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Molecular Endocrinology, doi:10.1210/me.2005-0291
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Molecular Endocrinology 20 (8): 1912-1923
Copyright © 2006 by The Endocrine Society

Ligand-Independent Homo- and Heterodimerization of Human Prolactin Receptor Variants: Inhibitory Action of the Short Forms by Heterodimerization

Aamer M. Qazi, Chon-Hwa Tsai-Morris and Maria L. Dufau

Section on Molecular Endocrinology, Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Maria L. Dufau, Building 49, Room 6A-36, 49 Convent Drive, MSC 4510, National Institutes of Health, Bethesda, Maryland 20892-4510. E-mail: dufaum{at}mail.nih.gov.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Prolactin (PRL) acts through the long form (LF) of the human PRL receptor (hPRLR) to cause differentiation of mammary epithelial cells through activation of the Janus kinase-2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) pathway and subsequent transcriptional events. To determine whether the inhibitory action of hPRLR short forms (SFs; S1a and S1b) on PRL-induced signal transduction through the LF results from heterodimerization, we studied complex formation among variant forms of the hPRLR. 3'-Tagged fusion constructs, with activities comparable to the wild-type species, were used to investigate homodimer and heterodimer formation. The LF and both SFs of the hPRLR formed homodimers under nonreducing conditions, independently of PRL, but formed only monomers under reducing conditions. Coimmunoprecipitation of the cotransfected LF with the SFs (S1a or S1b) in transfected cells showed ligand-independent heterodimerization of individual SFs with the LF. Bioluminescence resonance energy transfer analysis demonstrated homo- and heterodimeric associations of hPRLR variants in human embryonic kidney 293 cells. Biotin-avidin immunoprecipitation analysis revealed that hPRLR forms are cell surface receptors and that SFs do not influence the steady state or half-life of the LF. Significant homo- and heterodimerization of biotinylated membrane hPRLR forms was observed. These findings indicate that homo- and heterodimers of hPRLR are constitutively present, and that the bivalent hormone acts on the preformed LF homodimer to induce the active signal transduction configuration. Although SF homodimers and their heterodimers with LF mediate JAK2 activation, the SF heterodimer partner lacks cytoplasmic sequences essential for activation of the JAK2/signal transducer and activator of transcription 5 pathway. This prevents the heterodimeric LF from mediating activation of PRL-induced genes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE HIGHLY DIVERSIFIED actions of prolactin (PRL) are exerted through specific high-affinity receptors expressed in the cell membrane of several target tissues and mammary tumors (for review, see Ref. 1). Alternate splicing of the human PRL receptor (hPRLR) gene generates several receptor forms, including a long form (LF), an intermediate form, and short forms (SFs) (2, 3, 4, 5, 6, 7). These PRLR forms contain of an extracellular ligand domain, a single transmembrane region, and an intracellular domain of variable length and structure (see Fig. 1Go). The extracellular domain bearing the configuration for ligand interaction is shared for by all transmembrane forms. However, the intracellular domain that coordinates the signal transduction events leading to activation of gene transcription and cell proliferation by the LF, diverges in the intermediate and SFs. The SFs S1a and S1b (see Fig. 1Go) are cell surface-expressed receptors in normal tissue, breast tumors, and breast cancer cell lines (6, 7), and have affinities comparable with that of the LF. These short species can inhibit PRL-induced signal transduction activated by the LF (6).


Figure 1
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  		Fig. 1. Functional Characterization of hPRLR 3'-Tagged Constructs

A, Schematic structure of PRLR LF and SFs, wild-type and 3'-tagged constructs. The genomic region of the LF of hPRLR comprises of eight exons (exons 3–10) in which exons 3–7 code for the extracellular binding domain, exon 8 codes for the transmembrane domain (TM) , and exons 9 and 10 for the intracellular domain of the LF and most of the SFs (S1a and S1b; see below) (4 ). Exon 9 contains box 1 (267–275 aa), and box 2 (311–320 aa) is present in exon 10. Exon 11 in the human is distinct from other species, and is used in part to encode the SF of the receptor (6 ). In the present study, we used two novel forms of the hPRLR, S1a and S1b, which are derived from alternative splicing of exon 10 and exon 11 (6 ). S1a encodes 376 aa containing partial exon 10 and 39 aa of exon 11 (337–376 aa). S1b encodes 288 aa lacking exon 10 and contains 3 aa at C terminus derived from exon 11 (285–288 aa). Box 1 is present on all three forms, whereas Box 2 is missing in S1b form as illustrated. hPRLR variants were tagged with V5, Flag, GFP, YFP, and RL, and the expected molecular weights are also indicated (right). Amino acid number includes the signal peptide of 24 aa. B, Comparative functional studies on the effect of individual and cotransfected forms of hPRLR, wild-type (top panel), and 3'-tagged plasmids (middle and lower panels) on ß-casein promoter activity. The effect of SFs on ß-casein promoter activity induced by PRL via the hPRLR LF in HEK293 cells. HEK293 cells were transfected or cotransfected with SF S1a or S1b, and LF [wild type, or 3'-tagged (V5, Flag, GFP)] and the ß-casein promoter construct and incubated with or without PRL (100 ng/ml) for 24 h.

