seen with the 431 SS mutant. This shows,
in an independent manner, that the suppression of the three
phosphorylation sites indeed prevents agonist-induced receptor
internalization.

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Figure 6. Internalization of Wild Type and Mutant Receptors
A, [125I]GLP-1 was bound to fibroblasts expressing the
wild type or the mutant receptors for 4 h at 4 C. The cells were
then placed at 37 C for 0 or 5 min. Acid wash-resistant radioactivity
was measured and expressed as percent of total initial specific
binding. For all receptor forms, the results presented are mean ±
SEM of three experiments each performed in triplicate. The
inset shows the differences in acid wash-resistant
[125I]GLP-1 at 0 and 5 min and represents the amount of
peptide internalized over this period of time by each mutant.
Suppression of one phosphorylation site reduces the rate of
internalization. This rate is further reduced in double mutants and
suppressed in the triple mutant. Mutation of doublet 451/452 (451AA)
appears to have a lower effect in reducing internalization as compared
with the other phosphorylation sites. B, Internalization of the wild
type or 431SS receptors was measured after exposure of the
receptor-expressing cells to 10 nM GLP-1 for 15 min.
Endocytosis was assessed as the redistribution of the mature form of
the receptor from a plasma membrane-enriched (H) to an
endosome-enriched (L) vesicle fraction obtained by sucrose density
fractionation of cell homogenates prepared before (0) or after (15 )
addition of the peptide to the cells for 15 min. Upper
and lower arrows point to the mature and
core-glycosylated forms of the receptor, respectively. In wild type
receptor-expressing cells, GLP-1 binding induces an increase in
receptor in the endosomal compartment and a decrease from the plasma
membrane-enriched fraction. No such effect could be observed with the
phosphorylation-negative receptor (431SS). The core-glycosylated,
intracellular form of the receptor is not affected by GLP-1 binding.
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DISCUSSION
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In the present study we identified the sites phosphorylated after
GLP-1 binding to its receptor as being three serine doublets present in
the last segment of the receptor C tail. We showed that phosphorylation
of the receptor was responsible for most of the observed homologous
desensitization. The same three identified phosphorylation sites were
also required to control receptor endocytosis. However, studies with
phosphorylation site mutants suggest that desensitization and
internalization may be controlled by different molecular mechanisms.
These data are summarized in Fig. 7
.
Binding of GLP-1 to its receptor leads to activation of adenylyl
cyclase and the production of cAMP. This signaling pathway can be
attenuated by homologous desensitization but also after activation of
PKC (heterologous desensitization). We previously showed that both
forms of desensitization were additive in increasing the
EC50 and decreasing the Vmax for cAMP
production (30). Phosphorylation of the receptor induced by homologous
and heterologous desensitization was also additive, suggesting that
different amino acids were phosphorylated. PKC-induced desensitization
of the receptor involved phosphorylation on the four serine doublets
present in the last 32 amino acids of the receptor C tail (31). Here we
provide evidence that the last three serine doublets of the C tail are
the sites phosphorylated upon agonist binding. These sites thus overlap
those phosphorylated by PKC activation. As these sites are doublets of
serines, the additivity of the phosphorylation obtained in homologous
and heterologous desensitization may be explained by phosphorylation on
different serine residues at each of the doublets located at positions
441/442, 444/445, and 451/452 and by phosphorylation of doublet 431/432
uniquely by PKC. This is further supported by the fact that PKC induces
only a marginal internalization of the receptor (30) compared with that
induced by agonist binding, which requires receptor phosphorylation on
the distal three sites. We have previously shown that neither PKC nor
PKA were responsible for phosphorylation of the receptor after agonist
binding (30). Receptor-specific kinases of the GRK family may thus be
involved in this process. Studies in progress will indicate which, if
any, of the so far identified GRKs participate in this phosphorylation
process.
Implication of receptor phosphorylation in homologous desensitization
was studied after transfection of the wild type receptor or of mutants
thereof in fibroblasts using an expression vector containing the
metallothionein promoter. This promoter permits expression of the
receptor at a low level (20004000 receptors per cell), comparable to
the expression of the endogenous receptor in different insulinoma cell
lines (27). This is essential to perform desensitization experiments.
Indeed, in the presence of high surface expression, as obtained in COS
cells, the basal production of cAMP after a first stimulation of the
cells with GLP-1 is too high and does not permit generation of a
significant dose-response curve in a second exposure to the peptide.
