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. 2016 Mar 22;30(5):504–517. doi: 10.1210/me.2015-1319

GHR/PRLR Heteromultimer Is Composed of GHR Homodimers and PRLR Homodimers

Ying Liu 1, Yue Zhang 1, Jing Jiang 1, Peter E Lobie 1, Ramasamy Paulmurugan 1, John F Langenheim 1, Wen Y Chen 1, Kurt R Zinn 1, Stuart J Frank 1,✉
PMCID: PMC4853563  PMID: 27003442

Abstract

GH receptor (GHR) and prolactin (PRL) receptor (PRLR) are homologous transmembrane cytokine receptors. Each prehomodimerizes and ligand binding activates Janus Kinase 2 (JAK2)-signal transducer and activator of transcription (STAT) signaling pathways by inducing conformational changes within receptor homodimers. In humans, GHR is activated by GH, whereas PRLR is activated by both GH and PRL. We previously devised a split luciferase complementation assay, in which 1 receptor is fused to an N-terminal luciferase (Nluc) fragment, and the other receptor is fused to a C-terminal luciferase (Cluc) fragment. When receptors approximate, luciferase activity (complementation) results. Using this assay, we reported ligand-independent GHR-GHR complementation and GH-induced complementation changes characterized by acute augmentation above basal signal, consistent with induction of conformational changes that bring GHR cytoplasmic tails closer. We also demonstrated association between GHR and PRLR in T47D human breast cancer cells by coimmunoprecipitation, suggesting that, in addition to forming homodimers, these receptors form hetero-assemblages with functional consequences. We now extend these analyses to examine basal and ligand-induced complementation of coexpressed PRLR-Nluc and PRLR-Cluc chimeras and coexpressed GHR-Nluc and PRLR-Cluc chimeras. We find that PRLR-PRLR and GHR-PRLR form specifically interacting ligand-independent assemblages and that either GH or PRL augments PRLR-PRLR complementation, much like the GH-induced changes in GHR-GHR dimers. However, in contrast to the complementation patterns for GHR-GHR or PRLR-PRLR homomers, both GH and PRL caused decline in luciferase activity for GHR-PRLR heteromers. These and other data suggest that GHR and PRLR associate in complexes comprised of GHR-GHR/PRLR-PRLR heteromers consisting of GHR homodimers and PRLR homodimers, rather than GHR-PRLR heterodimers.

GH and prolactin (PRL) are approximately 22-kDa pituitary hormones that sometimes derive locally. Human GH (hGH) and human PRL (hPRL) are 21% homologous and both are class I cytokines (1, 2), each with multiple effects. GH is anabolic and regulates metabolism (3–6). PRL is critical in breast development and lactation (7, 8). Both may also impact cancer development or behavior (9–23). GH receptor (GHR) and PRL receptor (PRLR) are type 1 transmembrane glycoprotein cytokine receptor family members with similar overall structure and substantial homology in the extracellular domains (ECDs) (24). GHR and PRLR function as dimers, with emphasis to date mostly on GHR-GHR and PRLR-PRLR homodimers that are believed to form in the absence of ligand soon after protein synthesis (25–32). Although there are some differences in signal pathways accessed by the 2 receptors, they share the propensity to respond to ligand stimulation with activation of the cytoplasmic receptor-associated tyrosine kinase, Janus Kinase 2 (JAK2), and phosphorylation of signal transducer and activator of transcription (STAT) to effect changes in gene expression (33, 34).

In humans, GH binds and activates both GHR and PRLR, whereas PRL interacts with PRLR but not GHR (35–37). Although the impact of these observations is not yet fully appreciated, GH's ability to bind both receptors suggests physiological diversification of GH actions (35–39). A further interesting feature of these receptors is the observation that GHR and PRLR can physically and functionally interact when coexpressed in cells. Previous work suggested that placental lactogen (a GH/PRL family member) may foster GHR-PRLR interaction in the cellular context (40, 41). More recently, our work with human T47D breast cancer cells that endogenously express both receptors demonstrated specific ligand-independent coimmunoprecipitation of GHR with PRLR and vice versa (42). Notably, silencing of PRLR expression in these cells enhanced GHR abundance and GH sensitivity, likely by reducing GHR protein turnover, suggesting that GHR-PRLR association may posttranslationally reciprocally regulate each receptor's trafficking (43).

