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Molecular Endocrinology, doi:10.1210/me.2001-0033
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Molecular Endocrinology 16 (10): 2310-2322
Copyright © 2002 by The Endocrine Society

Characterization of a Novel and Functional Human Prolactin Receptor Isoform ({Delta}S1PRLr) Containing Only One Extracellular Fibronectin-Like Domain

J. Bradford Kline1, Michael A. Rycyzyn1 and Charles V. Clevenger

Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104

Address all correspondence and requests for reprints to: Charles V. Clevenger, M. D., Ph.D. Department of Pathology & Laboratory Medicine, University of Pennsylvania Medical Center, 513 Stellar-Chance Laboratories, 422 Curie Boulevard, Philadelphia, Pennsylvania 19104. E-mail: clevengc{at}mail.med.upenn.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 In Vitro Observations of...
 MATERIALS AND METHODS
 REFERENCES
 
Prolactin (PRL)-dependent signaling occurs as the result of ligand-induced homodimerization of the PRL receptor (PRLr). To date, short, intermediate, and long human PRLr isoforms have been characterized. To investigate the expression of other possible human PRLr isoforms, RT-PCR was performed on mRNA isolated from the breast carcinoma cell line T47D. A 1.5-kb PCR fragment was isolated, subcloned, and sequenced. The PCR product exhibited a nucleotide sequence 100% homologous to the human long isoform except bp 71–373 were deleted, which code for the S1 motif of the extracellular domain. Therefore, this isoform was designated the {Delta}S1 PRLr. Northern analysis revealed variable {Delta}S1 PRLr mRNA expression in a variety of tissues. Transfection of Chinese hamster ovary cells with {Delta}S1 cDNA showed the isoform is expressed at the protein level on the cell surface with a molecular mass of approximately 70 kDa. Kinetic studies indicated the {Delta}S1 isoform bound ligand at a lower affinity than wild-type receptor. The {Delta}S1 PRLr was also shown to activate the proximal signaling molecule Jak2 upon addition of ligand to transfected cells, and, unlike the long PRLr, high concentrations of ligand did not function as a self-antagonist to signaling during intervals of PRL serum elevation, i.e. stress and pregnancy. Given its apparent widespread expression, this PRLr isoform may contribute to PRL action. Furthermore, the functionality of this receptor raises interesting questions regarding the minimal extracellular domain necessary for ligand-induced receptor signaling.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 In Vitro Observations of...
 MATERIALS AND METHODS
 REFERENCES
 
THE NEUROENDOCRINE HORMONE PROLACTIN (PRL) exhibits high homology to GH as well as the peptide hormones of the interleukin family (1, 2). PRL has been implicated in several biological processes including proliferation and differentiation of mammary gland cells, as well as the initiation and maintenance of lactation (3, 4). Regulation by PRL also occurs at the autocrine level as the production and secretion of PRL by mitogen-stimulated T cells (5, 6) and breast epithelium (7, 8, 9) has been identified.

The biological effects of PRL are mediated at the molecular level by inducing the homodimerization of the PRL receptor (PRLr) (10). Lacking intrinsic enzymatic activity, the PRLr activates associated kinases and other signaling factors triggering several signaling cascades. Through Janus kinase 2 (Jak2), PRL stimulation activates signal transducer and activator of transcription family members in lymphocytes (11) and breast tissues (12, 13), resulting in the initiation of transcription for interferon-regulatory factor-1 and ß-casein gene products. PRL-induced signaling also induces the GRB2/SOS/Ras/Raf/MAPK kinase/MAPK signaling cascade, ultimately activating transcription factors involved in cell cycle progression including Myc, Jun, and ternary complex factor (14, 15, 16).

As a member of the cytokine receptor superfamily, the initially characterized human PRLr (hPRLr) (17) contains a conserved extracellular domain (ECD) of approximately 200 amino acids. This is characterized by four conserved cysteine residues in the amino-terminal half and the WSXWS box, a tryptophan-serine motif, in the carboxy-terminal half near the membrane-proximal end. The ECD is comprised of two 100-amino acid fibronectin type III motifs arranged into seven antiparallel ß-strands (1, 18, 19), the N- and C-terminal motifs termed S1 and S2, respectively. Connecting the S1 and S2 motifs is a five-residue linker (L4) that forms a single helical turn. While this ECD tertiary structure is similarly found in other members of the cytokine receptor family, it is important to note studies of receptor-hormone systems make clear that there are important differences in the structural details of how receptor activation and specificity are regulated among different receptor-ligand interactions (20).

Receptor dimerization by both GH and PRL has been shown to involve two different regions of the ligands referred to as binding sites I and II (21, 22). Like GH, PRL first binds to one PRLr via binding site I to form an inactive, intermediate 1:1 complex. PRL bound in this complex then binds to a second PRLr molecule via the site II binding site to create a 1:2 PRL-PRLr complex capable of initiating intracellular signaling cascades. While the 1:2 GH-GHR complex is generally stable, the PRL-PRLr complexes rapidly dissociate into 1:1 complexes (10). It is interesting to note, however, that cross-species 1:2 PRLr complexes are generally more stable than their same-species counterparts (10, 23, 24, 25, 26, 27). This suggests that the more physiologically relevant ternary complex is of a transient nature, and it has been suggested that optimal PRL-PRLr signaling must take into account not only the global affinity of the signaling complex, but also the relative affinities of the two binding sites (28).

While several different PRLr isoforms have been characterized in mammals (29, 30, 31, 32, 33, 34, 35, 36), only avian PRLr isoforms have exhibited notable differences in the tertiary structures of their ligand binding domains. Both pigeon (37) and chicken (38) PRLrs are unique in that they encode tandem repeated ECDs, which in the pigeon have been shown capable of binding ligand. hPRLr isoforms characterized thus far have been the long (17), intermediate (33), and short forms (34); however, studies have suggested the existence of a hPRLr isoform encoding an altered ECD (9, 33, 35), with widespread expression at the protein level in normal and malignant breast tissues.