 
The LF of the hPRLR, like other members of the cytokine receptor family, lacks intrinsic kinase activity. During hormonal stimulation, single PRL molecules bind to induced and/or preexistent receptor dimers to promote rapid tyrosine phosphorylation of the receptor-associated Janus kinase 2 (JAK2) kinase (8, 9). This enzyme in turn phosphorylates the receptor at a specific tyrosine residue (Tyr587) in its C-terminal region (10, 11), which is only present in the LF in the human receptor. This is followed by phosphorylation, dimerization, and nuclear translocation of transcription factor signal transducer and activator of transcription 5 (STAT5). The LF is the only PRLR form able to activate the JAK2/STAT5 pathway, which is essential for PRL-induced transcription of milk protein genes, differentiation of normal epithelial cells, and initiation and maintenance of lactation. In contrast to the LF, neither of the SFs mediates transcriptional activation of the ß-casein gene promoter induced by PRL. Instead, these forms exert dominant-negative effects forms on PRL-induced activation of transcription by the LF when coexpressed in transfected cells (6). PRL is a tumor promoter in rodents and stimulates the proliferation of human breast cancer cells (12). The role of PRLR variants in breast cancer is unknown, but there is increasing evidence that PRLR expression is altered in mammary neoplasms (13). A significant decrease in the ratio of the SFs S1a or S1b to LF is observed in the breast tumor tissue and cancer cell lines compared with the normal breast and control mammary cells (14). Accordingly, the relatively reduced expression of short PRLR forms in cancer could lead to gradations of unopposed PRL-induced LF stimulatory function and contribute to breast tumor development and progression.

The initiation of signal transduction in the cytokine receptor superfamily involves ligand-receptor dimer interaction (15). PRLR activation through the LF results from H1:R2 (H1, one PRL molecule; R2, one dimeric receptor) complex formation with homodimers of hPRLR (16, 17). The process of receptor dimerization can be either constitutive or ligand dependent. It is generally accepted that members of the tyrosine kinase and cytokine receptor families undergo agonist-promoted homo- and heterodimerization that is essential for activation of downstream signaling cascades (18, 19, 20, 21). However, this concept has been challenged by the recent demonstration that some receptors, including the erythropoietin and the GH receptors, can exist as preformed dimers (22, 23, 24, 25).

In view of the dimerization requirement for initiation of signal transduction by PRL, and because inhibitory actions of the short hPRLR forms on LF-induced transcriptional activation could result from heterodimerization, we have analyzed the associations between and among the hPRL LF and the SFs S1a and S1b. These studies have demonstrated that the formation of homo- and heterodimers of the hPRLR variants at the cell membrane is independent of the presence of hormone. Such findings provide an avenue for exploration of the structural requirements of these interactions, and definition of the initial steps participating in the activation and repression of signal transduction pathways induced by PRL through its hPRLR variants.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Characterization of hPRLR Forms Tagged Constructs Used for Homo- and Heterodimerization Studies
To assess the potential dimerization of hPRLR variants, expression constructs 3'-tagged receptors were prepared, and their function was evaluated. These included fusion constructs of LF with either V5 or Flag tag at the C terminus and of SFs (S1a and S1b) with green fluorescent protein (GFP) or Flag (Fig. 1AGo). The functional integrity of tagged constructs transfected in human embryonic kidney 293 (HEK293) cells were compared with the wild type. The LF 3'-tagged with V5 or Flag (LF-V5, LF-Flag) exhibited PRL-induced activation of ß-casein promoter activity (26) comparable with the wild-type LF pcDNA construct (Fig. 1BGo, middle and lower panels vs. control, top panel). In cells cotransfected with 3'-tagged LF (LF-Flag or LF-V5) and the 3'-tagged SFs (S1a-GFP or S1a-Flag, or S1b-Flag), either of the SFs caused major inhibition of PRL induced stimulation of promoter activity by the LF-V5 or LF-Flag form of the receptor that was comparable with the wild-type constructs. These results indicated that the function of LF and SF 3'-tagged constructs is preserved.

Homodimerization of LF, S1a, and S1b
To explore whether the individual PRLR variants undergo homodimerization, the individual forms LF and SFs (S1a or S1b) expressed in transfected HEK293 cells and resolved by PAGE under reducing and nonreducing conditions were evaluated by Western blot analysis using either PRLR antibody (PRLR Ab; Fig. 2AGo) or V5 antibody (Fig. 2AGo') and Flag antibody (Fig. 2BGo). Only the monomer of LF-V5 of 92 kDa was observed under reducing (R) condition. The presence of LF-V5 homodimers of 184 kDa in addition of the monomer was evident under nonreducing (NR) conditions using a specific PRLR antibody or V5 antibody (Fig. 2Go, A and A'). SFs S1a-Flag and S1b-Flag were resolved as monomers of 57 and 43 kDa, respectively, under reducing conditions (Fig. 2BGo). In addition, a small proportion of homodimers of these forms were detected under nonreducing conditions (Fig. 2BGo). PRL did not cause any increase in monomers or dimers of the hPRLR LF or SFs (Fig. 2Go, A, A', and B). These findings indicate that the formation of PRLR homodimers, either of LF or SFs, are independent of ligand binding.


