N-Glycosylation of the Prolactin Receptor Is Not Required for Activation of Gene Transcription but Is Crucial for Its Cell Surface Targeting
Hélène Buteau,
Alain Pezet,
Fatima Ferrag,
Martine Perrot-Applanat,
Paul A. Kelly and
Marc Edery
INSERM U344: Endocrinologie Moléculaire (H.B., A.P., F.F.,
P.A.K., M.E.) Faculté de Médecine Necker 75730
Paris, France
INSERM U460: Remodelage Vasculaire (M.P-A.)
CHU Xavier Bichat 75870 Paris, France
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ABSTRACT
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The functional importance of the three
oligosaccharide chains linked to Asn35,
Asn80 and Asn108, of
the long form of the PRL receptor (PRLR) was investigated by individual
or multiple substitutions of asparagyl residues using site-directed
mutagenesis and transient transfection of these mutated forms of PRLR
in monkey kidney cells, Chinese hamster ovary, and human 293 fibroblast
cells that exhibit different levels of protein expression. Scatchard
analysis performed on monkey kidney cells revealed that the mutants
possess the same affinity for PRL as compared with wild-type PRLR. A
strong reduction (90%) of the aglycosylated PRLR expression at the
cell surface of monkey kidney or human 293 cells was observed.
Immunohistochemistry experiments using an anti-PRLR monoclonal antibody
showed an accumulation of the deglycosylated receptor in the Golgi area
of transfected monkey kidney cells. Upon PRL stimulation, the
aglycosylated PRLR associated with Janus kinase 2 was phosphorylated
and was able to activate a ß-casein gene promoter in transfected 293
fibroblast cells. The active form of the PRLR was thus acquired
independently of glycosylation. By contrast, no functional activity was
detectable in transfected Chinese hamster ovary cells that expressed
low levels of PRLR. These studies demonstrate that the glycosylation on
the asparagyl residues of the extracellular domain of the PRLR is
crucial for its cell surface localization and may affect signal
transduction, depending on the cell line.
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INTRODUCTION
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PRL is involved in many biological functions in all
vertebrates (1), but its primary effect is on mammary gland development
and milk protein synthesis (2). Multiple forms of PRL receptor (PRLR)
cDNAs have been cloned in different tissues and species (3, 4, 5, 6) that
differ in the sequence and length of their cytoplasmic domains (7). The
PRLR belongs to the cytokine/hematopoietic receptor family defined on
the basis of several conserved features within extracellular, as well
as the cytoplasmic domains (8, 9). PRL binding to its receptor
activates the tyrosine Janus kinase 2 (JAK2), which has been shown to
phosphorylate the latent cytoplasmic transcription factor STAT (signal
transducer and activator of transcription) 5, required for the
transcriptional activation of milk protein genes (10, 11).
Previous studies have shown a differential contribution of the
carbohydrate groups of several cytokine receptors on their cellular
distribution, ligand binding, and functional activity. The
extracellular domain of the PRLR contains three N-linked glycosylation
sites (3, 12) defined by the presence of the consensus sequence
Asn-X-Ser/Thr-X, where X is any amino acid except a proline (13). First
reports demonstrated that deglycosylation by enzymatic treatment of the
short form of PRLR does not impair the PRL-binding capacity (14);
similarly, PRLRs presenting individual mutations of the three
asparagine (Asn) residues are correctly expressed at the cell surface
and exhibit a normal affinity (15). Using glycosylation blockers, it
has been shown that the glycosidic core of the PRLR appears necessary
to achieve an active conformation of the protein (14); however,
interaction of these inhibitors with cellular metabolism at levels
other than the glycosylation process is not excluded. Indeed,
tunicamycin has been shown to activate an endoplasmic reticulum stress
pathway, leading to induction of specific proteins, as CCAAT/enhancer
binding protein homologous proteins (16), or chaperones that are known
to influence the folding and/or trafficking of proteins. However, the
functional activity of deglycosylated PRLR has not been investigated.
Since specific signals emerging from the extracellular domain of
cytokine receptors have been demonstrated to modulate signaling (17, 18), the influence of N-glycosylation of the PRLR on signal
transduction is thus of potential interest.
To assess the functional importance of glycosylation of the PRLR,
different combinations of mutations were performed affecting the three
N-glycosylation sites of the long form of PRLR: Asn35,
Asn80, and Asn108. These mutated PRLRs were
expressed in several fibroblast cell lines (COS-7, 293, and CHO), and
their trafficking, ligand-binding capacity, and functional activity
were analyzed. Our results demonstrate that the carbohydrate groups are
not essential per se for functional activity, since the
aglycosylated PRLR, once partially addressed to the 293 cell membrane,
activates transcription of the ß-casein gene. The cell surface
targeting of the aglycosylated PRLR appears, however, to be
cell-dependent and may affect its biological activity.