In this study, we identify a novel isoform of the hPRLr cloned from the human breast cancer cell line T47D. The cDNA sequence reveals a deletion of PRLr gene exons 4 and 5, encoding for the S1 motif of the ECD.2 The isoform is analyzed for 1) in vivo expression and its ability to bind ligand, 2) the ability to activate the receptor-associated Jak2 kinase, 3) the relative levels of its corresponding mRNA in normal tissues, and 4) its ability to signal via various somatolactogenic hormone family members.


   RESULTS
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 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 In Vitro Observations of...
 MATERIALS AND METHODS
 REFERENCES
 
Isolation of the Human {Delta}S1 PRLr
While the presence and function of PRLr isoforms has been the focus of intense study in rats (39, 40), our understanding of the number and function of hPRLr isoforms remains incomplete. Previous studies have suggested the existence of at least two additional PRLr isoforms (9, 36), one of which has been reported on by our laboratory (33). To investigate further the existence of additional hPRLr isoforms, RT-PCR was performed on cDNA generated from the breast cancer cell line T47D using oligonucleotides homologous to the 5'- and 3'-ends of the human long PRLr. While 1.8- and 1.3-kb fragments were identified as the previously characterized long (17) and intermediate (33) PRLr isoforms, respectively, an additional 1.5-kb fragment was found to encode a novel hPRLr isoform. DNA sequencing of the cloned 1.5-kb fragment revealed a PRLr form coding for a truncated ECD (Fig. 1AGo). This DNA sequence is most likely the result of an RNA splicing event, since a consensus splice site was present at the juncture between bp 70 and 374. The open reading frame is homologous to the long isoform up to the end of the sequence coding for the leader peptide (bp 70), and the deletion results in the removal of 100 amino acid residues from the translated peptide. This region also spans exons 4 and 5, which encodes the S1 motif of the PRLr ECD (Fig. 1BGo). Based on this deletion, the isoform was termed the {Delta}S1 PRLr.



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  		Figure 1. Complete Coding Sequence of the {Delta}S1 PRLr Isoform

A, DNA sequence alignment of the 5'-end of the {Delta}S1 open reading frame with long PRLr isoform. Leader sequence is shaded. Arrows indicate splice junctions between exons 3 and 6. B, Schematic representation of the {Delta}S1 PRLr isoform. Start codon (ATG), stop codon (TGA), WS motif, transmembrane domain (TM), intracytoplasmic domain (ICD), and leader peptide are indicated. S1 and S2 designate ECD motifs.

 
Expression and Ligand Binding Analysis of the {Delta}S1 PRLr Isoform
To investigate whether the {Delta}S1 isoform was efficiently translated, cDNAs of the {Delta}S1 PRLr and the long PRLr (positive control) were subcloned into the vector pEF1V5/HisA, which enabled the addition of a V5 epitope tag to the carboxyl-terminal ends of both PRLr variants. Immunoblotting of lysates from Chinese hamster ovary (CHO) cells transiently transfected with the constructs revealed proteins of the correct molecular mass previously reported for the long form (85 kDa) (17) and approximately 70 kDa for the {Delta}S1 isoform (Fig. 2Go). When transfectants were incubated with radiolabeled human PRL (hPRL), ligand binding was observed with the {Delta}S1 transfectants, albeit at lower levels than the long transfectant. In contrast, cells transfected with vector alone showed no binding of 125I-hPRL (data not shown). This implied the {Delta}S1 PRLr was capable of binding ligand and that the receptor was expressed on the cell surface.



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  		Figure 2. Western Blot of Recombinant {Delta}S1 Isoform

CHO cells (2 x 105) were transiently transfected for 48 h with pEF1-V5/HisA vector containing either the long or {Delta}S1 isoform cDNAs. Lysates were separated by 8% SDS-PAGE, transferred to nitrocellulose, and probed with anti-V5/HRP antibody (1:1000). Relative migration of molecular mass standards is indicated in kilodaltons (kDa).

 
To further investigate the PRL binding properties of the {Delta}S1 PRLr, recombinant forms of the {Delta}S1 and long PRLr ECDs were generated in Escherichia coli for subsequent analysis by surface plasmon resonance. This technique enables a determination of kinetic constants and the stoichiometry of interaction of a complex in which one component is immobilized on a flexible dextran matrix, whereas the other is free in solution. Previous studies revealed PRL could be immobilized via its amino groups without compromising its ability to bind soluble PRLr ECD (10). Therefore, hPRL was coupled to a dextran matrix to investigate the binding of the soluble PRLr ECDs to hormone. Using various concentrations of recombinant ECDs, the dissociation constants (Kds) of the {Delta}S1 PRLr hormone binding sites I and II were found to be 1265 nM and 5000 nM, respectively (Fig. 3Go, B and D). This translated into a 94-fold lower affinity of the {Delta}S1 ECD site I contact vs. the long PRLr ECD (Kd site I = 13.4 nM) and a 30-fold lower affinity site II contact vs. the long PRLr ECD (Kd site II = 157.6 nM) (Fig. 3Go, A and D). The differences in affinity can be attributed to the slower kon and faster koff rates observed for the {Delta}S1 PRLr ECD in relation to the long ECD binding rates. The kon rates for both sites I and II were 12- to 13-fold faster for the long ECD vs. the {Delta}S1 ECD. Additionally, the {Delta}S1 site I koff rate was 7-fold faster than the long ECD, while the site II koff rate was approximately 2-fold faster. In contrast, a negative control protein (Vav1) was incapable of binding hormone (Fig. 3CGo). To confirm the Kd values generated by surface plasmon resonance, Scatchard analysis using hPRL and transiently transfected CHO cells was employed. These results demonstrated a Kd of 1.4 nM for the long PRLr and 8.02 nM for {Delta}S1 (Fig. 3EGo). The discrepancy in the values generated between these two assays may be due to improper folding or coupling of the bacterially expressed {Delta}S1 ECD to the surface plasmon resonance chip. Nevertheless, when taken together, the kinetic data suggest the {Delta}S1 PRLr is capable of binding ligand, but at a lower affinity than the long PRLr.