Figure 2
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  		Fig. 2. Dimerization of hPRLR Forms Expressed in HEK293 Cells

A, A', and B, Homodimer of hPRLR forms. Western blot analysis of extracts from HEK293 cells expressing hPRLR LF V5 tagged (LF-V5) using antibody specific against PRLR: U5 (PRLR Ab) (A), V5 Ab (A'), and SFs S1a- Flag and S1b-Flag with Flag antibody (Flag Ab) (B) incubated in the presence or absence of PRL for 24 h. hPRLR constructs used are indicated below. NR, Nonreducing; R, reducing condition. A, V5 cDNA: vector only (negative control). C, D, and E, Heterodimer species of hPRLR forms. Western blot analysis of extract from HEK293 cells coexpressing hPRLR LF Flag tagged (LF-Flag) and S1a-GFP (C) and hPRLR LF V5 tagged (LF-V5) and S1b-Flag (D and E) or S1a-Flag (E) incubated in the presence or absence of PRL for 24 h. CO-IP was performed using anti-Flag antibody (C and E) or anti-V5 antibody (D) and Western blot using anti-GFP antibody (C), anti-Flag antibody (D), and anti-V5 antibody (E), respectively. WB, Western blot analysis; F, anti-Flag antibody; V, anti-V5 antibody; G, anti-GFP antibody. Arrows indicate the antibody used in the study. HC, Heavy chain *. LC, Light chain.

 
Heterodimerization of Cotransfected Long and Short Forms of hPRLR
To explore the potential physical association between hPRLR variants, coimmunoprecipitates of the hPRLR pair expressed in HEK293 cells cotransfected with the LF construct along with either SF bearing a different tag at the 3' end were subsequently analyzed by Western blot. When cells were cotransfected with LF-Flag and S1a-GFP, and coimmunoprecipitated with anti-Flag antibody, an 83-kDa S1a-GFP protein band was detected in Western blots by the anti-GFP antibody (Fig. 2CGo, lane 2). No difference in band intensity was observed in cells incubated in the absence or presence of PRL (lanes 2 and 3). The size of the band was similar to that of the protein from cells transfected only with S1a-GFP and analyzed by Western blot either directly or after immunoprecipitation (IP) with anti-GFP (lanes 1 and 5). No S1a-GFP band was present in the negative control groups, which included cells that were transfected only with LF-Flag (lane 4), or pcDNA (lane 6) or when IgG was used in coimmunoprecipitation (CO-IP) of hPRLR from cells cotransfected with LF-Flag and S1a-GFP (lane 7). Similarly, in cells transfected with both S1b-Flag and LF-V5 (Fig. 2DGo), the formation of LF-V5/S1b complex was evident by the presence in Western blots of the 43 kDa S1b-Flag protein revealed by anti-Flag antibody, after CO-IP of LF-V5 by the V5 antibody (Fig. 2DGo, lanes 2 and 3). PRL did not exhibit any effect in the dimerization between LF and S1b (lanes 2 vs. 3). The 43-kDa S1b-Flag protein band was observed in cells transfected only with S1b-Flag and either analyzed by Western blot directly (lane 1) or after CO-IP with anti-Flag antibody (lane 5). No S1b-Flag was present in the negative control groups where only LF-V5 or pcDNA was transfected (lanes 4 and 7), or IgG was used in the CO-IP of cells cotransfected with LF-V5 and S1b-Flag (lane 8). Heterodimerization was confirmed by IP of either SF forms (S1a-Flag, S1b-Flag), which revealed the presence of LF-V5 in Western blots using the V5 antibody (Fig. 2EGo).

Assessment of hPRLR Variants Dimerization in Vivo Using Bioluminescence Resonance Energy Transfer (BRET) Method
To further study the heterodimers of hPRLRs forms demonstrated above, we examined the expressed receptors by BRET analysis. BRET is a resonance energy transfer technique for monitoring dynamic protein-protein interactions in living cells and depends on the close proximity (<100Å) of donor and acceptor partners (27). A dose-dependent increase BRET signal was observed with increasing doses of LF-Y, S1a-Y, or S1b-Y when cotransfected with either fixed amount of LF-RL, S1a-RL, or S1b-RL, respectively (homodimer), and similar increases were observed for heterodimers of LF-RL with SFs by varying concentrations of S1a-Y or S1b-Y. Based on these dose-response curves (data not shown) RL and the yellow fluorescent protein (YFP) constructs were contrasfected at optimal ratios of 1:3 (RL:YFP) in HEK293 cells (Fig. 3Go). The heterodimers of LF-RL/S1a-Y and LF-RL/S1b-Y showed similar BRET ratios, whereas the ratios of homodimers of the LF (LF-RL/LF-Y) were significantly higher than those of the SF S1a (S1a-RL/S1a-Y) (P < 0.01). Of note is the high ratio observed for S1b homodimer (S1b-RL/S1b-Y), which indicated stronger interaction or closer proximity between S1b forms than for other variant forms of hPRLR. This is consistent with the results obtained from S1b homodimers in SDS-PAGE under nonreducing conditions (Fig. 2Go). In all instances, no significant differences were observed in cells incubated in presence and absence of the hormone. BRET signals were undetectable in cells transfected with LF-RL, S1a-RL, or S1b-RL. Furthermore, BRET ratios were less than 10% of those obtained with interacting homo- or heterodimers when the LF-RL, S1a-RL, or S1b-RL constructs were cotransfected with the YFP construct. These results revealed the interaction between RL and Y moieties of the fusion proteins in intact cells and further support the existence of constitutive homodimers of the individual forms and complex formation between LF and SFs of hPRLR.


Figure 3
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  		Fig. 3. BRET Analysis in HEK293 Cells Expressing hPRLR Variants

BRET ratio was measured in 100,000 cells (100K) expressing the indicated hPRLRs DNA-fusion constructs (LF, 666 ng; SFs, 2000 ng) incubated in the presence or absence of 100 ng PRL. RL, Renilla luciferase; Y, Aequora enhanced YFP. hPRLR LF and SFs (S1a and S1b) were in-frame with RL (LF-RL, S1a-RL, S1b-RL) or Y (S1a-Y or S1b-Y), respectively. RL constructs were also used as negative controls along with YFP plasmid. Results represent the mean ± SEM of four independent experiments performed in triplicate.

