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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go) 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. 2Go); 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. 6AGo).



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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.

 
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. 3Go, 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 1Go 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 (10,000–20,000 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

 
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. 4Go, 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. 4Go, 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. 4fGo) without any significant immunostaining at the level of the plasma membrane (Fig. 4cGo). 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. 4iGo). Immunostaining at the cell surface observed in cells transfected with the WT receptor (Fig. 4gGo) or the N80,108D receptor (Fig. 4hGo) 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.

 
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. 5Go, b and c). In some cells, staining was also visible in the rough endoplasmic reticulum (Fig. 5dGo). 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. 5fGo): the pattern of endoplasmic immunostaining markedly differs from the perinuclear staining pattern observed for Golgi protein (Fig. 5Go, c and e) or the triple mutant (Fig. 5aGo).



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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.

 
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 2Go.


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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

 
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. 6AGo and 7AGo). After stripping the membrane and reprobing with anti-JAK2 antibody, JAK2 (130 kDa) was shown to associate with all forms of PRLR (Figs. 6BGo and 7BGo). 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. 7CGo). However, we observed reduced amounts of N35,80,108D receptor (Fig. 7AGo) and JAK2 protein (Fig. 7BGo). 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. 6Go, 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.

 
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 (80–90% 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. 8AGo). 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. 8BGo); 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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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-{gamma} 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-{alpha} (GM-CSF) receptor (32) or the interleukin-2{alpha} 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 9450–1-7). Antibodies were obtained from commercial sources: mouse monoclonal anti-phosphotyrosine 4G10 (Upstate Biotechnology, Lake Placid, NY) at dilution 1:10, rabbit polyclonal anti-JAK2 ({alpha}-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 Ham’s F-12 medium containing 10% FCS. Before transfection, cells were starved in GC3 serum-free medium (1:1 mixture of MEM and Ham’s 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 80–140 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 {gamma}-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 (0–1 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 3–5 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.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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