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RESULTS
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Contribution of Carbohydrate Groups to the Molecular Mass of the
PRLR
Monkey kidney cells (COS-7) expressing wild-type (WT) PRLR and
double and triple mutants (described in Fig. 1
) were incubated with iodinated human GH
(hGH) in the presence of disuccinyl suberate (DSS), and cross-linked
complexes were analyzed by SDS-PAGE. A specifically radiolabeled band
with a molecular mass of 117 kDa was apparent for the WT PRLR (Fig. 2
); N35,80,108D and N80,108D receptors
migrated as smaller specific bands of apparent molecular mass of 109
and 113 kDa, respectively. Excess of unlabeled ovine PRL (oPRL) was
able to compete with the [125I]hGH binding of all
mutants. After the molecular mass of the complex was corrected by
subtracting the mass of the hGH (22 kDa), the molecular mass for WT
PRLR, N80,108D, and N35,80,108D receptors was determined to be,
respectively, 95, 91, and 87 kDa. These results suggest that each of
the three N-linked glycosylation sites of the PRLR undergoes
glycosylation in COS-7 cells and that
8 kDa of N-glycosylation
contributes to the molecular mass of the PRLR, as previously described
(15). Four kilodaltons of glycosylation occur at Asn35 (difference
between the molecular mass of the WT PRLR and the N80,108D mutant), and
2 kDa for each of the two other sites as shown by individual mutations
(see below in Fig. 6A
).

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Figure 1. The Long Form of the WT PRLR and Glycosylation-Site
Mutated PRLRs
A, Schematic representation of the long form of the rat PRLR: the
transmembrane domain is represented by a black box;
numbers to the left indicate the first and last amino
acids of the mature protein. Positions of the three potential N-linked
glycosylation sites are indicated to the right. B,
Single, double, and triple mutants of the long form of the rat PRLR.
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Figure 2. Cross-Linking of [125I]hGH with
Mutated PRLRs
COS-7 cells expressing WT or mutant PRLRs were incubated with
[125I]hGH in the absence (-) or presence of excess of
unlabeled oPRL (2.5 µg or 5 µg). The cross-linking agent DSS (0.5
mM) was added to the incubation medium; the reaction was
stopped by addition of 50 mM of Tris-HCl, pH 7.4. Cells
were solubilized in 2x Laemmli sample buffer and cross-linked
receptors analyzed by a 7.5% SDS-PAGE. Molecular mass standards are
indicated to the left.
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Figure 6. Tyrosine Phosphorylation of JAK2 by N35D, N80D, and
N108D Mutants in Response to PRL
Cells were cotransfected with the cDNA encoding JAK2 and the native or
mutated forms of PRLR and stimulated with biotinylated oPRL (+) or
nonlactogenic biotinylated bGH (-). A, Expression of mutant forms of
PRLR. Cell lysates were processed as described in Materials and
Methods, and membranes were immunoblotted with the monoclonal
U5 antibody. B, JAK2 association with the different mutant forms of
PRLR. The membrane described in panel A was stripped and reprobed with
an anti-JAK2 antibody. C, Tyrosine phosphorylation of the kinase and
the receptors. Immunoblotting was performed with an
anti-phosphotyrosine antibody. Positions of the JAK2 and WT PRLR are
indicated to the right.
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Mutation of All Glycosylation Sites Alters Cell Surface Expression
of the PRLR, But Not Affinity of the Receptor for PRL
To determine the influence of carbohydrate groups on cellular
expression of the PRLR, transiently transfected COS-7 expressing WT or
mutant PRLR forms were assessed for cell surface binding at 4 C with
[125I]hGH. As shown in Fig. 3
, mutants N35D, N80D, N108D, and
N80,108D showed a similar level of binding to that of the WT PRLR
(100% activity of the native receptor corresponds to
20% specific
binding of the ligand), which suggests that about the same number of
receptors are expressed at the cell surface. In contrast, a 90%
decrease in binding of the N35,80,108D mutant was observed at 4 C. To
determine whether this altered binding was due to a modification of
binding affinity, competition studies of oPRL binding were performed in
COS-7 cells expressing the mutated forms of the PRLR. Experiments were
performed at 20 C to access to the intracellular pool of receptors.
Table 1
shows that the mutants exhibited
the same relative affinity for oPRL compared with the wild-type PRLR
[association constant (Ka) = 3.82
nM-1]; this constant was similar to the
affinity constant deduced from Scatchard analysis performed on
microsomal preparations from COS-7 or 293 cells (data not shown).
Similar levels of ligand-binding sites were detected in cells
expressing WT PRLR and single and double mutants (10,00020,000 sites
per cell); the weaker (but not significantly different) number of
N35,80,108D receptors can be explained by variations of the level of
expression in transiently transfected cells. Taken together, these
results indicate that the aglycosylated PRLRs appear to be normally
expressed in the cell and exhibit a high-affinity binding site similar
to the WT receptor. The altered cell surface binding observed at 4 C
may thus reflect a defect in the transfer of the aglycosylated receptor
to the cell surface.

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Figure 3. Reduction of Cell Surface Transport of the
Aglycosylated PRLR
COS-7 cells transiently expressing the different PRLR mutants were
incubated with [125I]hGH overnight at 4 C in the presence
or absence of an excess of unlabeled oPRL. Specific binding was
determined as the difference between total and nonspecific binding.
Similar transfection efficiencies were obtained with the three
different forms. Maximal specific binding was obtained with WT PRLR
(100%): the percentage of activity of the mutant PRLRs was calcutated
from this data. Values represent the mean ± SEM of
three independent experiments.