 
To exclude the possibility that the BRET signals observed above could result from overexpression, BRET experiments were carried out with increasing doses of cotransfected hPRLR variants (Fig. 4Go, top panel; LF-RL with either S1a-Y, left, or S1b-Y, right) at two different cell densities (50K and 100K). The BRET ratios of SF receptor-YFP to LF receptor-RL were maintained constant (Fig. 4Go, top panel), and these were independent of their receptor expression level, which increased in a dose-dependent manner with increased transfection of the DNA (Fig. 4Go, lower panel).


Figure 4
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  		Fig. 4. BRET Signal Is Independent of hPRLR Isoform Expression Level in HEK293 Cells

LF-RL/S1a-Y or LF-RL/S1b-Y were cotransfected with increasing DNA doses (150–2000 ng LF-RL, 50–666 ng SF (S1a-Y or S1b-Y) in HEK293 cells. Top panel, BRET signal. Lower panel, Fluorescence emitted by GFP protein (S1a-Y or S1b-Y).

 
hPRLR Variants Are Cell Surface-Targeted Receptors
To further establish that dimerization of PRLR forms occurs at the cell membrane, cell surface biotinylation was performed on HEK293 cells transfected with defined combinations of hPRLR forms (Fig. 5AGo) followed by avidin IP and Western analysis under reducing and nonreducing conditions. Under reducing condition, the expected molecular weight of each form was clearly noted using either anti-luciferase antibody to detect luciferase fusion protein (LF-RL, 124 kDa; S1a-RL, 90 kDa; or S1b-RL, 76 kDa) or anti-GFP antibody to detect YFP fusion protein (LF-Y, 117 kDa; S1a-Y, 83 kDa; S1b-Y, 69 kDa) (Fig. 5AGo). No PRLR forms were detected after trypsin treatment before the biotinylation, confirming that LF and the SFs of PRLR were cell surface-targeted receptors. Further analysis of these biotin-avidin IP products under nonreducing condition using an anti-GFP antibody (Fig. 5Go, B–D) revealed in addition of the monomeric forms, dimerized bands of each form, and heterodimers of LF with S1a and S1b as noted in earlier studies (Fig. 2Go) (LF-RL/LF-Y, 241 kDa; LF-RL/S1aY, 207 kDa; LF-RL/S1b-Y, 193 kDa; S1a-RL/S1a-Y, 173 kDa; S1b-RL/S1b-Y, 145 kDa). The steady-state level of LF expressed at the cell surface was not significantly changed upon cotransfection with S1a or S1b in 1- to 8-fold molar excess (Fig. 6AGo). Furthermore, pulse-chase 35S labeling of cells transfected with LF to examine its surface half-life in the absence or presence of S1a or S1b showed no significant effect of the SFs. The observed surface half-life of derived from exponential decay analysis was comparable for LF in the presence or absence of either of the SFs (LF, 2.3 ± 0.2 h; LF/S1a, 1.9 ± 0.2 h; LF/S1b, 2.1± 0.1 h) (Fig. 6BGo). The relatively short half-life of LF in these studies in transfected HEK293 cells was consistent with those derived for total PRLR receptors in mice mammary explants in a previous study (28). Overall, our results demonstrate the presence of dimerized hPRLR forms at the cell surface, and the lack of effect of coexpressed SFs on the turnover of the LF (Figs. 5Go and 6Go).


Figure 5
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  		Fig. 5. Dimers of Cell Surface Expressed hPRLR Variants in HEK293 Cells

A, Cell surface expression of hPRLR variants. Biotin-avidin IP analysis of transiently expressed PRLR forms in HEK293 cells. Western analysis was performed in the reducing condition using either anti-luciferase (RL) or GFP antibody. Trypsin digestion was used as a negative control. *, Expected protein band. B, C, and D, Dimerization of hPRLR forms on the cell surface. Biotin-avidin IP products of transiently expressed PRLR forms in HEK293 cells were analyzed by Western blot using anti-GFP antibody (Ab) under nonreducing condition (B) in 4–20% PAGE and in 6% PAGE (C and D). Expected monomer size of each isoform ({square}). LF-Y, 117 kDa; S1a-Y, 83 kDa; S1b-Y, 69 kDa. Expected molecular weight for the homodimer and heterodimer hPRLR forms. Homodimer ({circ}); heterodimer (arrow) (LF-RL/LF-Y, 241 kDa; LF-RL/S1aY, 207 kDa; LF-RL/S1b-Y, 193 kDa; S1a-RL/S1a-Y, 173 kDa; S1b-RL/S1b-Y, 145 kDa).

 

Figure 6
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  		Fig. 6. Effect of SF on LF Expression, Half-Life and JAK2 Activation by PRL

Steady-state LF expression in the presence or absence of SF (A). Half-Life of LF in the membrane in the absence and presence of S1a or S1b (B). JAK2 activation (C). Cells were transfected with either 3:1 ratio of SF to LF (A) or 2:1 (1X), 4:1 (4X), or 8:1 (8X) of SF to LF to HEK293 cells. V, Vector with pCMV-driven YFP only. Biotin-avidin IP products of transiently expressed PRLR forms in HEK293 cells were analyzed by Western blot using anti-GFP antibody under reducing conditions (A). Pulse-chase experiment of 35S-PRLR-labeled forms biotinylated and double immunoprecipitated with GFP and avidin and resolved in 4–20% PAGE. The relative amount of 35S-labeled LF was expressed as percentage of that of the control cells (B). Cells cotransfected with PRLR variants and JAK2 incubated in presence or absence of PRL for 24 h were analyzed by Western blot analysis using an anti-JAK2 antibody (JAK) (to determine expression) (C, above), or anti-phopho-JAK2 (JAK-P) (to assess activation) (C, below).