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  		Figure 3. Real-Time Kinetic Measurement of the Interaction Between PRL and the ECD of the {Delta}S1 PRLr

hPRL and protein G were covalently linked to a dextran matrix via amino groups at a concentration yielding 1000 RU. Five concentrations of recombinant human {Delta}S1 PRLr-ECD (A), long PRLr-ECD (B), or Vav1 (C, neg. cont.) were injected in PBS/Tween and then washed out before regeneration. Bulk refractive indices were corrected by subtracting the RU values in the protein G flow cell from the PRL flow cell. BIA Evaluation software (Pharmacia Biotech, Inc., Uppsala, Sweden) was used to calculate kinetic constants. Representative of one of three experiments. D, Summary of kinetic data obtained from panels A and B. E, Representative data from radioligand binding studies and subsequent Scatchard plot. Specific activity of 125I-PRL was 79,206 cpm/nmol. Representative of one of three experiments.

 
The {Delta}S1 Isoform Is Capable of Activating Proximal PRLr Signaling Molecules
Since the ECD of the {Delta}S1 PRLr was observed to bind hPRL, we sought to test whether the proximal signaling pathways known to be activated by the long PRLr were also induced via the {Delta}S1 PRLr. Jak2 is one such protein known to be activated upon ligand stimulation of the long PRLr (41). To investigate whether the {Delta}S1 PRLr isoform was capable of activating this proximal signaling molecule, CHO cells were transiently transfected with constructs expressing the long or {Delta}S1 PRLr isoforms and stimulated with 5 nM hPRL. CHO cells were used for these assays because they contain endogenous Jak2 that can be activated by PRL without the need for overexpression, approximately 1.8 times the amount present in the human breast cancer line T47D (data not shown). Furthermore, the per cell levels of both the long and {Delta}S1 PRLr isoforms in the CHO transfectants were comparable or slightly lower than that observed in T47D (PRLr no./T47D ~30,000; data not shown). The {Delta}S1 PRLr was capable of activating Jak2 (Fig. 4AGo) in response to ligand, with maximal activation occurring about 15 min after addition of 5 nM hPRL. This is in contrast to the long PRLr transfectants, whose maximal activation of Jak2 (Fig. 4AGo) occurred 7.5 min after the addition of ligand. Comparable results were obtained when cells were treated with 500 pM hPRL (data not shown). Thus, the {Delta}S1 PRLr isoform appeared capable of activating proximal PRLr-associated signaling kinases in response to ligand, with kinetics of activation delayed from those seen for the long hPRLr isoform. To analyze what effect the coexpression of the {Delta}S1 and long PRLr isoforms had on Jak2 activation, CHO cells were transiently transfected with both constructs before stimulation with hPRL. As observed in Fig. 4BGo, a composite pattern of Jak2 phosphorylation was observed at both 7.5 and 15 min. All of the aforementioned studies were additionally continued out to 45 min. These data demonstrated no evidence of Jak2 activation through the {Delta}S1 receptor past 15 min in the presence or absence of the long PRLr (data not shown). Coimmunoprecipitation studies analyzing interactions between the {Delta}S1 and long PRLr isoforms did not reveal any heterodimer formation upon ligand addition (data not shown) and therefore, the phosphorylated Jak2 observed at both time points is most likely due to signaling through each respective homodimer.



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  		Figure 4. The {Delta}S1 PRLr Isoform Induces the Activation of Proximal Signaling Proteins upon Ligand Binding

CHO cells were transiently transfected with 2 µg of the long isoform (2:0), 2 µg {Delta}S1 isoform (0:2), or 2 µg of each isoform (2:2) before resting for 24 h and stimulation with 5 nM hPRL. Harvested lysates were immunoprecipitated with anti-Jak2 and resolved by SDS-PAGE. Phosphorylation of signaling molecules was visualized by immunoblot analysis with anti-phospho-Jak2. Equal PRLr expression was observed between samples (data not shown). Representative of one of three experiments.

 
The Human {Delta}S1 PRLr Shows Variable Tissue Expression
Previous studies have shown that the number of isoforms and levels of PRLr expression vary between tissues in different species (29, 33, 35, 37, 42). To determine the variability of {Delta}S1 PRLr transcript expression between tissues, two independent dot blots containing mRNA isolated from a variety of human tissues were probed with cDNA fragments specific for the {Delta}S1 isoform or the long PRLr. To avoid cross-hybridization, the {Delta}S1 probe was generated from the splice junction region of cDNA, allowing the hybridization to only the truncated ECD transcript (Fig. 5AGo). Relative levels of expression were compared with those of the pituitary (Fig. 5BGo) (33). As previously observed in rat tissues (42), the levels of long PRLr transcript varied greatly between human tissues. The highest levels of {Delta}S1 transcript expression occurred in the placenta and kidney, with wide variability in transcript levels observed among the remaining tissues. Additionally, it is important to note that the comparative mRNA expression in the isoforms differed between tissues. For example, the testis expressed barely detectable levels of {Delta}S1 transcript, while the long transcript levels were somewhat comparable to those of the pituitary (~80%).



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  		Figure 5. Expression of Long and {Delta}S1 Isoform mRNA Transcripts

A, {Delta}S1 cDNA probe does not cross-hybridize with long PRLr mRNA transcripts. A cDNA probe specific for the {Delta}S1 form was hybridized to a slot blot of RNA purified from T47D cells or CHO transfectants expressing vector, {Delta}S1, or long PRLr constructs (upper panel). Equal loading is represented by ethidium bromide staining of the blot (lower panel). B, Differential expression of long and {Delta}S1 isoform mRNA transcripts. cDNA probes specific for the long (black bars) and {Delta}S1 forms (white bars) were hybridized to a whole-tissue mRNA dot blot. Autoradiographs of the blot were scanned, and the intensity of the signals was quantitated using ImageQuant densitometry software (Molecular Dynamics, Inc.). Signals were set relative to expression observed in the pituitary gland. Representative of one of two experiments.