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Table 1. Glycosylation Site Mutated PRLRs Exhibit
High-Affinity Binding Sites for PRL Similar to the Wild-Type
PRLR
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Intracellular Localization of the Aglycosylated PRLR
To test the hypothesis of intracellular retention of the triple
mutant, the cellular distributions of WT or mutated forms of PRLR were
analyzed in COS-7 cells transfected with PRLR cDNAs by indirect
immunofluorescence study using the specific mouse monoclonal antibody
U5 directed against the extracellular domain of the PRLR. In
nonpermeabilized cells, the U5 antibody revealed the presence of WT
PRLR at the cell surface; strong immunostaining was observed both at
the cell surface and within the cytoplasm (rough endoplasmic reticulum,
Golgi apparatus, and vesicles) when cells were permeabilized (Fig. 4
, a and d), confirming previous results
(19). The localization at the plasma membrane level was also observed
in nonpermeabilized cells with the N80,108D receptor mutant, whereas in
permeabilized cells, immunostaining was observed in the endoplasmic
reticulum, with a partial accumulation in the juxtanuclear area
depending on the cell and in vesicles (Fig. 4
, b and e).
Immunofluorescence results for N35D, N80D, and N108D receptors showed
localization (data not shown) and cell surface binding similar to that
of the WT and N80,108D PRLRs. In contrast, in COS-7 cells expressing
the N35,80,108D receptor, we detected immunostaining only in the
juxtanuclear area (Fig. 4f
) without any significant immunostaining at
the level of the plasma membrane (Fig. 4c
). This staining was lower
than that observed for the WT PRLR or single mutant receptor,
suggesting a lower level of protein expressed. It also suggests that
this receptor is located in or close to the Golgi compartment. Similar
results were obtained in 293 cells transfected with this mutant,
especially the absence of immunofluorescence in the cell surface of
nonpermeabilized transfected cells (Fig. 4i
). Immunostaining at the
cell surface observed in cells transfected with the WT receptor (Fig. 4g
) or the N80,108D receptor (Fig. 4h
) confirms results obtained with
the COS-7 cell line.

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Figure 4. Perinuclear Localization of the Aglycosylated PRLR
COS-7 cells transfected with WT (a and d), N80,108D (b and e), or
N35,80,108D (c and f) PRLR cDNAs were fixed in 4% paraformaldehyde (a,
b, and c: nonpermeabilized conditions) or in methanol, -20 C (d, e,
and f: permeabilized conditions). Mouse monoclonal antibody U5 (160
µg/ml) followed by FITC goat anti-IgG (dilution 1:40) were used, as
described in Materials and Methods. Note the
immunostaining in the juxtanuclear area of the aglycosylated PRLR in
permeabilized cells (f) and the absence of expression at the plasma
membrane level in nonpermeabilized cells (c). 293 cells transfected
with WT (g), N80,108D (h), or N35,80,108D (i) PRLR cDNAs were processed
for fixation (4% paraformaldehyde) and immunofluorescence staining, as
described for COS-7 cells. Note the absence of immunostaining at the
cell surface and low perinuclear staining of the aglycosylated receptor
in nonpermeabilized conditions (i). Magnification, x400.
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To pinpoint the intracellular localization of the aglycosylated PRLR,
colocalization experiments within the same cells using the monoclonal
anti-PRLR antibody (U5) and the polyclonal anti-rab6 (directed against
an ubiquitous ras-like GTPase involved in intra-Golgi transport) were
performed and confirm a mainly Golgi compartmentalization of the
N35,80,108D receptor: indeed, similar staining patterns were observed
with the triple mutant and the Golgi rab6 (Fig. 5
, b and c). In some cells, staining was
also visible in the rough endoplasmic reticulum (Fig. 5d
).
Immunofluorescence experiment using antibody directed against the
protein disulfide isomerase [PDI, a luminal rough endoplasmic
reticulum foldase (21)] was performed in parallel on COS-7 cells (Fig. 5f
): the pattern of endoplasmic immunostaining markedly differs from
the perinuclear staining pattern observed for Golgi protein (Fig. 5
, c
and e) or the triple mutant (Fig. 5a
).

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Figure 5. Deglycosylated PRLR Colocalizes with the Golgi rab6
in COS-7 Cells
a, Labeling of fixed permeabilized COS-7 cells transfected with
N35,80,108D receptor using U5 antibody. Panels b and c and panels d and
e show a double- labeling experiment of fixed permeabilized COS-7 cells
transfected with the N35,80,108D mutant, with anti-PRLR antibody
[visualized with a secondary anti-mouse FITC secondary antibody (b and
d)], and anti-Golgi antibody [anti-rab6 visualized with biotinylated
secondary anti-rabbit IgG followed by Texas-Red-conjugated streptavidin
(c and e)]. The choice of mouse anti-PRLR and rabbit anti-Golgi IgG as
primary antibodies allows accurate double-labeling experiments. Panels
b-c and d-e show two different COS-7 cells. f, Micrographs of COS-7
cells stained for anti-protein disulfide isomerase (endoplasmic
reticulum protein) antibody, visualized with Texas-Red-conjugated
anti-mouse secondary antibody. Magnification, x400.
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The level of expression, as determined by binding studies, and the
cellular distribution, as assessed by immunofluorescence studies of the
different forms of the receptors expressed in COS-7 and 293 cells, are
summarized in Table 2
.