 
Activation of JAK2 by Homodimers and Heterodimers
Other studies demonstrated PRL-induced activation of JAK2 phosphorylation in cells transfected with either SF (Fig. 6CGo). The activation was comparable with that induced by LF transfection. In contrast, when LF and SF were cotransfected, a reduction of basal was observed; however, the relative JAK2 activation by PRL was comparable. The basal levels of JAK2 phosphorylation likely result from autophosphorylation (29). These findings indicate that PRL induce activation of JAK2 through SF homodimers and also of heterodimers with LF (Fig. 6CGo).

Site-Directed Mutagenesis of Unpaired Cysteines
Three adjacent unpaired cysteines in extracellular (EC) (Cys208), transmembrane (TM) (Cys249), and intracellular (IC) (Cys266) domain of PRLR were selected to analyze their potential contribution to dimerization. Because S1b displayed the highest dimerization capacity and also possessed these three conserved cysteine residues, point mutation from cysteine to serine was performed in S1b fusion protein containing either luciferase (RL) or YFP (Y) for further BRET analysis (Fig. 7Go). The cell surface expression of all PRLR mutants was confirmed by biotinylation (data not shown). Substitution of Cys266 by Ser (S1b266x-R/S1b266x-Y) did not alter the BRET signal compared with the wild type (S1b-RL/S1b-Y). A small but significant decrease in the BRET signal was noted with Cys249 mutant located in the TM (S1b 249x-RL/S1b249x-Y; P < 0.01). In contrast, a marked increase in the BRET ratio occurred with the EC Cys208 mutant (S1b208x-RL/S1b208x-Y; P < 0.01). Additional mutation at Cys249 in S1b208X construct (S1b208, 249x-RL/S1b208, 249x-Y) abolished the increased BRET signal observed in the S1b208x. Although this result suggested the TM cysteine could partially contribute to the dimerization process while the EC cysteine inhibits association, the studies indicated that intermolecular disulfide bonds were not involved in dimerization.


Figure 7
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  		Fig. 7. Site-Directed Mutagenesis of Unpaired Cysteines

BRET analysis using wild type of S1b and mutated unpaired cysteines. Three adjacent cysteines (C) shared by all forms LF and SF: extracellular domain (EC), transmembrane domain (Tm), and intracellular domain (IC) of S1b were mutated to serine (S) at aa 208, 249, and 266. S1b (#X)-RL or -Y: #, aa number of Cys location; x, mutation; RL, luciferase; Y, YFP. Wild and mutant S1b-RL and S1b-Y were cotransfected in HEK293 cells.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this study, differentially epitope-tagged constructs of the hPRLR forms including LF and SFs, S1a and S1b, were expressed in HEK293 cells, and analyzed by Western blots, CO-IP, BRET, and biotinylation approaches to investigate homo- and heterodimerization of hPRLR variants and the effects of ligand. The formation of homodimers of LF, S1a, and S1b was revealed by their respective anti-tag antibodies in Western blots only under nonreducing conditions. CO-IP studies using combinations of specific tagged variants demonstrated the presence of heterodimerized complexes between LF and the S1a or S1b SFs. Using BRET analysis as an alternative tool to detect protein association, we further demonstrated the physical association of hPRLR variants in HEK293 cells and its occurrence in vivo. All of the expressed hPRLR forms are located at the cell surface membrane. We also demonstrated that the formation of homo- and heterodimerization of hPRLR variants is independent of PRL. These studies suggest that the functional inhibition of PRL activation through the LF of the receptor by the SFs results from heterodimerization between these forms. Although such heterodimers are competent to bind hormone, subsequent signal transduction events via the JAK/STAT pathway is not operative, and as consequence, the activation of transcription of relevant genes is reduced or abolished.

The presence of preexisting homodimers of the activating LF of the receptor in the absence of lactogenic hormone (Figs. 2Go and 3Go) differs from the generally recognized mechanism of ligand-induced binding of PRLR receptors (1). This modifies the concept of the initial step of PRL’s action from dimer-inducer to a conformational modifier. The lack of hormone dependence is also observed in the formation of homodimers of the SFs. Although to date the only recognized and demonstrated function of the SFs is the inhibition of PRL’s activating function mediated by the LF, probably through heterodimerization, it is conceivable that homodimers of the SFs can regulate intrinsic signaling mechanism(s).