Although GHR-GHR dimers and PRLR-PRLR dimers have been intensely studied, the nature of the GHR-PRLR heteromeric assemblage is less clear. We recently adapted the split luciferase complementation assay to study GHR-GHR dimers (44). In this assay, 1 protein is molecularly fused to an N-terminal part (residues 1–398) of firefly luciferase (N-terminal luciferase [Nluc]) and the other is fused to C-terminal luciferase residues (394–550) (C-terminal luciferase [Cluc]); interaction yields luciferase activity from close approximation of the enzyme parts, neither being active alone (45–47). Expression of GHR-Nluc with GHR-Cluc yielded specific complementation that was acutely augmented in response to GH treatment, consistent with preformed GHR homodimers undergoing GH-induced conformational changes that further approximate the intracellular domains (ICDs) of the receptor dimer partners (44).

In the current study, we extend these analyses to examine basal and ligand-induced complementation of coexpressed PRLR-Nluc and PRLR-Cluc chimeras and coexpressed GHR-Nluc and PRLR-Cluc chimeras. We find that both PRLR-PRLR and GHR-PRLR form specifically interacting ligand-independent assemblages and that either GH or PRL causes PRLR-PRLR dimers to undergo augmented complementation, much like the GH-induced changes in GHR-GHR dimers. However, in contrast to the complementation patterns for GHR-GHR or PRLR-PRLR homomers, both GH and PRL cause decline in luciferase activity for GHR-PRLR heteromers. These and other data suggest that GHR and PRLR associate in complexes comprised of GHR-GHR/PRLR-PRLR heteromers consisting of GHR homodimers and PRLR homodimers, rather than GHR-PRLR heterodimers.

Materials and Methods

Materials

Common molecular biology reagents were purchased from Sigma-Aldrich Corp unless otherwise noted. Restriction endonucleases were obtained from New England Biolabs. Fetal bovine serum was purchased from Atlanta Biologicals. Gentamicin sulfate, zeocin, penicillin, and streptomycin were purchased from Mediatech. Recombinant hGH was kindly provided by Eli Lilly Co. Recombinant hPRL was obtained from the National Hormone and Pituitary Program. B2036 was obtained from Pfizer, Inc. Recombinant G120R, G129R, and B2036-G129R (B-G) were produced and prepared as previously described (48, 49).

Antibodies

4G10 monoclonal antiphosphotyrosine antibody was purchased from Upstate Biotechnology, Inc, as was the anti-phospho (p)-JAK2 state-specific antibody reactive with JAK2 that is phosphorylated at residues Y1007 and Y1008. Polyclonal antiphospho-STAT5 antibody was purchased from Zymed Laboratories. Polyclonal anti-STAT5 and polyclonal anti-PRLR (H300, against hPRLR residues 322–622) antibodies were from Santa Cruz Biotechnology, Inc. Polyclonal antiphospho-STAT3 (Y701) and anti-STAT3 antibodies were purchased from Cell Signaling Technology, Inc. Polyclonal antisera against GHR (anti-GHRcyt-AL47) (50), JAK2 (anti-JAK2AL33) (51), and PRLR (anti-PRLRcyt-AL84, against the hPRLR ICD) (52) have been previously described.

Construction of PRLR, GHR, erythropoietin receptor (EPOR), estrogen receptor (ER), and luciferase chimera expression plasmids

The hPRLR cDNA in pEF/V5/HIS was a kind gift provided by Dr C. Clevenger. The full-length firefly luciferase-encoding plasmid has been described (47). The Nluc cDNA construct encoding residues 1–398 with a flexible linker (AAAGSGGGGS) on the amino terminus was created by PCR (primers available upon request) with full-length firefly luciferase as a template. This fragment was subcloned downstream of wild-type hPRLR in the context of pcDNA3.1(+)/zeo. The Cluc cDNA construct encoding firefly luciferase residues 394–550 was created in an analogous fashion and similarly subcloned downstream of wild-type hPRLR. GHR-Nluc, GHR-Cluc, EPOR-Cluc, and ER-Cluc have been previously described (44).