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Table 2. Relationship between the Level of Expression
of Mutated Receptors and Their Cellular Distribution in COS-7 and 293
Cell Lines
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The Aglycosylated PRLR Activates JAK2 and Is Tyrosine
Phosphorylated in Response to PRL
The tyrosine phosphorylation status of the mutants and of JAK2
kinase was assessed in 293 fibroblast cells, which allow a high level
of expression of transfected cDNA. Biotinylated PRL was used to
stimulate the cells and specifically precipitate cell surface
receptors. Specific bands corresponding to mutated PRLRs were detected
using the monoclonal U5 antibody (Figs. 6A
and 7A
). After stripping the membrane
and reprobing with anti-JAK2 antibody, JAK2 (130 kDa) was shown to
associate with all forms of PRLR (Figs. 6B
and 7B
). In response to PRL,
all receptors are phosphorylated and JAK2 kinase is activated. The
intensity of phosphorylation of both N80,108D receptor and associated
JAK2 was similar to the WT PRLR (Fig. 7C
). However, we observed reduced amounts
of N35,80,108D receptor (Fig. 7A
) and JAK2 protein (Fig. 7B
). These
results may reflect a lower expression of the aglycosylated receptor
and, consequently, lower activation of the kinase. Thus, aglycosylated
PRLR retains active conformation for its phosphorylation and
association with JAK2, but its intracellular retention can indirectly
prevent coupling of the receptor with JAK2 kinase.

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Figure 7. Tyrosine Phosphorylation of JAK2 by the N80,108D
and N35,80,108D Mutants
Cells were cotransfected with the cDNA encoding JAK2 and the different
forms of PRLR and then stimulated with biotinylated oPRL (+) or
nonlactogenic biotinylated bGH (-). PRLR complexes were purified as
described in the legend to Fig. 6 , and the membrane was immunoblotted
with U5 (A), anti-JAK2 (B), and anti-phosphotyrosine (C) antibodies,
respectively. Position and molecular mass of the JAK2 and WT PRLR are
indicated to the right.
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Aglycosylated PRLR Triggers Specific Transcriptional Activation of
the ß-Casein Gene Promoter in 293 Cells
Transcriptional activity of mutants using the PRL-inducible
ß-casein gene promoter (21) was compared with that of WT PRLR. All
mutants were able to trigger PRL-dependent transcriptional induction of
ß-casein promoter (8090% of the activity of the WT receptor) in
Chinese hamster ovary cells (CHO), except for the aglycosylated PRLR,
which was devoid of any activity (Fig. 8A
). Since this receptor was shown to be
phosphorylated and could associate with the JAK2 protein in 293
transfected cells, this suggested that the aglycosylated receptor could
be functional in this cell line. This mutant was thus expressed in 293
cells to test its functional activity; a significant activity (40% of
the WT activity) was observed when cells were transfected with 0.1 µg
of N35,80,108D mutant cDNA (Fig. 8B
); the overexpression of this
receptor (0.5 µg) permitted an almost complete rescue of activity.
Our hypothesis is that even though only a small fraction of
aglycosylated PRLRs are transported to the cell surface of 293 cells,
these receptors are able to mediate the milk protein gene
transcription, while the absence of detectable transcriptional activity
in CHO cells could be due to the inhibition of the transport of the
triple mutant, resulting in the almost complete absence of receptors at
the cell surface, as compared with 293 cells. The cell surface
targeting of the aglycosylated receptor is thus differently regulated
in these two cell lines and may influence its further signaling.

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Figure 8. Aglycosylated PRLR Specifically Triggers the
Transcription of a ß-Casein Gene Reporter When Overexpressed in 293
Cells
A, CHO cells were transiently cotransfected with 1 µg WT or mutated
PRLR cDNAs, 0.5 µg of the ßCas-luc reporter construct, and 1 µg
of the ß-galactosidase plasmid and stimulated 48 h with 20
nM oPRL and 250 nM dexamethasone. Supernatants
of lysed cells were then assayed for luciferase and ß-galactosidase
activities. Maximal activity was obtained with WT PRLR (100%
corresponds to a 8.7-fold induction): activities of the mutant PRLRs
were calculated from these data. Values are mean ±
SEM of five independent experiments. B, 293 cells were
transiently cotransfected with 0.1 or 0.5 µg of N35,80,108D or WT
PRLR cDNA, 0.1 µg of ßCas-luc, and 0.5 µg of ß-galactosidase
expression plasmids. The activity of the mutant was calculated by
comparison with WT PRLR (100% activity corresponds to a 7.7-fold
induction). Values are mean ± SEM of three
independent experiments.
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DISCUSSION
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In the present study, the use of site-directed mutagenesis of the
PRLR has permitted us to determine the contribution of the glycosidic
chains to the cell membrane expression, ligand binding, and
transcriptional activity of the receptor. Disadvantages associated with
chemical or enzymatic deglycosylation, such as incomplete removal of
the oligosaccharides or perturbation of the cell metabolism, were thus
avoided.
Hormone Binding of Glycosylation Site Mutated PRLRs
Our cross-linking studies suggest that the three Asn-linked
carbohydrate moieties contributed 8 kDa of glycosylation to the total
molecular mass of the long form of PRLR. If the theoretical molecular
mass of the nonglycosylated PRLR is considered to be
67 kDa, the
molecular mass of the N-glycosylated form should be 75 kDa. The
difference in the molecular mass between the predicted (75 kDa) and
apparent (95 kDa) glycosylated form observed on SDS gel may be due to
the presence of ubiquitin chains, as it was recently described for the
rabbit PRLR (22) or to other posttranscriptional modifications such as
phosphorylation. Our results suggest a contribution of
4 kDa of
the Asn35-linked glycosylation chain, and
2 kDa,
respectively, for Asn80 and Asn108-linked
glycosylation chains to the molecular mass of the long form of the
PRLR, in agreement with previous results obtained with the short form
of PRLR (15). The glycosidic core of the receptor is in fact composed
of similar tri- or tetra-antennary complex carbohydrates, with terminal
sialic acid residues (14): the higher molecular mass of glycosylated
chain linked to Asn35 compared with those linked to
Asn80 or Asn108 can be attributed to a
different number of antennae.