 
The {Delta}S1 PRLr Signals via Lactogenic and Somatogenic Hormones
Whereas various species of PRL interact with the PRLr, human GH (hGH) binds to both PRL and GH receptors (43), whereas human placental lactogen (hPL) has been shown to bind to the PRLr (44) and ovine PL to heteromers of hGH and hPRL receptors (45). It should be noted that the presence of 50 µM ZnCl2 is necessary to stabilize the binding of both GH and PL to the PRLr. Based on these findings, we wished to determine whether both lactogenic and somatogenic hormones were capable of signaling through the {Delta}S1 PRLr. To this end, NIH 3T3 cells transiently transfected with Jak2 and the {Delta}S1 isoform were stimulated with a variety of hormones at a concentration previously demonstrated to induce maximal activation of the long PRLr and analyzed for their ability to activate Jak2 (Fig. 6AGo). No species specificity was observed, as both hPRL and bovine PRL (bPRL) showed comparable levels of Jak2 phosphorylation as quantitated in Fig. 6BGo. As with the long PRLr, both hGH and hPL were also capable of inducing Jak2 autophosphorylation via the {Delta}S1 isoform, albeit at levels approximately half those observed for the long isoform (data not shown).



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  		Figure 6. The {Delta}S1 PRLr Isoform Signals via Different Hormones

A, Activation of Jak2 by different ligands. NIH 3T3 cells were transiently transfected in triplicate with the {Delta}S1 isoform in conjunction with Jak2 cDNA. Cells were rested for 24 h and then stimulated with 11 nM of various hormones for 10 min. Harvested lysates were immunoprecipitated with anti-Jak2 antiserum and immunoblotted with anti-phospho-Jak2 antibody. hGH, human GH + 50 µM ZnCl2; hPL, human PL + 50 µM ZnCl2. B, Quantitation of Jak2 activation via the {Delta}S1 PRLr. Three independent experiments as in panel A were scanned, and the intensity of the signals was quantitated using ImageQuant densitometry software (mean ± SEM). Fold induction was set relative to the levels of phosphorylated Jak2 observed in resting cells.

 
PRL Does Not Exhibit Self-Antagonistic Activity via {Delta}S1 PRLr Signaling
It is believed that the bell-shaped curves obtained in both PRLr (46) and GHR-mediated (47) bioassays illustrate how hormone concentrations can affect receptor dimerization. At lower hormone concentrations active 1:2 complexes (one ligand, two receptors) are favored. At high hormone concentrations self-antagonism is observed, resulting in inactive 1:1 complexes. This is due to the hormone having a binding site I of higher affinity as compared with site II. Since the {Delta}S1 PRLr isoform showed an overall weaker affinity for hPRL as compared with the long PRLr, we wished to compare the signaling properties of both isoforms using a large range of hormone concentrations. To that end, NIH 3T3 cells transiently transfected with Jak2 in conjunction with either the long or {Delta}S1 PRLr were stimulated with 0–4.5 µM bPRL and analyzed for their ability to activate Jak2 (Fig. 7AGo). bPRL was used instead of hPRL as it was shown to signal through the {Delta}S1 PRLr at levels comparable to hPRL (see Fig. 6Go) and was available in sizable quantities necessary to carry out the experiment. Quantitation of these experiments illustrated that bPRL functioned as an antagonist via long PRLr signaling as the level of hormone increased (Fig. 7BGo). In contrast, no antagonistic affects were observed via {Delta}S1 PRLr signaling at the highest hormone concentrations tested.



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  		Figure 7. PRL Does Not Exhibit Self-Antagonistic Activity During {Delta}S1 PRLr Signaling

A, Activation of Jak2 at increasing hormone concentrations. NIH 3T3 cells were transiently transfected with Jak2 in conjunction with long or {Delta}S1 PRLr cDNA. Cells were rested 24 h and then stimulated with 0–4.5 µM bPRL for 15 min (long PRLr) or 10 min ({Delta}S1 PRLr). These were times of maximal activation as determined in Fig. 4Go. Harvested lysates were immunoprecipitated with anti-Jak2 antiserum and immunoblotted with anti-phospho-Jak2 antibody. Equal receptor expression was observed in all transfectants (data not shown). B, Quantitation of Jak2 activation. Three independent experiments as in panel A were scanned, and the intensity of the signals quantitated using ImageQuant densitometry software. Fold induction was set relative to the levels of phosphorylated Jak2 observed in resting cells.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
In Vitro Observations of...
 MATERIALS AND METHODS
 REFERENCES
 
Previous reports have suggested the existence of a membrane-bound hPRLr with an altered ECD (33, 35). In this study, we present evidence for a novel hPRLr isoform with altered ligand binding and signaling kinetics. Both chickens (48) and pigeons (38) have been shown to express membrane-bound PRLr isoforms with alterations in their ECDs differing from other known mammalian forms. Unlike the human {Delta}S1 PRLr, these avian forms contain ECD duplications rather than deletions. The pigeon PRLr has two highly homologous units in its ECD, which are 64% identical to each other. Interestingly, the pigeon PRLr was found to bind rat PRL with high affinity, illustrating that hormone-receptor interactions can vary widely from conventional models.