Cells, cell culture, and transfection

γ2A-JAK2 cells were generated by transfection of γ2A cells (53) (gift of Dr George Stark, Cleveland Clinic) with pcDNA3.1(+)/zeo-JAK2 and carried in culture, as described (54, 55). γ2A-JAK2-PRLR-Nluc cells were generated by cotransfection of γ2A-JAK2 cells with pcDNA3.1(+)/zeo-PRLR-Nluc and a hygromycin-encoding plasmid at a weight ratio of 20:1 and followed by hygromycin selection and single clone amplification. γ2A-JAK2-PRLR-Nluc/PRLR-Cluc cells were generated by cotransfection of pcDNA3.1(+)/zeo-PRLR-Nluc, pcDNA3.1(+)/zeo-PRLR-Cluc, and a hygromycin-encoding plasmid at a weight ratio of 9.5:9.5:1 and followed by hygromycin selection and single clone amplification. γ2A-JAK2-GHR-Nluc/PRLR-Cluc cells were generated by cotransfection of pcDNA3.1(+)/zeo-GHR-Nluc, pcDNA3.1(+)/zeo-PRLR-Cluc, and a hygromycin-encoding plasmid at a weight ratio of 9.5:9.5:1 and followed by hygromycin selection and single clone amplification. γ2A-JAK2-PRLR-Nluc cells, γ2A-JAK2-PRLR-Nluc/PRLR-Cluc cells, and γ2A-JAK2-GHR-Nluc/PRLR-Cluc cells were maintained in 200-μg/mL hygromycin-containing γ2A-JAK2 cell medium. Transient transfection was achieved by introducing pcDNA 3.1-driven plasmids encoding receptor-luciferase (luc) chimeras (1 pmol DNA for single plasmid transfection and 0.7 pmol DNA for receptor-Nluc-encoding plasmid/0.35 pmol DNA for receptor-Cluc-encoding plasmid for cotransfection per transfection in a 6-cm2 dish), using Lipofectamine LTX Plus (Invitrogen) according to the manufacturer's instructions.

Bioluminescence imaging

Transfected cells expressing the indicated receptor-luc chimeras were seeded in 96-well black wall plates (3.6 × 104 cells/well). Six hours before experimentation, medium was replaced by serum-free medium, which was changed just before addition of reagents and imaging to imaging medium (175 μL/well; composed of phenol red-free DMEM, 1-g/L glucose, 1-mg/mL D-luciferin, 25mM HEPES [pH 7.5], and 0.1% wt/vol BSA) at room temperature for 10 minutes. Baseline bioluminescence signal (photons/s per cm2/steradian) was collected at 5-minute intervals for 20 minutes at 37°C using an IVIS 100 system (PerkinElmer; no filter; F-stop, 1; field of view, level B; 5-min exposure with Bin 8). Ligand (GH, PRL, or B-G) was added in a volume of 25 μL/well to its indicated final concentration, and sequential bioluminescence imaging commenced immediately thereafter at 5-minute intervals, as described above. For addition of antagonists, cells were preincubated for 30 minutes with B2036 (20 μg/mL), G129R (20 μg/mL), or G120R (20 μg/mL) and followed by the same experimental procedures.

Protein extraction, immunoprecipitation, and immunoblotting

Cells were serum starved for 6 hours, with or without preincubation of B2036 (20 μg/mL), G129R (20 μg/mL), or G120R (20 μg/mL) for 30 minutes, treated with GH (500 ng/mL), PRL (500 ng/mL), or B-G (5 μg/mL) at 37°C for 10 minutes, unless otherwise indicated, and lysed. Cell lysates were immunoprecipitated with antibody against GHR (anti-GHRcyt-AL47) or PRLR (anti-GHRcyt-AL84) with preimmune serum as control. Eluates were resolved by SDS-PAGE, and immunoblotted, as indicated. Cells prepared in parallel with those described in bioluminescence assay were plated in 6-well plates (5.4 × 105 cells/well) and serum starved before treatment with antagonists, as above, and stimulation with GH, PRL, or B-G, as described. Detergent extraction, electrophoresis, and immunoblotting of tissue culture cells was performed as described previously (56, 57).