Our results showing that aglycosylated PRLR binds ligand with a high
affinity, similar to that of the glycosylated form (Ka =
1.8 nM-1) indicate that the binding of the
hormone to these receptors does not depend on the glycosylation. This
is in agreement with Lascols (24), who showed that deglycosylation of
mature mouse PRLR did not affect its ligand-binding properties. On the
other hand, deglycosylated hepatic rat receptor resulting from
tunicamycin treatment is not able to bind PRL (14). In fact,
tunicamycin may act at other levels, more important for cellular
processing than glycosylation, and thus may indirectly modify other
processes, such as trafficking of the receptor. This result may also be
explained by a specific process in liver cells. Nevertheless, the
site-directed mutagenesis approach used in this study as well as
previous results indicate clearly that Asn-linked glycosylation of
PRLR is not implicated in the binding of the hormone to the PRLR.
Cellular Expression of N-Glycosylation Site Mutated PRLRs
Cell surface PRL binding experiments showed that removal of all
N-glycosylation sites from the PRLR resulted in alteration in its
distribution, since low levels of aglycosylated PRLR are present at the
cell surface; the majority of this receptor accumulates instead in the
perinuclear Golgi-like compartment, as shown by indirect
immunofluorescence studies. Presence of vesicles are never observed in
cells transfected with this mutant using immunofluorescence, but this
does not exclude that a small undetectable fraction is targeted to the
cell surface and reinternalized after which it accumulates in the Golgi
compartment.
Although the translocation of the PRLR from the endoplasmic reticulum
to the Golgi apparatus occurs in the absence of N-glycosylation,
further transport to the cell membrane seems to depend essentially on
the presence of carbohydrate groups on the receptor. Several studies
using N-glycosylation inhibitors, such as tunicamycin, have also
demonstrated that deglycosylated receptors cannot be transported to the
cell surface and accumulate in the endoplasmic reticulum and pre-Golgi
compartment (14). This selective retention is accounted for by a
quality control mechanism present in the endoplasmic reticulum (24), by
which the transport of incompletely folded or misassembled proteins
into the secretory pathway is inhibited; these defective proteins
retained in the endoplasmic reticulum or in other early compartments of
the secretory pathway accumulate and aggregate and are usually degraded
(25, 26). Absence of glycosylation may thus result in protein
alteration at the level of folding of the PRLR in the endoplasmic
reticulum, which is probably further targeted to a degradation process,
as suggested by the lower cytoplasmic immunostaining observed with the
aglycosylated PRLR. The glycosylated chains of the receptor may
interact with specific chaperone proteins, promoting correct folding of
the receptor for its cell membrane targeting. Inhibition of such
association may lead to an unstable protein that may be targeted to a
degradation process. On the other hand, it cannot be excluded that the
substitutions of Asn residues result in some modifications of the
three- dimensional structure of the aglycosylated receptor that
consequently prevent its surface expression. Thus, glycosylation
appears crucial for the correct folding of the PRLR, allowing transport
to the plasma membrane.
The double mutant, glycosylated only on Asn35 residue,
showed no significant difference in its ligand binding or cellular
localization compared with the native PRLR and is still targeted to the
cell surface along with partial accumulation in a perinuclear
compartment, depending on the cell (and possibly on the level of
expression). Individual mutations confirmed that no specific
glycosylated chain is involved in the cellular localization of the long
form of the PRLR; however, comparison of the expression of the N35D and
the N80,108D mutants demonstrates that correct expression of the
protein can be achieved with partial glycosylation equivalent to 4 kDa
either on Asn35 or on both Asn80 and
Asn108.
Of interest is the residual cell surface expression of aglycosylated
PRLR (only detectable in COS-7 and 293 cells using binding
experiments), which is sufficient to allow the functional activity of
this mutant in 293 cells. This activity cannot be explained by
existence of other glycosylation chains associated with the PRLR, such
as O-glycosylation, since it has been shown that this receptor is only
N-glycosylated (12, 14). Overexpression of receptor, which leads to the
saturation of the endoplasmic reticulum and Golgi compartments, could
modify the environment conducive to conformational rescue of the
misfolded protein necessary for its cell surface expression. Other
motifs in the primary sequence of the PRLR could also be implicated in
this export process, e.g. the conserved WSXWS motif of the
extracellular domain (15, 27) or potential cytoplasmic sequences, as
has been described for other receptors (28). Interaction of the
aglycosylated receptor with other chaperones that might assist the
protein to its cell membrane localization cannot be excluded. Lastly,
the cell surface expression of an aglycosylated protein may reflect a
more specific cell line-dependent mechanism.
The influence of N-glycosylation on the transport of receptors has been
described for other members of the cytokine receptor superfamily; for
example, the erythropoietin receptor is more abundantly expressed than
a glycosylation-defective erythropoietin receptor mutant in a lymphoid
BA/F3 cell line (29); interferon-
receptor and GH receptor transport
also appears to be glycosylation-dependent (30, 31). On the other hand,
N-glycosylation of the human granulocyte macrophage-colony
stimulating factor-
(GM-CSF) receptor (32) or the interleukin-2
receptor (33) does not participate in cell surface receptor expression.