With the presently used fibroblast cell lines, we could study the
effect of mutating the three phosphorylation sites together or in
different combinations. Our results indicated that desensitization
could be mostly, but not completely, suppressed by mutating the three
sites. This indicates that phosphorylation plays a major role in
inducing homologous desensitization but also suggests that additional
modifications may also participate in the observed desensitization.
These results are comparable to those obtained with the
CT431
receptor (30), which is not phosphorylated but still partially
desensitized. Interestingly, however, we observed that the extent of
desensitization was dependent on the number of phosphorylation sites
present. When one site was removed, desensitization was intermediate,
and when two sites were mutated desensitization was as impaired as with
the triple mutant. Homologous desensitization is thought to be mediated
by ß-arrestin binding to phosphorylated receptors. Although the exact
mechanism for GLP-1 receptor desensitization is not known, our data
suggest that phosphorylation at multiple, adjacent sites is required to
generate high affinity binding sites for ß-arrestins.
Internalization of receptor-ligand complexes is an essential aspect of
the function of G protein-coupled receptors. It is required for
dissociation of the ligand from its receptor but also for
resensitization of the receptor (15, 33), probably by dephosphorylation
of the receptor by phosphatases encountered in the transit through the
endosomal compartment. Signals for internalization of this class of
receptors are not well defined. In the present study, we showed, using
point mutants, that removing the three phosphorylation sites led to a
complete suppression of receptor internalization. Mutation of one or
two sites, however, led to rates of internalization that were
intermediate between that of the wild type receptor and that of the
triple mutant. The different phosphorylation sites, however, appear to
contribute differentially to receptor endocytosis. This is especially
noticeable for the site at position 451/452, which has a significantly
smaller effect in reducing the internalization rate than mutation of
the other sites (see Fig. 7
). Altogether, these data show that there is
thus a strict correlation between internalization and phosphorylation.
However, this correlation is qualitatively different from that observed
between phosphorylation and desensitization. This is evident especially
for the double and triple mutants. Whereas these two classes of mutants
are equally resistant to desensitization, there is a marked difference
in the ability of the double mutants to be internalized, at a reduced
but still significant rate, compared with the triple mutant, which is
not internalized at all. Also, mutation of site 451/452 has a much
lower effect in reducing internalization compared with mutation of
either of the two other phosphorylation sites. No such differential
effect on desensitization can be observed with the single mutants.
Together, these data indicate that phosphorylation of the receptor at
the identified sites by a kinase activated after agonist binding is
responsible for two events: homologous desensitization and receptor
internalization. The exact contribution of each phosphorylation site to
both mechanisms appears to be, at least in part, distinct, suggesting
that the molecular basis for the control of receptor desensitization
and endocytosis is different.
In pancreatic ß-cells, glucose-induced insulin secretion can be
potentiated by activation of receptors linked to the adenylyl cyclase
or the phospholipase C pathways. Activation of muscarinic receptors by
carbachol strongly stimulates the insulin-secretory response but, at
the same time, it induces a strong desensitization of the GLP-1
receptor (C. Widmann and B. Thorens, unpublished observations). By its
poor ability to induce receptor internalization, PKC may desensitize
the receptor for a longer period of time compared with homologous
desensitization if internalization is primarily required for receptor
resensitization. This may be of functional significance in the
integration by ß-cells of different signals modulating insulin
secretion, in particular in the postprandial state when cholinergic and
gluco-incretin (GLP-1, glucose-dependent insulinotropic polypeptide)
signals converge to the ß-cells to stimulate their secretory
activity.
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MATERIALS AND METHODS
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Cells and Cell Culture
COS cells and Chinese Hamster Lung (CHL) fibroblasts were
cultured as described (34). Clone 5 is a CHL fibroblast cell line
stably transfected with the rat GLP-1 receptor cDNA (32, 34).
Transformation of COS cells and generation of stable CHL transformants
were performed as described earlier (34). The number of cell
surface-expressed receptors in stably transfected fibroblasts
expressing the wild type or mutant forms of the receptor has been
established by saturation binding experiments (20004000 receptors per
cell) and was published previously (31).
Mutagenesis
The different GLP-1 receptor mutants used in this study are
described in Fig. 2
. The deletion mutant
CT431 mutant was described
previously (30). The other mutations were generated by PCR
amplification, as described (31, 35), and each mutant was verified by
DNA sequencing. The mutant GLP-1 receptor cDNAs were subcloned in the
pcDNA-3 vector (Invitrogen, Leek, The Netherlands) and in the pmlMTIi
vector (30). The cDNAs subcloned in the pcDNA-3 and pmlMTIi vectors are
under the control of the cytomegalovirus and metallothionein promoters,
respectively.