Statistical analysis and figure preparation

For bioluminescence complementation, each experimental condition was assessed in triplicate wells or indicated wells of 96-well plate. Each well was defined as a region of interest that generates a bioluminescence value expressed as total flux (photons per second). The percentage change of complementation signal was calculated by dividing the total flux value from GH-, PRL-, or B-G-treated wells by baseline total flux value from this same set of wells with or without antagonist preincubation. Data are expressed as mean ± SE of ligand-induced signal as a percentage above baseline signal (n = 3 unless otherwise indicated). Two sample t tests with equal variance and pooled SE were employed for statistical analysis. P < .05 was considered as significant. Immunoblots and split luciferase complementation data shown are in all instances representative of at least 3 experiments. In generating figures, irrelevant intervening lanes from original immunoblots have been cropped for clarity of presentation. In these cases, a space is maintained where intervening lanes were cropped. In all cases, only data from the same original blots are incorporated in figures with consistent brightness/contrast adjustment made across each blot.

Results

Specific PRLR-PRLR interaction is detected by the split luciferase complementation assay

Parallel to our studies of GHR-GHR complementation, we sought to employ the split luciferase complementation assay (45) to study PRLR-PRLR association. We first created hPRLR-Nluc and PRLR-Cluc chimera proteins with a flexible 10-residue linker (AAAGSGGGGS) between receptor and luc fragments (Figure 1A). For comparison, our previously described GHR-Nluc, GHR-Cluc, EPOR-Cluc, and ER fragment-Cluc (44) are also shown in Figure 1A. As previously, we employed the JAK2-expressing human fibrosarcoma cell line, γ2A-JAK2 (53–55), as a host for expression of the chimeras. An expression vector encoding wild-type PRLR was used as a positive control and vector-only as a negative control for experiments comparing transient transfection of vectors encoding PRLR-Nluc and PRLR-Cluc. Immunoblotting of detergent extracts of transfected cells with anti-PRLR (Figure 1B) revealed that both chimeras were expressed and detected, as expected, at the appropriate Mr. Further, both GH and PRL caused acute activation of signaling, as detected by immunoblotting with anti-pSTAT3/anti-STAT3 (Figure 1C), indicating appropriate folding and cell surface presentation of PRLR-luc chimera receptors, as previously shown for the GHR-luc and EPOR-luc chimeras (44).

Figure 1.

Figure 1.

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Specific luciferase complementation of PRLR-Nluc with PRLR-Cluc. A, Schematic diagram of luciferase fragment chimeras used in this study. All constructs described in the text and in Materials and Methods. PRLR-Nluc and PRLR-Cluc: full-length hPRLR fused at the end of the ICD to either Nluc or Cluc, respectively, via a linker peptide. GHR-Nluc and GHR-Cluc: full-length human GHR fused at the end of the ICD to either Nluc or Cluc via a linker peptide. EPOR-Cluc: full-length human EPOR fused at the end of the ICD to Cluc via a linker peptide. ER-Cluc: human ER fragment fused to Cluc. B, PRLR-Nluc and PRLR-Cluc are specifically immunodetectable and exhibit expected SDS-PAGE migration. γ2A-JAK2 cells were transiently transfected to express PRLR, PRLR-Nluc, PRLR-Cluc or with empty vector pcDNA3.1(+)/zeo. Detergent cell extracts were resolved by SDS-PAGE and immunoblotted with anti-PRLR (H-300). PRLR and PRLR-luc chimeras are indicated by an asterisk and exhibit the expected migration. NS, nonspecific. C, PRLR-Nluc and PRLR-Cluc allow normal acute ligand-induced signaling. γ2A-JAK2 cells were transfected to express PRLR, PRLR-Nluc, PRLR-Cluc, or with empty vector pcDNA3.1(+)/zeo. Treatment: ±GH or PRL. Immunoblot of cell extracts for phosphorylated and total STAT3. D, Specific luciferase complementation of PRLR-Nluc with PRLR-Cluc. γ2A-JAK2 cells were transiently transfected with expression plasmids encoding the indicated chimeras. Bioluminescence was determined in triplicate (inset shows actual color-coded signals) and is displayed graphically as mean ± SE of total flux (photons/s × 1000). For PRLR-Nluc/PRLR-Cluc vs PRLR-Nluc/EPOR-Cluc or PRLR-Nluc/ER-Cluc, P < .05, respectively. See Materials and Methods for details.