The {Delta}S1 PRLr derives its name from the RNA processing event that results in the deletion of exons 4 and 5, coding for the N-terminal S1 motif of the ECD. Immunoblotting of transiently transfected CHO cells revealed a molecular mass of approximately 70 kDa, compared with the 85–90 kDa long PRLr observed by us and others (17). Because the three N-linked glycosylation sites of the long PRLr are within the deleted S1 motif, this observed molecular mass is consistent with an unglycosylated receptor isoform. Indeed, deglycosidase treatment did not modify the electrophoretic mobility of the {Delta}S1 PRLr (data not shown). Preliminary experiments revealed that the {Delta}S1 transfectants were capable of binding radiolabeled ligand, albeit at lower levels than the long PRLr transfectant. To investigate this observation further, surface plasmon resonance experiments were performed to quantitate the affinity of the {Delta}S1 ECD for hPRL as compared with the long PRLr ECD. The Kd values observed for the interactions between hPRL contact sites I and II of the long PRLr ECD were 13.4 nM and 157.6 nM, respectively. This was consistent with previously reported hormone-receptor interactions such as those observed by Gertler et al. (10) in which a 5- to 10-fold difference in Kd values was observed between sites I and II. In contrast to the long ECD, the {Delta}S1 ECD exhibited a 94-fold lower affinity for site I binding and a 30-fold lower affinity for site II. However, given that the ECD portions used in this assay were expressed in E. coli and may not be entirely folded in the proper orientation upon binding to the dextran surface, Scatchard analysis was also employed. This too demonstrated the PRL could bind to the {Delta}S1 isoform with a lower affinity (8.02 nM compared with 1.4 nM for the long PRLr isoform). Regardless of the assay employed, a decreased binding affinity for {Delta}S1 was observed, and this lessened affinity may relate to the intrinsic structures of the {Delta}S1 vs. the long PRLr isoform. Interspecies signaling was observed, as bPRL activated Jak2 via the {Delta}S1 PRLr comparable to hPRL. Additionally, both hGH and hPL were also capable of signaling albeit at lowered activity. The hGH-hPRLr crystal structure has been solved and reveals the involvement of six intra-ß-strand loops of the PRLr in hormone-receptor binding (49). Assuming that the S2 domain is capable of acquiring a folded structure similar to wild type in the absence of the S1 domain, then only the L5 and L6 loops would be available for the {Delta}S1 PRLr to bind hGH. This is effectively nine of 24 residues (38%) present in the long PRLr, as well as three of eight of 8 (38%) of the hydrogen bonds. Theoretically, it is therefore possible that enough contact points may exist to generate the interaction of hPRL with the {Delta}S1 PRLr that occurs with reduced affinity.


   In Vitro Observations of {Delta}S1 hPRLr
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 In Vitro Observations of...
MATERIALS AND METHODS
 REFERENCES
 
hPRL binding kinetics prompted us to further characterize the signaling capabilities of the isoform. Jak2 phosphorylation assays of transiently transfected CHO cells revealed that the {Delta}S1 PRLr was capable of activating the proximal signaling molecule Jak2 upon addition of hPRL with delayed kinetics vs. those observed through the long PRLr. This may be related to the differences in affinity observed between the two isoforms. It has been shown that the affinity of GH for its receptor may be decreased up to 30-fold with no change in maximal Jak2 activation (50). Thus, hPRL-long PRLr site I binding affinities may surpass those required for maximal cellular activity. Additionally, a recombinant epidermal growth factor mutant with a 50-fold reduced affinity for receptor was found to be a more potent mitogenic stimulus for fibroblasts that wt epidermal growth factor (51).

Our results with the widely expressed {Delta}S1 isoform differed from previous studies in which a mutant rabbit PRLr (rbPRLr) not naturally occurring was generated with a deletion of the S1 motif and was found to be functionally inactive (52) (Fig. 8Go, rbPRLr {Delta}3–103). Importantly, the mutated rbPRLr contained two N-terminal S1 residues before the deletion of the remaining S1 motif. It is possible that these residues may have sterically affected the signaling capabilities of their construct, as suggested by unpublished data from our laboratory. While performing kinetic studies on hPRL-{Delta}S1 ECD interactions, one recombinant {Delta}S1 ECD construct initially used by our laboratory contained four heterologous N-terminal residues that completely abrogated hormone binding (data not shown). Only after reengineering the construct to remove these four extra residues, did we observe interactions of the {Delta}S1 ECD with hPRL. Therefore, we believe differences in the N-terminal contact region between the construct generated by Gourdou et al. (52) and the {Delta}S1 PRLr may explain the differences observed in signaling capability.



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  		Figure 8. Schematic Representation of the Truncated ECDs of Native and Recombinant PRLrs

Activity in the absence (constitutive activation) or presence (PRL-induced activation) of hormone is indicated on the right. The S1 (dark gray), S2 (light gray), WSXWS motif (white box), and transmembrane domain (TM) are indicated. The long and {Delta}S1 PRLr isoform ECDs are at the top. hPRLr {Delta}178, ECD deletion mutant of the first 178 residues of the hPRLr (66 ); rbPRLr {Delta}103–203, rbPRLr deletion of the S2 domain (55 ); rbPRLr {Delta}3–103, rbPRLr deletion of the S1 domain (55 ). Amino acid residues are indicated.

 
Other mutational analyses of the PRLr have analyzed the function of the five cysteine residues of the ECD (53). Single cysteine substitutions in the rat PRLr S1 motif (C12A, C22A, C51A, C62A) all resulted in the abrogation of PRL binding. As suggested by the authors, this was presumably due to improper folding as monoclonal antibodies generated against the ECD failed to interact with the mutant receptors. We speculate that lacking the cysteine-rich S1 region, the S2 region is able to fold in a manner to enable productive ligand binding.

Examination of the mRNA levels of {Delta}S1 PRLr, as compared with the long PRLr, revealed variable levels of PRLr isoform expression within different human tissues. Of the tissues examined, the highest levels of both long and {Delta}S1 PRLr mRNA were observed in the placenta and kidney. Maaskant et al. (54) previously reported PRLr gene expression in placental trophoblast and immunoblot analysis detected six molecular species, one of which was approximately the size of the {Delta}S1 PRLr. Given that the long and {Delta}S1 PRL were found at high levels in the placenta, both isoforms may contribute to the regulation of hormone action during pregnancy (55, 56). In the kidney, PRL is known to induce ornithine decarboxylase activity (57), and there is also evidence of PRL binding sites in the kidney (58, 59, 60, 61), as both hormone and receptor can be localized on the epithelium of Bowman’s capsule and the proximal tubules (62). These findings suggest a role for PRL in modulating renal function, to which the {Delta}S1 PRLr may contribute. Whether the RNA levels observed in these various human tissues will translate into corresponding protein levels of {Delta}S1 isoform remains to be determined. To that end, we are currently collecting data regarding the protein expression of the {Delta}S1 PRLr. However, previously published data from our laboratory (9) would indicate that the {Delta}S1 PRLr is a predominant receptor isoform at the protein level in both normal and malignant breast tissue.