The finding of a small fraction of higher molecular weight species corresponding to homodimerization of LF and SFs under nonreducing conditions (Fig. 2Go, A and B) suggested the involvement of disulfide bonding in the association of monomeric receptors. Two paired cysteine residues located at amino acids (aa) 36/46 and 75/86 of hPRLR could form intramolecular disulfide bonds. Although these may not be directly associated with the dimerization between hPRLR receptors, we cannot exclude their contribution to the stability of dimer conformation. Four other unpaired cysteine residues observed at aa 20, 208, 249, and 266 are shared by LF and SFs. Three of these residues are located adjacent to and on the transmembrane of hPRLR (extracellular Cys208, transmembrane Cys249, and intracellular Cys266). These residues, which are not present in the related cytokine receptor family members like GH receptor or erythropoietin receptor, could be available for disulfide-bond formation between the monomeric forms. However, this is unlikely because studies on the crystal structure of ovine placental lactogen bound to PRLR extracellular domain dimers have indicated that the cysteine residues reside at distance incompatible with disulfide bonding (30). Our mutagenesis studies indicated that Cys residues in the TM and EC do not contribute to hPRLR dimerization through covalent bonding. Mutation of TM Cys249 minimally reduced the formation of S1b dimmer, whereas mutation of its adjacent Cys208 in the EC domain enhanced monomeric PRLR interaction. The diverse effects of these two Cys were indirectly confirmed by double mutation, where BRET signals returned to levels observed for wild-type PRLR interactions (Fig. 7Go). The significance of the inhibitory action of Cys208 residue in EC domain for PRLR dimerization is not clear. TM Cys249 appears to have a minor role in receptor interaction, although it could be of relevance to PRLR dimerization. In this regard, there is evidence that the transmembrane domain is essential for ligand-independent dimerization observed in erythropoietin receptors (24).

Because there is only a small but significant contribution of the TM Cys249 to PRLR dimerization, it is probable that other motifs must participate in this process. The extracellular domains of cytokine receptors have been implicated as receptor dimer partners by crystallographic and mutagenetic studies. Six intermolecular receptor-receptor hydrogen bonding interactions within subdomain 2 of the GH receptor were shown to be essential for forming the dimerization interface monomeric units (30). Studies on the crystal structure of ovine placental lactogen bound to PRLR extracellular domain dimers demonstrated four H-bonding interactions between monomers (30). Furthermore, crystallographic evidence for preformed dimers of the erythropoietin receptor revealed an extracellular dimer formed by self-association of at least three key residues within the binding site for the ligand (22). It is possible that such monomer interactions could occur in the context of H-bond contribution to the homo- and heterodimerization of hPRLR variants demonstrated in our studies.

The demonstration of heterodimers between LF and SFs (S1a and S1b) shown in this study has indicated that sequences downstream of Box 1 or the unique C-terminal sequences of S1a (36 aa) or S1b (contributed by exon 10 in the case of LF or exon 11 of S1a or S1b) are not appear to be involved in dimer/heterodimer formation. S1b, which lacks amino acids derived from exon 10 sequences and only includes box 1, showed efficient dimerization and the highest BRET signal of all the three homodimers of the hPRLR forms studied. This could indicate that sequences downstream of the transmembrane region, can reduce interactions among monomers. In this regard, BRET signals for homodimers of the LF and S1a forms were comparable and significantly lower than those of S1b, whereas heterodimers exhibited similar BRET signals.

Assessment of biochemical surface biotinylation (Fig. 5AGo) confirmed that hPRLR forms are expressed at the cell surface. The lack of signal observed in Western blots from cells treated with trypsin before biotinylation further indicated that the hPRLR dimers were derived from the cell surface. Similar to the results obtained from CO-IP analysis (Fig. 2Go), homo- or heterodimeric complexes (Fig. 5Go, B–D) were resolved in the nonreducing gel after biotinylation. This finding supports the hypothesis that hPRLR forms interact at the cell surface. Furthermore, the steady-state cell surface expression and half-life of the LF were not altered by the overexpression of SF in the transient transfection system (Fig. 6Go, A and B). This implies that the inhibitory action of SF on functional LF-mediated signal transduction is caused by heterodimerization of the LF with SFs. The biological relevance of the protein-protein interaction of the hPRLR forms was further indicated by their independence from receptor density, as indicated by the constant BRET signal with increasing expression levels of receptors in living cells.

This study has shown significant hPRLR homodimer/heterodimer formation that is independent of the hormone, which does not induce additional dimeric forms. Furthermore, the inhibitory effect of the SFs on LF stimulatory activity induced by PRL can be attributed to the formation of heterodimers. SF homodimers and the heterodimeric forms of LF with SF (LF/S1a, LF/S1b) mediated PRL-induced JAK2 phosphorylation, due to the presence of Box 1 in these species. However, further signaling events of the LF through STAT cannot proceed due to the absence of relevant downstream sequences (i.e. Y587) in the SF heterodimer partner.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Construction of Eukaryotic Expression 3'-Tagged Plasmids Used for Homo- and Heterodimerization Studies
The full-length coding region of hPRLR variants, LF and SFs S1a and S1b (6), were subcloned in different expression plasmids including pcDNA3.1/V5-His-A (Invitrogen, Carlsbad, CA) at XhoI site for LF (LF-V5) and pEGFP-N2 vector (BD Biosciences, Palo Alto, CA) at KpnI/ApaI for S1a (S1a-GFP), and pCMV4A-Flag (Stratagene, La Jolla, CA) at XhoI site for LF, S1a, and S1b (LF-Flag, S1a-Flag, and S1b-Flag). All hPRLR variants constructs with mutation of their respective termination codons and in frame with the tag-marker used were verified by restriction enzyme mapping, sequencing, and expression (Western blot).

Cell Culture and Transfection
HEK293 (American Type Culture Collection, Manassas, VA) cells were maintained in DMEM (Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), antimycotic antibiotic at 37 C in a CO2 incubator. The hPRLR variant constructs in different combination (LF:S1a, 1:64, and LF:S1b, 1:8; 4 µg total) were transiently cotransfected along with ß-Casein-luciferase reporter plasmid (0.1 µg) (kindly provided by Dr. Warren Leonard, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD), ß-galactosidase reporter gene (0.1 µg, for normalization), and pcDNA (empty vector for equalization of DNA transfection) using LipofectAMINE Plus reagent (Invitrogen) as described in the manufacturer’s protocol. Twenty-four hours after transfection, media were replaced with serum-free DMEM and cells were incubated with or without hPRL (NIDDK S1AFP-B2, AFP-2969A; 0.1 µg/ml) for 24 h followed by measurement of luciferase activity using a luminometer (Autolumat Plus LB 953; Birthold Technologies, Bad Wildbad, Germany) and luciferase reagent kit (Promega, Madison, WI). The optimal ratio of LF/SF was previously determined for maximal inhibitory of LF activation by PRL and to account for differences in the turnover rate of SFs (6).