To assess interaction of receptors, we transiently coexpressed PRLR-Nluc with PRLR-Cluc in γ2A-JAK2 and measured reconstitution of luciferase activity (complementation of Nluc with Cluc) using IVIS 100 bioluminescence imaging of living transfected cells in the presence of D-luciferin (Figure 1D). Substantial complementation resulted with PRLR-Nluc/PRLR-Cluc coexpression, whereas very little signal was detected with PRLR-Nluc/EPOR-Cluc or PRLR-Nluc/ER-Cluc coexpression. This result suggests ligand-independent specific preassociation of PRLRs, consistent with studies using different methods that demonstrated ligand-independent PRLR dimer formation (25, 31).

Effects of ligand treatment on PRLR-PRLR complementation

To investigate ligand effects on PRLR-PRLR complementation, we isolated γ2A-JAK2-PRLR-Nluc/PRLR-Cluc cells, in which PRLR-Nluc and PRLR-Cluc are stably coexpressed in the γ2A-JAK2 cell background. As in the transient transfections in Figure 1, PRLR-Nluc and PRLR-Cluc were detected in γ2A-JAK2-PRLR-Nluc/PRLR-Cluc cells by anti-PRLR immunoprecipitation and immunoblotting, and both chimeras were detected by anti-pY immunoblotting after brief treatment of the cells with PRL (Figure 2A, upper panel). In this stable clone, PRLR-Nluc was of slightly greater abundance and was correspondingly more inducibly phosphorylated in response to PRL than was PRLR-Cluc. As expected, PRL also caused acute phosphorylation of both JAK2 and STAT5, as detected by immunoblotting of cell extracts (Figure 2A, lower panel).

Figure 2.

Figure 2.

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PRL and GH specifically induce temporal changes in PRLR-PRLR complementation. A, PRL signaling in γ2A-JAK2-PRLR-Nluc/PRLR-Cluc cells, in which PRLR-Nluc and PRLR-Cluc are stably expressed in the γ2A-JAK2 background. Treatment: ±PRL. Upper panel, Detergent cell extracts were immuno-precipitated with anti-PRLR (anti-PRLRcyt-AL84), resolved by SDS-PAGE, and sequentially immunoblotted with anti-pY and anti-PRLR (anti-PRLRcyt-AL84). Lower panel, Immunoblot of cell extracts for anti-pJAK2, anti-JAK2, anti-pSTAT5, and anti-STAT5. The black arrow heads indicate phosphorylated or total PRLR-Nluc (upper band) and phosphorylated or total PRLR-Cluc (lower band). B, PRL concentration-dependent changes in complementation. After basal bioluminescence was determined in γ2A-JAK2-PRLR-Nluc/PRLR-Cluc cells, PRL at indicated concentrations was added, and bioluminescence was serially determined at 5-minute intervals over 40 minutes. Data are expressed as mean ± SE of PRL-induced signal as a percentage above baseline signal (n = 3 per condition). For 100-ng/mL PRL vs 250-ng/mL PRL or 500-ng/mL PRL, P < .05 at each time point, respectively. For 250-ng/mL PRL vs 500-ng/mL PRL, P < .05 at 35 and 40 minutes. C, GH concentration-dependent changes in complementation. After basal bioluminescence was determined in γ2A-JAK2-PRLR-Nluc/PRLR-Cluc cells, GH at indicated concentrations was added, and bioluminescence was serially determined. See Materials and Methods for details. For 100-ng/mL GH vs 500-ng/mL GH, P < .05 at each time point. For 100-ng/mL GH vs 250-ng/mL GH, P < .05 at 25 and 30 minutes. D, PRLR-specific antagonist, G129R, inhibits GH- and PRL-induced JAK2 activation in γ2A-JAK2-PRLR-Nluc/PRLR-Cluc cells. Preincubation: ±G129R (20 μg/mL). Treatment: ±GH or PRL. Immunoblot of cell extracts for phosphorylated and total JAK2. E, G129R alone does not affect PRLR-PRLR basal complementation. γ2A-JAK2-PRLR-Nluc/PRLR-Cluc cells were preincubated with vehicle or G129R for 30 minutes, after which bioluminescence was determined. Data are displayed graphically as mean ± SE total flux (photons/s × 1000). No significant difference was detected (n = 12 per condition). F, PRLR-specific antagonist, G129R, inhibits PRL-induced changes in PRLR-PRLR complementation. γ2A-JAK2-PRLR-Nluc/PRLR-Cluc cells were preincubated ± G129R and followed by PRL (500 ng/ml) treatment, after which bioluminescence was serially determined. See Materials and Methods for details. For PRL vs PRL+G129R, P < .05 at each time point. G, PRLR-specific antagonist, G129R, inhibits GH-induced changes in PRLR-PRLR complementation. γ2A-JAK2-PRLR-Nluc/PRLR-Cluc cells were preincubated ± G129R and followed by GH (500 ng/ml) treatment, after which bioluminescence was serially determined. See Materials and Methods for details. For GH vs GH+G129R, P < .05 at each time point.