It is important to note the significance attributed to the WSXWS motif located near the C-terminal end of the ECDs of both the long and {Delta}S1 PRLr isoforms. The WSXWS sequence is thought to be critical in maintaining the structure of the S2 motif adjacent to the plasma membrane (49). Previous deletion mutagenesis studies generated a constitutively active PRLr in which residues 1–178 had been deleted N-terminal to the WSXWS motif (63) (Fig. 8Go). This mutation essentially removed the S1 and most of the S2 motif. It was hypothesized that the constitutive activity of this receptor was driven by the hydrophobic interactions between the WSXWS domains of two receptors. Therefore, the structure of this mutant PRLr does not require a ligand-induced conformational change in one receptor to allow binding at site II and subsequent receptor dimerization. In contrast, Gourdou et al. (52) reported the generation of a constitutively active PRLr mutant in which the S2 motif was deleted (Fig. 8Go). Based upon these data, they suggested that the S2 region functions to suppress the self-dimerizing properties of S1. Our observations of {Delta}S1 PRLr activity lead us to speculate that the N-terminal region of the S2 domain not only is involved in ligand binding, but functions to sterically suppress constitutive receptor-receptor association via the WSXWS box and the S1 region.

We examined the ability of PRL to self-antagonize {Delta}S1-associated signaling at high hormone concentrations. As expected, at pharmacological concentrations of bPRL the long PRLr showed a diminution in its ability to activate Jak2. In contrast, the {Delta}S1 PRLr exhibited no reduction in signaling at all hormone concentrations tested. This is consistent with previous studies suggesting that while the overall affinity of the 2 PRLr:1 PRL ternary complex is important, the ability of hormones to induce a biological response via their receptors must also take into account the relative affinities of their two binding sites (28). Our kinetic studies indicate a 12-fold difference in the relative affinities of site I and site II for hPRL on the long PRLr ECD. Therefore, at high concentrations of hormone, preferential site I binding occurs, resulting in the formation of inactive 1:1 complexes and lower Jak2 activation. In contrast, less than a 4-fold difference in the lower relative affinities of sites I and II on the {Delta}S1 PRLr ECD was observed. This decrease in the relative affinities of site I vs. site II measured with the {Delta}S1 ECD presumably lowers the level of preferential ligand binding to site I over site II, enabling the formation of productive 1:2 complexes and subsequent Jak2 activation at high concentrations of bPRL.

What are the possible physiological functions of the {Delta}S1 isoform? Given its reduced affinity for ligand, we speculate that the {Delta}S1 isoform may contribute to effects observed with PRL during states of higher serum concentration, such as the pregnancy-associated alveolar differentiation of the breast. Thus, given its ability to signal at higher ligand concentrations, the widely expressed {Delta}S1 PRLr isoform may significantly contribute to the pleiotropic actions of PRL.


   MATERIALS AND METHODS
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 In Vitro Observations of...
 MATERIALS AND METHODS
REFERENCES
 
mRNA Isolation and RT-PCR
T47D cells, an estrogen receptor/PRLr-positive human breast cancer cell line, were used for mRNA isolation. Whole RNA was purified from 107 washed cells using trizol-reagent (Life Technologies, Inc., Gaithersburg, MD) as described previously (9). mRNA was then purified from the whole RNA preparation with oligo-dT cellulose (Invitrogen, San Diego, CA). T47D mRNA (5 µg) was used for first-strand synthesis of cDNA using the Superscript II RT cDNA kit (Life Technologies, Inc.). Negative controls consisted of reactions containing no T47D mRNA or no reverse transcriptase. A positive control reaction consisted of chloramphenicol acetyltransferase mRNA template. For PCR, 2 µl of the corresponding cDNA reactions were added to a 50-µl reaction containing 5 µl 10x PCR buffer, 3 µl 25 mM MgCl2, 1 µl 10 mM deoxynucleoside triphosphate mix, 5 U of TAQ polymerase (Life Technologies, Inc.), and primers for amplification. As the positive control reaction, primers A (5'-GACATGGAAGCCATCACAGAC-3') and B (5'-CGACCGTTCAGCTGGATATTA-3') were used to amplify a fragment of the chloramphenicol acetyltransferase gene from control cDNA. The PRLr gene amplification reaction contained primers PRLR-F3 (5'ATGAAGGAAAATGTGGCA-3') and PRLR-1 (5'-TCAGTGAAAGGAGTGTGT-3'), which correspond to the 5'- and 3'-ends of the human long PRLr open reading frame. The primary cycle of the reaction consisted of 94 C for 2 min, 42 C for 1 min, 72 C for 3 min, and 94 C for 2 min, which was followed by 30 cycles of 94 C for 30 sec, 47 C for 30 sec, and 72 C for 2 min. It was then extended at 72 C for 3 min. Isolated PCR fragments were subcloned into the TA vector pCR 2.1 (Invitrogen, San Diego, CA) and analyzed by dideoxynucleotide sequencing. For eukaryotic expression of the {Delta}S1 PRLr isoform, the gene was reamplified by PCR with primers PRLR-Kl (5'-CGAATTCCACCATGAAGGAAAATGTGGCA-3') and PRLR-599' (5'-GCGCTCGAGTCAGTGAAAGGAGTGTGTAAA-3'), which contain a 5'-EcoRI restriction site and Kozak initiation sequence, and a 3'-XhoI restriction site, respectively. An alternative 3'-primer, PRLR-LONG (5'-CGCTCGAGGTGAAAGGAGTGTGTAAA-3'), was also used to remove the tertiary stop codon from the open reading frames of the isoforms, allowing the addition of a carboxy-terminal V5 epitope tag when ligated into vector pEF1-V5/HisA (Invitrogen). The DNA fragments were digested with EcoRI and XhoI and ligated into the corresponding restriction sites of pEF1-V5/HisA. The clones were subsequently checked for amplification errors by dideoxynucleotide sequencing.