These latter studies have not focused, however, either on the
importance of other glycosylated receptor-associated chains in the
trafficking of the receptor or in differences in the cell lines
used.
Previous data have suggested a role of N-glycosylation in the
internalization process; for example, all N-linked glycosylation chains
of the ectodomain of the ß-subunit of insulin receptor apparently
participate in the control of receptor internalization (34); total
inhibition of N-glycosylation of the GH receptor also results in a more
efficient internalization of the protein in stably transfected mouse T
cells (31). In the present report, the low level of aglycosylated
receptors present at the cell surface at 4 C make the internalization
study difficult to analyze (data not shown); nevertheless, a
modification in recycling of the receptor cannot be excluded.
JAK2 Activation and Transcriptional Activity of Glycosylation Site
Mutants
Recently, it has been demonstrated that the extracellular domain
of cytokine receptors can modulate the activity of the cytoplasmic
domain (17, 18). Several studies have analyzed the implication of
glycosylation of the extracellular domain on receptor function.
N-Glycosylation does not appear to be critical to elicit either
tyrosine phosphorylation of proteins after growth hormone/receptor
interaction (31) or the erythropoietin-induced cell proliferation
signal (29). However, the absence of glycosylation of the GM-CSF
receptor markedly decreased GM-CSF-induced deoxyglucose uptake and
protein tyrosine phosphorylation in HL-60 (eos) cells (32). Our
experiments on the activation and association of the tyrosine kinase
JAK2, the initial step in PRLR signaling, indicate that all mutant
receptors were able to be phosphorylated and to activate the kinase in
a PRL-dependent manner. This indicates that the absence of
N-glycosylation does not interfere with the first key steps in PRLR
signaling. These results imply that homodimerization of the
aglycosylated PRLR occurs. In fact, previous studies have demonstrated
the formation of a complex consisting of one molecule of hormone and
two molecules of recombinant nonglycosylated extracellular domain of
the rat PRLR, expressed in a prokaryotic cell system (35). Functional
activity of the mutant receptors was further analyzed by testing their
ability to activate a PRL-dependent ß-casein gene promoter. Partially
deglycosylated receptors were all functional when tested in CHO cells
and also in 293 cells (data not shown); on the other hand, the
aglycosylated mutant, inactive in CHO cells, was fully active when
overexpressed in 293 cells. Trafficking and subsequent cell surface
expression of this mutated receptor appear to be different between
these two cell lines. The biological response may be elicited by a
limited number of receptors present at the cell surface as it has been
shown for insulin receptor (36). 293 cells do express limited but
detectable levels of aglycosylated receptors at the cell surface; it is
thus conceivable that this limited number of receptors is sufficient to
transduce the PRL signal into the cell. The situation is quite
different in CHO cells where much lower levels of receptors are
expressed (37, 38), and thus the level of aglycosylated receptors
present at the cell membrane may be below the threshold level required
for a biological response. Moreover, in 293 or COS cells,
overamplification of multiple cDNAs can create perturbations,
especially in the endoplasmic reticulum, as was recently described for
the expression of the human calcitonin receptor in COS cells (39). The
cell surface expression of the aglycosylated receptor can thus be
explained by an escape process linked to this overexpression, which
appears specific to the cell line.
Taken together, these results demonstrate that the N-glycosylation of
the extracellular domain of the PRLR is not implicated in the
acquisition of a correct conformation of the receptor essential for
ligand binding, but its absence leads to different alterations in cell
surface translocation of the protein, depending on the cell line, which
can affect further signal transduction.
 |
MATERIALS AND METHODS
|
---|
Hormones and Antibodies
Ovine PRL (oPRL) was obtained from the National Hormone and
Pituitary Program/NIDDK (Baltimore, MD) and recombinant hGH was kindly
provided by Ares-Serono Laboratories (Geneva, Switzerland). Recombinant
bovine GH (bGH) was kindly provided by Dr. W. Baumbach (American
Cyanamid Co., Princeton, NJ; ref. no. 94501-7). Antibodies were
obtained from commercial sources: mouse monoclonal anti-phosphotyrosine
4G10 (Upstate Biotechnology, Lake Placid, NY) at dilution 1:10, rabbit
polyclonal anti-JAK2 (
-JAK2, Upstate Biotechnology, dilution
1:4000). Mouse monoclonal anti-PRLR U5 has been described previously
and used at a concentration of 0.5 µg/ml in Western blot (12).
Monoclonal mouse antibodies specific for the 58K protein (Sigma, St.
Louis, MO; dilution 1:40) and the protein disulfide isomerase (PDI,
Stressgen Biotech Corp., Victoria, British Columbia, Canada; dilution
1:100) were used to localize the Golgi apparatus and the rough
endoplasmic reticulum, respectively. The rabbit polyclonal anti-rab6
antibody was generously provided by Dr. B. Goud (UMR 144, CNRS,
Institut Curie, Paris, France) (40). The secondary antibodies used in
these studies were the Texas-Red-conjugated and
fluorescein-isothiocyanate-conjugated (FITC) goat anti-mouse IgG
(dilution 1:40) (Vector Laboratories, Burlingame, CA; and Biosys,
Compiègne, France), the biotinylated donkey anti-rabbit IgG and
the Texas-Red-conjugated streptavidin (1:100; Amersham International,
Aylesbury, UK).