Because hPRLR is engaged by both PRL and GH (Table 1), we tested ligand effects on PRLR-Nluc/PRLR-Cluc complementation in time-course experiments with varying concentrations of PRL (Figure 2B) and GH (Figure 2C). In each case, after detection of basal bioluminescence, ligand was added to γ2A-JAK2-PRLR-Nluc/PRLR-Cluc cells and bioluminescence was sequentially measured at 5-minute intervals for 40 minutes. PRL rapidly augmented basal bioluminescence in a dose-dependent fashion, increasing on average by approximately 45% after 15 minutes with 500-ng/mL PRL. Thereafter, the signal decreased to approximately 30%–35% above basal by 30–40 minutes. (Figure 2B). Like PRL, GH treatment also dose dependently augmented PRLR-Nluc/PRLR-Cluc complementation by nearly 70% after 15 minutes with 500-ng/mL GH (Figure 2C). These patterns were reminiscent of that previously seen for GH treatment of cells expressing GHR-Nluc/GHR-Cluc (44) and likewise suggest that both PRL and GH enhance the approximation of the C-terminal tails of PRLR-Nluc and PRLR-Cluc within a preformed PRLR dimer.

Table 1.

Binding Sites of Ligands and Antagonists Against Receptor Dimers

graphic file with name zmg00516-5669-t01.jpg

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G129R is a hPRL mutant with the glycine residue at position 129 of PRL substituted with arginine; the net effect is to reduce PRL's site 2 binding affinity to PRLR and thus G129R behaves as a PRLR-specific antagonist (58, 59), both for GH and PRL (Table 1). As expected, treatment of γ2A-JAK2-PRLR-Nluc/PRLR-Cluc cells with G129R itself did not trigger signaling; however, G129R completely blocked both GH- and PRL-induced JAK2 phosphorylation (Figure 2D). Consistent with these signaling data, G129R itself did not alter PRLR-Nluc/PRLR-Cluc basal complementation (Figure 2E) but completely blocked both PRL-induced (Figure 2F) and GH-induced (Figure 2G) augmentation of PRLR-Nluc/PRLR-Cluc complementation. This strongly suggests that the ligand-induced augmentation of PRLR-Nluc/PRLR-Cluc complementation reflects the proximal events of PRLR activation upon ligand-binding.

GHR-PRLR interaction and hetero-complementation

In T47D human breast cancer cells that endogenously express both GHR and PRLR, the 2 receptors are specifically coimmunoprecipitated independent of ligand binding (42), suggesting that GHR and PRLR interact in intact cells, although the stoichiometry and architecture of GHR-PRLR complexes are unknown. We pursued this association further with our split luciferase complementation assay, employing γ2A-JAK2 cells that stably express PRLR-Nluc (γ2A-JAK2-PRLR-Nluc) (Figure 3A). γ2A-JAK2-PRLR-Nluc cells were transiently transfected with either GHR-Cluc, PRLR-Cluc, EPOR-Cluc, or ER-Cluc. As expected, PRLR-Cluc expression resulted in substantial specific PRLR-Nluc/PRLR-Cluc complementation. Notably, similar complementation was observed with PRLR-Nluc/GHR-Cluc coexpression. This is consistent with our previous GHR-PRLR coimmunoprecipitation findings.