CO-IP and Western Blot Analysis
HEK293 cells were transiently transfected with the LF or S1a or S1b 3'-tagged constructs or cotransfected with LF and S1a or S1b 3'-tagged variants for 24 h in presence of DMEM/10% FBS. Subsequently, cells were incubated for additional 24 h in presence or absence of hPRL (100 ng/ml). Cells were analyzed using 50 mM HEPES buffer, 1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, and 0.1 mM Na3VO3 in the presence of protease inhibitor cocktail containing broad-spectrum serine, cysteine, and metalloproteases and calpains (Roche Diagnostics, Mannheim, Germany). After the preclearing steps, recovered supernatants were incubated in the presence of protease inhibitors with their corresponding anti-tag antibodies (2 µg) or normal mouse Ig (IgG) for 1 h at 4 C on rotary shaker. Protein G-Sepharose beads (Invitrogen) were added for overnight incubation at 4 C with rotation. The protein G-protein complex recovered by brief centrifugation was washed five times (three times with lysis buffer and two times with 1x PBS). The beads were resuspended in 20 µl of SDS-PAGE loading buffer, denatured by heating at 95 C for 10 min in the presence of 5% ß-mercaptoethanol (Sigma-Aldrich, St. Louis, MO). In nonreducing condition, samples were heated at 70 C for 5 min in the absence of 5% ß-mercaptoethanol. Samples were resolved by 10% SDS-PAGE and transferred onto nitrocellulose membrane (Invitrogen). The membrane was briefly rinsed with PBST (PBS with 1% Tween 20) and incubated with appropriate primary anti-tag antibody indicated in the specific experiments. The immunoreactivity was revealed by using horseradish peroxidase-coupled anti-mouse IgG second antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The immunosignals were detected using SuperSignal West Pico Chemiluminescent substrate (Pierce Biotechnology, Rockford, IL). The primary antibodies used in the IP or Western analysis included anti-V5 antibody (monoclonal; Invitrogen), anti-GFP antibody (monoclonal, JL-8; BD Biosciences), anti-FLAG (M2, monoclonal; Stratagene), and anti-PRLR (U5, monoclonal; Affinity Bioreagents, Golden, CO).

Construction of Constructs Used in BRET Assays; BRET Analysis
Coding regions of LF, S1a, and S1b fragments with mutated termination codon generated by PCR were ligated in-frame into the mammalian expression vector pRL-CMV-RLU (Promega) at NheI site for LF (LF-RL) or SF (S1a-RL and S1b-RL) and yellow variant GFP-topaz vector (pEYFP-NI; BD Biosciences) at KpnI site for LF (LF-Y) or SFs (S1a-Y and S1b-Y); 935 bp of the luciferase coding region with mutated termination codon generated by PCR was also subcloned in-frame into yellow variant GFP-topaz vector (pEYFP-NI) at ApaI site for positive control (RLY). All of the plasmids were verified by sequencing. Twenty-four hours after transient transfection in DMEM/10% FBS in T75 flasks, medium was replaced with serum-free DMEM/0.1% BSA and incubated in the presence or absence of PRL for 24 h. HEK293 cells were detached and washed twice in PBS. Approximately 100,000 cells per well were distributed in a 96-well microplate (black, flat-bottom, tissue culture treated; Costar, Corning, NY) and either incubated in DMEM for additional 2 h with or without PRL. Coelenterazine was added at a final concentration of 5 µM, and the reading was taken for the light emitted between 400 and 600 nm using Mithras LB940 (Berthold Technologies). BRET ratio was calculated using the following formula: [(emission at 510–590) – emission at 440–500 x Cf]/(emission at 440–500), where Cf corresponds to (emission at 510–590)/(emission at 440–500) (12) for the LF-RL or S1a-RL or S1b-RL construct transfected individually in parallel experiments.

Cell Surface Biotinylation
HEK293 cells (2.5 x 107) transiently transfected with hPRLR forms were washed with cold PBS (6x) and incubated with 0.5 mg of hexanoate [Ez-Link Sulfo-NHS-LC-Biotin (sulfosuccinimidyl-6-(biotin-amido)hexanoate); catalog no. 21335; Pierce] for 30 min at room temperature. Biotinylated cells were washed with 50 mM Tris-HCl (pH 8.0) (1x) and cold PBS (3x) and lysed in 1x PBS containing 1% (vol/vol) Nonidet P-40, 0.5% (wt/vol) sodium deoxycholate, and 0.1% (wt/vol) sodium dodecyl sulfate at 4 C for 30 min and centrifuged. Fifty microliters of streptavidin-agarose (ImmunoPure Immobilized Streptavidin Gel; catalog no. 20349; Pierce) were added to the supernatants and incubated at 4 C with rotation overnight. The streptavidin-agarose was pelleted by centrifuging at 3000 rpm, 4 C, resuspended in 50 µl loading buffer (2x; SDS Protein Gel Loading Solution; catalog no. 351-082-031; Quality Biological, Gaithersburg, MD) and boiled for 10 min in reducing experiment. In nonreducing experiment, 5% ß-mercaptoethanol was eliminated from the loading buffer, and the samples were incubated at 70 C for 5 min. In a parallel group, trypsin (0.2%, 15 min, 37 C) was used to eliminate cell surface protein before the biotinylation procedure as a negative control.