Construction of the Mutants
Short-form rat PRLRs presenting individual substitutions of the
asparagyl residues (Asn35, Asn80, and
Asn108) by aspartic residues were previously characterized
(15); the negative charge present on terminal sialic acid residues of
glycosylation chains of the native PRLR was then conserved.
NcoI inserts encoding modified sequences were subcloned into
the pECE or pcDNA3 (Invitrogen, San Diego, CA) plasmids containing the
long form of the rat PRLR. The double mutant (N80,108D) was obtained
after BamHI digestion of pcDNA3 mutants. From this mutant
plasmid, a single-stranded DNA was generated by using the origin of
replication of the M13 phage present in the pcDNA3 vector, in the
Escherichia coli CJ236 strain in the presence of M13K07
helper phage. This single-stranded DNA was then used as a template for
mutagenesis with the primer 5'-CAGTGAATAATCGGTAAGGAAG-3' (Genosys
Biotechnologies, Cambridge, UK) where the Asn35 residue was
replaced by an aspartate residue, to obtain the triple mutant. The
modified region was verified by sequencing.
Cell Culture
Kidney monkey cells (COS-7) were grown as monolayers in DMEM nut
F12 medium containing 10% FCS. Cells were transiently transfected at
60% confluence using the diethylaminoethyl-dextran-chloroquine
procedure (38). A 10% solution of Me2SO4 in
HBSS was applied 4 h after transfection, after which cells were
incubated with complete medium. This cell line, known to generate a
high level of expression of recombinant receptors, was used for binding
and immunofluorescence studies. Chinese hamster ovary (CHO K1) cells,
routinely used for transcriptional assays (37, 38), were maintained in
Hams F-12 medium containing 10% FCS. Before transfection, cells were
starved in GC3 serum-free medium (1:1 mixture of MEM and Hams F-12
supplemented with 10 µg/ml transferrin, 3 µg/ml insulin, 2.5
mM glutamine, and 1x nonessential amino acids). 293
fibroblast cells were maintained in DMEM nut F12 containing 10% FCS.
Cells were transfected by the calcium phosphate technique in a rich
medium (2/3 DMEM nut F12, 1/3 DMEM containing 4.5 g/liter glucose with
10% FCS) and used for Western blot studies and transcriptional
assays.
Determination of Cell Surface Ligand Binding
[125I]hGH was prepared using chloramine T to a
specific activity of 80140 µCi/µg (41). COS-7 (106)
cells were transfected with 1 µg of cDNAs encoding WT or mutant PRLR
in six-well plates. After 48 h, cells were starved in GC3 medium
for several hours and then incubated with 50,000 cpm of
[125I]hGH in the presence or absence of excess of
unlabeled oPRL (2.5 µg), in 1 ml PBS, 0.5% BSA overnight at 4 C.
After being washed twice with chilled PBS, cells were solubilized with
1 ml of 1 M NaOH, and cell-associated radioactivity was
counted using a
-counter. Specific binding was determined by the
difference between total and nonspecific binding.
Competition Curves
Transfected COS-7 cells were incubated with 100,000 cpm of
[125I]hGH and increasing concentrations of unlabeled oPRL
(01 mg) in 1 ml of PBS/BSA 0.5% overnight at 20 C. Cells were washed
twice with chilled PBS and solubilized with 1 ml of 1 M
NaOH, after which radioactivity was counted. Binding data were analyzed
using the LIGAND program (Elsevier Biosoft, Cambridge, UK).
Indirect Immunofluorescence
COS-7 (2 x 105) or 293 (2 x 105) cells
transfected with 0.5 µg of WT or mutant PRLR cDNAs were grown as
subconfluent monolayer cultures in Labtek chambers for 2 days. Cells
were fixed with 4% paraformaldehyde in 0.1 M phosphate
buffer, pH 7.4, for 20 min at room temperature and permeabilized or not
with methanol (-20 C) for 5 min. Antibody dilutions were made in PBS
containing 0.1% of BSA. The nonspecific sites were saturated with
blocking solution (goat serum diluted 1:40). Slides were incubated with
the U5 antibody at 160 µg IgG/ml overnight at 4 C and then
extensively washed in PBS and further incubated with a 1:40 dilution of
FITC goat anti-mouse IgG, as previously described (19), for 1 h at
room temperature. Cells were incubated overnight at 4 C with 1:100
dilution of mouse monoclonal anti-PDI antibody to localize the rough
endoplasmic reticulum, and then extensively washed and further
incubated with a 1:40 dilution of Texas-Red-conjugated goat anti-mouse
IgG, for 1 h at room temperature. For the double labeling
experiment, transfected cells fixed with 4% paraformaldehyde were
permeabilized with Triton 0.1% for 3 min at room temperature. After
washing, donkey serum diluted in PBS-BSA 2.5% was applied during 15
min. Cells were then successively exposed to the anti-rab6 antibody
(1:40) overnight at 4 C followed by 1 h exposure at room
temperature to biotinylated donkey anti-rabbit IgG, and then washed and
incubated 1 h at room temperature with Texas-Red-conjugated
streptavidin. The same cells were then successively incubated with goat
serum for 15 min at room temperature, with anti-PRLR U5 antibody for
3 h at room temperature, and with the FITC goat anti-mouse IgG for
1 h at room temperature. Once washed in PBS, slides were mounted
in 50% glycerol in PBS and observed on a Zeiss microscope (Carl Zeiss,
Thornwood, NY). No immunofluorescence was detected in any of the
specificity control experiments (including incubation of cells in the
absence of primary and/or secondary antibodies) or with IgG control
monoclonal antibodies.