Hormones
hPL, bPRL, and hGH were gifts from the National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases. hPRL was recombinantly expressed in the Drosophila Expression System (Invitrogen).

Cell Culture and Transfection
T47D cells were maintained in DMEM (Life Technologies, Inc.) supplemented with 10% fetal bovine serum, 50 U/ml penicillin, and 50 µg/ml streptomycin. CHO-K1 cells were maintained in Ham’s F-12 medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum, 50 U/ml penicillin, and 50 µg/ml streptomycin. NIH 3T3 cells were maintained in DMEM supplemented with 10% calf serum, 50 U/ml penicillin, and 50 µg/ml streptomycin. CHO and NIH 3T3 cells (2 x 105) were transiently transfected with 2 µg {Delta}S1 or long isoform cDNA clones in pEF1-V5/HisA in conjunction with 2 µg of human Jak2 (gift of Dr. Roy Duhe, University of Mississippi Medical Center, Jackson, MS) cDNA in pEF1-V5/HisA using Fugene 6 (Roche Molecular Biochemicals, Indianapolis, IN) as instructed. Cells were incubated 48 h before use.

Jak2 Phosphorylation Analysis
CHO cells (2 x 105), transfected with the PRLr isoforms, remained overnight in F-12/0.1% BSA and were then stimulated with hPRL (500 pM to 5 nM) for 0–45 min. Cells were lysed in lysis buffer (0.5% Nonidet P-40; 50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 10% glycerol; 1 mM phenylmethylsulfonyl fluoride and protease inhibitors) and immunoprecipitated overnight as previously described (64) using 5 µl anti-Jak2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) antibodies. Antigen-antibody complexes were isolated by the addition of 50 µl protein-A beads, washed three times with lysis buffer, and then suspended in 50 µl 2x Laemmli buffer with 2-mercaptoethanol. Boiled samples were analyzed by 8% SDS-PAGE followed by immunoblot analysis. Phosphorylated Jak2 protein was evaluated with a 1:1000 dilution of antiphospho-Jak2 antiserum (Upstate Biotechnology, Inc., Lake Placid, NY) followed by a 1:2500 dilution of mouse antirabbit monoclonal antibody (Sigma-Aldrich Corp., St. Louis, MO) and visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Arlington Heights, IL). Jak2 and phosphorylated Jak2 levels were quantitated by scanning densitometry using ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA). To confirm {Delta}S1 PRLr expression, transient CHO cell transfectants were lysed in Laemmli buffer containing sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (65). Lysates were electrophoresed through an 8% SDS-polyacrylamide gel and transferred to nitrocellulose. Nonspecific binding was blocked with 5% milk in PBS/Tween 20. Antigen-antibody complexes were labeled with 1 µg horseradish peroxidase-conjugated anti-V5 antibody (Invitrogen) per ml. Jak2 expression was determined by incubation with a 1:1000 dilution of anti-Jak2 antiserum (Santa Cruz Biotechnology, Inc.) followed by a 1:2500 dilution of mouse antirabbit monoclonal antibody (Sigma-Aldrich Corp.).

Generation of Recombinant PRLr ECDs and Surface Plasmon Resonance Analysis of {Delta}S1 hPRLr:hPRL Interaction
The recombinant ECDs of the {Delta}S1 and long PRLr isoforms were generated by the insertion of their respective cDNAs into the pGEX-4T expression vector (Pharmacia Biotech, Piscataway, NJ), enabling expression of these domains as glutathione-S-transferase (GST) fusion proteins. Briefly, the cDNAs encoding for the ECDs of both isoforms were amplified by PCR using primers PRLR-EK (5'-GCGAATTCGACGATGACGATAAGCAGTTACCTCCTGGAAAACCTGAG-3') and PRLR-211' (5'-GCGCTCGAGTCAATCATTCATGGTGAAGTC-3'). Primer PRLR-EK encodes an EcoRI restriction site and an enterokinase cleavage site, allowing the complete removal of the GST tag, producing amino termini homologous to the amino termini of the mature receptors after cleavage of their leader peptides. PRLR-211' encodes an XhoI restriction site and a stop codon. Accordingly, both cDNAs were cloned into the EcoRI and XhoI sites of pGEX-4T. The chimeras were expressed as instructed for 4 h. Pelleted cells were lysed and refolded as previously described (66). Refolded fusion proteins were conjugated to glutathione beads (Pharmacia Biotech) for 30 min and washed with lysis buffer three times, followed by three times with PBS. Proteins were eluted in EKMax buffer (Invitrogen) containing 50 mM glutathione for 20 min, and then incubated overnight at room temperature with enterokinase (Invitrogen) to cleave the fusion proteins. Preparations were dialyzed overnight in 4 liters PBS to exchange buffer and remove the glutathione. Proteins were then incubated several times with fresh glutathione beads to remove the GST, leaving pure, monomeric recombinant ECDs, as evidenced by analysis on reducing and nonreducing SDS-PAGE gels (data not shown).