Pulse-Chase Experiments; Determination of LF Half-Life at the Cell Surface
HEK293 cells were transiently transfected with LF-YFP, S1a-RL, and S1b-RL for 24 h and briefly pulse-chase labeled with L-[35S]methionine (100 µCi/ml; Amersham Biosciences, Piscataway, NJ) for 20 min after the cells were depleted of methionine for 1 h, using the procedure described earlier (6). After the addition of unlabeled L-methionine (2 mM) to the culture, incubations were continued for the specified times before termination. Cell surface biotinylation was performed as described above. Cell lysates were immunoprecipitated with GFP antibody first. Complexes were dissociated from the primary IP by incubation with 5 mM dithiothreitol at 37 C for 20 min. Samples were reimmunoprecipitated with 50 µl streptavidin-agarose as described above. The cell surface-labeled LF-YFP were resolved by 4–20% SDS-PAGE. The specific 35S-labeling of LF was quantified by densitometry using Molecular Imager GS-800 (Bio-Rad, Hercules, CA). Individual points are the mean of three experiments. Half-lives were derived by an Exponential Decay Analysis Program.

Mutagenesis
Point mutation of cysteine in human S1b was prepared following the protocol described in the QuikChange Site-Directed Mutagenesis kit (Stratagene; catalog no. 200518). Briefly, PCR was performed using mutagenesis-grade PfuTurbo DNA polymerase with the oligonucleotide primers containing the required point mutation: C208S (forward, 5'-CCTTGTCCAGGTTCGCAGCAAACCAGACC-3'; reverse, 5'-GGT-CTGGTTTGCTGCGAACCTGGACAAGG-3'), C249S (forward, 5'-CCTTTCTGCTGTCATCAGTTTGATTATTGTCTGGGC-3'; reverse, 5'-GCCCAGACAATAATCAAACTGATGACAGCAGAAAGG-3') and C266S (forward, 5'-CTATAGCATGGTGACCAGCATCTTTCCGCC-3'; reverse, 5'-GGCGGAAAGATGCTGGTCACCATGCTATAGC-3'). The PCR product was treated with DpnI. The DpnI was used to digest the methylated parental DNA template and select for the newly synthesized DNA containing mutations. The nicked vector DNA incorporating the desired mutations was then transformed into XL1-Blue cells. The mutated plasmid was isolated, sequenced, and checked for expression.

JAK2 Phosphorylation
JAK2 phosphorylation was performed as previously described (31) with modifications. Briefly, HEK293 cells (ATCC CRL-1573) were grown in DMEM, phenol red-free medium (Invitrogen) containing 10% FBS. At 90% confluency, the cells were transiently cotransfected with the cDNAs of PRLRs (LF-RL, 0.5 µg; S1a-Y, 1 µg; and S1b-Y, 0.5 µg) and JAK2 (0.5 µg) (Homo sapiens Janus Kinase 2; Origene, Rockville, MD). Twenty-four hours after transfection, the cells were washed once with DMEM containing 0.1% BSA and then replaced with the same medium with or without PRL with a final concentration of 30 nM for 24 h. Forty-eight hours from the start of transfection, cells were washed twice with 1x PBS, and lysates were prepared using radioimmunoprecipitation assay buffer (Upstate, Charlottesville, VA) [Tris-HCl (50 mM; pH 7.4), Nonidet P-40 (1%), Na-deoxycholate (0.25%), NaCl (150 mM), EDTA (1 mM), phenylmethylsulfonyl fluoride (1 mM), aprotinin, leupeptin, pepstatin (1 µg/ml each), including phosphatase inhibitors Na3VO4 (1 mM), NaF (1 mM)]. Lysates were incubated on ice for 20 min and then centrifuged at maximum speed (16,000 x g) for 10 min at 4 C. Supernatants were taken for protein estimation and subjected to Western blot analysis using specific antibodies (anti-JAK2; Santa Cruz Biotechnology) for determination of total JAK expression and anti-phospho-JAK2 (Upstate) for assessment of JAK2 activation. Immunosignals were detected as described above.

Statistical Analysis
The significance of the differences in the BRET analyses among wild-type and Cys-mutated SFs (Cys208, Cys249, Cys266, and Cys208, 249) was determined by Dunnett’s multiple-comparison test (one-way ANOVA analysis), respectively.


   ACKNOWLEDGMENTS
 
We thank Dr. Jean Garnier (Institut National de la Recherche Agronomique, Unit Mathematique Informatique et Genome, Jouy en Josas, France) for helpful discussions during the preparation of this manuscript.


   FOOTNOTES
 
This work was supported by the Intramural Research Program of the National Institute of Child Health and Human Development of the National Institutes of Health.

First Published Online March 23, 2006

Abbreviations: aa, Amino acid; BRET, bioluminescence resonance energy transfer; CO-IP, coimmunoprecipitation; FBS, fetal bovine serum; GFP, green fluorescent protein; hPRLR, human prolactin receptor; JAK2, Janus kinase 2; LF, long form; PRL, prolactin; SF, short form; STAT5, signal transducer and activator of transcription 5; YFP, yellow fluorescent protein.

Received for publication July 15, 2005. Accepted for publication March 13, 2006.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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