Desensitization of the GLP-1-Induced cAMP Production
Desensitization was assessed as described earlier (30), by
comparing the dose-response curves of cAMP production as a function of
increasing GLP-1 concentrations for control cells and cells pretreated
for 15 min with 10 nM GLP-1 at 37 C.
Phosphorylation and Phosphoamino Acid Analysis
GLP-1-induced phosphorylation of the wild type or mutant GLP-1
receptors expressed transiently in COS cells was assessed as described
earlier (30). Briefly, COS cells were transiently transfected with the
different receptor cDNA constructs by the diethylaminoethyl-dextran
technique. For radiolabeling, cells were incubated for 23 h in the
presence of 500 µCi/ml of [32P]orthophosphate. GLP-1
(10 nM) was then added to the cells for 15 min, and the
cells were quickly washed in ice-cold PBS (8 g/liter NaCl, 0.2 g/liter
KCl, 1.44 g/liter Na2HPO4·2 H2O,
0.2 g/liter KH2PO4, pH 7.4). The cells were
detached from the culture dishes after a 10-min incubation with 1.52
ml of PBS, 1 mM EDTA and collected in an Eppendorf tube.
The cells were lysed in PBS containing 1% Triton X-100, 5
mM EDTA, 1 mM N-ethylmaleimide, 2
mM phenylmethylsulfonyl fluoride, 25 mM NaF,
and 1 mM NaVO4 for 10 min at 4 C.
Immunoprecipitation of the receptor and analysis by gel electrophoresis
were then performed exactly as described (31).
Phosphoamino acid analysis of the gel-purified receptor was performed
by TLC using a HTLE 7000 electrophoresis system (C.B.S. Scientific
Company, Del Mar, CA) according to the manufacturers protocol and
Copper et al. (36).
Internalization of Receptor-Ligand Complex
Measurements of ligand-receptor internalization were performed
as previously described (32). Briefly, for evaluation of
[125I]GLP-1 endocytosis, the radiolabeled peptide (400
pM) was first bound at 4 C for 46 h, after which the
cells were washed with ice-cold HBSS containing 20 mM
HEPES, pH 7.4, and returned to 37 C for the indicated periods of time.
The cells were then washed with the HBSS buffer and lysed with 0.2
N NaOH/1% SDS, and the radioactivity was counted (total
binding). Alternatively, a duplicate set of cells was washed and
incubated for 2 min in an acidic solution (50 mM glycine,
150 mM NaCl, pH 3) to remove surface-bound radioactivity.
The cell-associated radioactivity that remained was considered to
represent the internalized peptide. A fraction of surface-bound
[125I]GLP-1 could not be removed at pH 3 before
incubation at 37 C (
30%).
Analysis of GLP-1 receptor redistribution to endosomal compartments
after GLP-1 binding was studied exactly as described (32). Briefly,
cells expressing the wild type receptor or different mutants were
exposed for 15 min to GLP-1 at 37 C. The cells were then treated with
500 µg/ml Concanavalin A and lysed in a hypotonic lysis buffer (1
mM Tris-HCl, pH 7.4, 2 mM EDTA). The cells were
then scraped with a rubber policeman, and the total cell lysate was
loaded on a discontinuous sucrose gradient consisting of 4 ml 60%
sucrose, 4 ml 38% sucrose, and 4 ml 15% sucrose all made up in 20
mM Tris-HCl, pH 7.4. After centrifugation at 112,000
x g for 1 h at 2 C in a Beckman SW40 Ti rotor, the
membrane fractions at the 2538% sucrose interface (endosomal
fraction) and 3860% sucrose interface (plasma membrane fraction)
were collected and analyzed for the presence of the receptor by Western
blot analysis using receptor-specific antibodies (32).
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FOOTNOTES
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Address requests for reprints to: Bernard Thorens, Insitute of Pharmacology and Toxicology, University of Lausanne, 27, rue du Bugnon, 1005 Lausanne, Switzerland.
B.T. was supported by a Career Development Award from the Swiss
National Science Foundation. This work was supported by Grant
3130313-90 from the Swiss National Science Foundation.
1 Present address: National Jewish Center for Immunology and Respiratory
Medicine, Pediatrics Department, 1400 Jackson Street, Denver, Colorado
80206. 
Received for publication January 14, 1997.
Revision received March 11, 1997.
Accepted for publication April 16, 1997.
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