hPRL and protein G (negative control, Sigma-Aldrich Corp.) were covalently linked to a dextran matrix via amino groups according to Johnsson et al. (67). Briefly, HEPES-buffered saline was injected at 5 µl/min, and activation with 0.05 M N-ethyl-N'-(3-diethylaminopropyl) carbodiimide/N-hydroxysuccinimide was carried out for 6 min. hPRL was then injected at 5 µl/min in 10 mM sodium acetate at pH 4.5 until coupling yielded 1000 reactive units (RU). Unreacted sites were blocked with an 8-min injection of 1 M ethanolamine hydrochloride (pH 8.5). Five concentrations of recombinant human {Delta}S1 PRLr-ECD, long PRLr-ECD, or a negative control protein (Vav1) were injected in PBS/0.01% Tween 20 for 2 min, followed by washing with PBS/0.01% Tween 20 for 5 min. Regeneration between runs was achieved with treatment of the biosensor chip with 1 M NaCl, pH 3.0, followed by 3 min with PBS/0.01% Tween 20. Bulk refractive indices were corrected by subtracting the RU values in the protein G flow cell from the PRL flow cell. BIA Evaluation software version 3.0.2 (Pharmacia Biotech) was used to calculate kinetic constants and fit experimental curves with both 1:1 and 1:2 association-dissociation models to calculate the most accurate representation of the data. Reverse verification of the calculated data was performed by simulating the interaction with BIA Simulation version 2.0 (Pharmacia Biotech). Scatchard analysis was performed as previously described (33).

Northern Analysis of {Delta}S1 PRLr Isoform Expression in Various Tissues
Master blots of human total mRNA (CLONTECH Laboratories, Inc., Palo Alto, CA) were probed with cDNAs specific for either the wild-type or {Delta}S1 alternatively spliced PRLr ECDs. Equal loading of mRNAs was confirmed by the quantitation of eight distinct housekeeping genes. The cDNA probe for the wild-type ECD was composed of nucleotides 1–140 of the long PRLr open reading frame (17). The probe for the {Delta}S1 isoform spans the 300-bp deletion of the ECD that is generated by alternative splicing. This corresponds to nucleotides 1–70 and 374–444 of the long open reading frame. Hybridizations were performed as instructed by CLONTECH Laboratories, Inc. Under these conditions, no cross-hybridization was observed between isoforms. The blot was exposed to x-ray film for 2 d, and signal intensities were obtained using ImageQuant densitometry software (Molecular Dynamics, Inc.).


   ACKNOWLEDGMENTS
 
We wish to thank Irwin Chaiken and Gabriella Canziani of the University of Pennsylvania Biosensor Core Facility (Philadelphia, PA) for their help in generating and analyzing kinetic binding data for the {Delta}S1 PRLr. We thank Dr. A. F. Parlow and the National Hormone Pituitary Program for providing the hPL, bPRL, hGH, and recombinant hPRL.


   FOOTNOTES
 
1 J.B.L. and M.A.R contributed equally to this work. Back

This study was supported in part by NIH Grants 2R01CA-69294 and 1R01DK-50771 (to C.V.C.) and 1F32DK-09727 (to J.B.K.).

Abbreviations: bPRL, Bovine PRL; CHO, Chinese hamster ovary; ECD, extracellular domain; GST, glutathione-S-transferase; hGH, human GH; hPL, human placental lactogen; hPRL, human PRL; hPRLr, human PRL receptor; Jak2, Janus kinase 2; PL, placental lactogen; PRL, prolactin; PRLr, PRL receptor; rbPRL, rabbit PRL; RU, reactive units.

2 The nucleotide sequence reported in this paper has been submitted to the DNA Databank of Japan/European Molecular Biology Laboratory/GenBank databases under accession no. AF349939. Back

Received for publication April 30, 2001. Accepted for publication July 16, 2002.


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N. Ben-Jonathan, C. R. LaPensee, and E. W. LaPensee
What Can We Learn from Rodents about Prolactin in Humans?
Endocr. Rev., February 1, 2008; 29(1): 1 - 41.
[Abstract] [Full Text] [PDF]

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 Mol. Endocrinol.Home page
S. L. Gadd and C. V. Clevenger
Ligand-Independent Dimerization of the Human Prolactin Receptor Isoforms: Functional Implications
Mol. Endocrinol., November 1, 2006; 20(11): 2734 - 2746.
[Abstract] [Full Text] [PDF]

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 Endocr Relat CancerHome page
E. Ueda, U. Ozerdem, Y.-H. Chen, M. Yao, K. T. Huang, H. Sun, M. Martins-Green, P. Bartolini, and A. M Walker
A molecular mimic demonstrates that phosphorylated human prolactin is a potent anti-angiogenic hormone.
Endocr. Relat. Cancer, March 1, 2006; 13(1): 95 - 111.
[Abstract] [Full Text] [PDF]

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 Mol. Cell. Biol.Home page
Y. Li, K. G. Suresh Kumar, W. Tang, V. S. Spiegelman, and S. Y. Fuchs
Negative Regulation of Prolactin Receptor Stability and Signaling Mediated by SCF{beta}-TrCP E3 Ubiquitin Ligase
Mol. Cell. Biol., May 1, 2004; 24(9): 4038 - 4048.
[Abstract] [Full Text] [PDF]

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 EndocrinologyHome page
M. D. Schroeder, J. L. Brockman, A. M. Walker, and L. A. Schuler
Inhibition of Prolactin (PRL)-Induced Proliferative Signals in Breast Cancer Cells by a Molecular Mimic of Phosphorylated PRL, S179D-PRL
Endocrinology, December 1, 2003; 144(12): 5300 - 5307.
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M. A. Olayioye, M. A. Guthridge, F. C. Stomski, A. F. Lopez, J. E. Visvader, and G. J. Lindeman
Threonine 391 Phosphorylation of the Human Prolactin Receptor Mediates a Novel Interaction with 14-3-3 Proteins
J. Biol. Chem., August 29, 2003; 278(35): 32929 - 32935.
[Abstract] [Full Text] [PDF]

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 Endocr. Rev.Home page
C. V. Clevenger, P. A. Furth, S. E. Hankinson, and L. A. Schuler
The Role of Prolactin in Mammary Carcinoma
Endocr. Rev., February 1, 2003; 24(1): 1 - 27.
[Abstract] [Full Text] [PDF]

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