Cross-Linking Experiments
COS-7 cells (106 cells per plate) were transfected
with WT or mutant PRLR cDNAs and then incubated with 1 ml of DMEM/0.1%
BSA containing 500,000 cpm of [125I]hGH in the absence or
presence of excess of unlabeled oPRL (2.5 or 5 µg) at 37 C for 30
min. Cells were then incubated with 0.5 mM of the
cross-linking agent DSS at room temperature for 20 min. The
cross-linking reaction was quenched with 50 mM Tris-HCl, pH
7.4, 150 mM NaCl. Cells were lysed in 50 µl 2x Laemmli
buffer (42) for 15 min at 4 C. Protein complexes were electrophoresed
on a 7.5% SDS-PAGE. Gels were dried and submitted to autoradiography
for 35 days.
Purification of PRLR Complexes by Streptavidin-Agarose
293 cells (107/100 mm dish) were transfected with 4
µg cDNA encoding wild type or mutated forms of PRLR and 2 µg cDNA
encoding the human tyrosine JAK2, using the calcium phosphate
procedure. Twenty four hours after transfection, cells were deprived of
serum for 16 h, and then stimulated with 20 nM
biotinylated oPRL or bGH for 10 min at 37 C (Biotinylation kit,
Boehringer Mannheim, Mannheim, Germany). Cellular proteins were
extracted in 1 ml lysis buffer (150 mM NaCl, 50
mM Tris-HCl, pH 7.5, 5 mM EDTA, 1
mM Na3VO4, 30 mM Na
pyrophosphate, 50 mM NaF, 1 mM
phenylmethylsulfonylfluoride, 5 µg/ml aprotinin, 2 µg/ml leupeptin,
1 µg/ml pepstatin, 10% glycerol, and 0.2% Triton X-100) for 10 min
at 4 C. Equivalent quantities of proteins were then mixed with 20 µl
of streptavidin-agarose beads (50% vol/vol) 2 h at 4 C. The
agarose beads were washed three times with lysis buffer, eluted in SDS
sample buffer, and subjected to a 7.5% SDS-PAGE.
Western Blot
Proteins were transferred to a nitrocellulose membrane at room
temperature for 1 h, incubated in TBS (50 mM Tris HCl,
pH 7.5, 200 mM NaCl, 1% BSA) for 1 h, and then
incubated with the first antibody in TBS complemented with 0.05%
Tween-20 for 90 min. After washing, membranes were incubated for 1
h at room temperature with anti-rabbit or anti-mouse IgG conjugated to
horseradish peroxidase (Amersham Corp., Arlington Heights, IL) diluted
at 1:4000. Proteins were visualized using the enhanced
chemiluminescence detection system (Amersham International, Aylesbury,
UK). Membranes were stripped overnight at 4 C in an acid solution (0.1
M glycine, pH 3, 0.1 M NaCl) and reprobed.
Transcriptional Assays
CHO cells (8 x 105) were transiently transfected in
six-well plates by the calcium phosphate procedure with 1 µg of cDNAs
encoding the WT or mutant PRLR, 0.5 µg of the ßCas-luc reporter
construct [containing the luciferase-coding sequence linked to the
sequence -2300/+490 of rat ß-casein gene promoter (21)], and 1 µg
of pCH110 (ß-galactosidase expression vector, Pharmacia, Bromma,
Sweden). Cells were submitted to a glycerol shock after 4 h of
incubation, and then incubated for 48 h in GC3 medium containing
20 nM oPRL and 250 nM dexamethasone (Sigma
Chemical Co.). 293 cells (106) were also transiently
transfected with 0.1 µg of receptor cDNA, 0.1 µg of reporter
construct, and 0.5 µg of pCH110 using the same precipitation
procedure. After an overnight 3% CO2 shock, cells were
starved in serum-free medium for several hours, and then stimulated
with 20 nM oPRL and 250 nM dexamethasone for
24 h. Cells were then lysed in lysis buffer (1% Triton X-100, 25
mM Tris-phosphate, pH 7.8, 2 mM dithiothreitol,
2 mM
1.2-diaminocyclohexane-N,N,N',N'-tetraacetic
acid, 10% glycerol). After centrifugation, 20 µl of supernatant were
used for determination of luciferase and ß-galactosidase activities.
A LUMAT LB9501 (Berthold, Wildbad, Germany) was used to quantify
luciferase activity into relative light units. Arbitrary luciferase
units were normalized for ß-galactosidase activity in each
experiment. Fold induction is calculated as the ratio between the
normalized light units of stimulated vs. unstimulated cells.
Activities of the mutants were calculated from the maximal activity
obtained with WT PRLR. Values are mean ± SEM of five
or three independent experiments performed on CHO cells or 293 cells,
respectively.
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to Dr. J. Rosen for providing the ß-casein
promoter, and Dr. J. Ihle for the JAK2 cDNA. We thank Dr. B. Goud for
generously providing the polyclonal anti-rab6 antibody used for the
double-labeling experiment.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Marc Edery, Inserm U344, Endocrinologie Moléculaire, Faculté de Médecine Necker, 156 rue de Vaugirard, 75730 Paris, France.
Received for publication July 28, 1997.
Revision received December 3, 1997.
Accepted for publication December 24, 1997.
 |
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