Figure 3.

Figure 3.

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Specific luciferase hetero-complementation of PRLR-luc with GHR-luc and the ligands effect. A, Specific luciferase complementation of PRLR-Nluc with GHR-Cluc. γ2A-JAK2-PRLR-Nluc cells were transiently transfected with expression plasmids encoding the indicated chimeras. Bioluminescence was determined in triplicate (inset shows actual color-coded signals) and is displayed graphically as mean ± SE total flux (photons/s × 1000). For GHR-Cluc vs EPOR-Cluc or ER-Cluc, P < .05. For PRLR-Cluc vs EPOR-Cluc or ER-Cluc, the value was P < .05. See Materials and Methods for details. B, Characterization of GNR-Nluc and PRLR-Cluc double stable cells. Serum-starved γ2A-JAK2-GHR-Nluc/PRLR-Cluc cells were treated ± GH or PRL. Left panel, Detergent cell extracts were immuno-precipitated with anti-GHR (anti-GHRcyt-AL47) or anti-PRLR (anti-PRLRcyt-AL84), resolved by SDS-PAGE and sequentially immunoblotted with anti-pY and anti-GHR (anti-GHRcyt-AL47) or anti-PRLR (anti-PRLRcyt-AL84). Right panel, Immunoblot of cell extracts for phosphorylated and total STAT5 and phosphorylated and total STAT3. C, GH concentration-dependent changes in hetero-complementation. After basal bioluminescence was determined in γ2A-JAK2-GHR-Nluc/PRLR-Cluc cells, GH at indicated concentrations was added, and bioluminescence was serially determined. See Materials and Methods for details. For 50-ng/mL GH vs 100-ng/mL GH, P < .05 at each time point from 20 to 40 minutes. For 50-ng/mL GH vs 250-ng/mL GH, P < .05 at each time point except 10 and 15 minutes. For 50-ng/mL GH vs 500-ng/mL GH, P < .05 at each time point except 5 minutes. For 50-ng/mL GH vs 1000-ng/mL GH, P < .05 at each time point except 5 minutes. For 100-ng/mL GH vs 250-ng/mL GH, P < .05 at each time point except 10 and 15 minutes. For 100-ng/mL GH vs 500-ng/mL GH, P < .05 at each time point except 5 minutes. For 100-ng/mL GH vs 1000-ng/mL GH, P < .05 at each time point except 5 minutes. For 250-ng/mL GH vs 500-ng/mL GH or 1000-ng/mL GH, P < .05 at each time point. D, PRL concentration-dependent changes in hetero-complementation. After basal bioluminescence was determined in γ2A-JAK2-GHR-Nluc/PRLR-Cluc cells, PRL at indicated concentrations was added and bioluminescence was serially determined. See Materials and Methods for details. For 50-ng/mL PRL vs 100–ng/mL PRL, P < .05 at 25, 30, and 40 minutes. For 50-ng/mL PRL vs 250-ng/mL PRL or 500-ng/mL PRL, P < .05 at each time point except 5 minutes. For 50-ng/mL PRL vs 1000-ng/mL PRL, P < .05 at each time point. For 100-ng/mL PRL vs 250-ng/mL PRL or 500-ng/mL PRL or 1000-ng/mL PRL, P < .05 at each time point. For 250-ng/mL PRL vs 500-ng/mL PRL, P < .05 at 20 and 25 minutes. For 250-ng/mL PRL vs 1000-ng/mL PRL, P < .05 from 10 to 25 minutes.

Because GH activates both GHR and PRLR, we reason that the GHR-PRLR association may have biologically relevant consequences. To better understand the nature of the GHR-PRLR heteromer and the effects of ligand treatment, we isolated γ2A-JAK2-GHR-Nluc/PRLR-Cluc cells that stably express GHR-Nluc and PRLR-Cluc and exhibit substantial basal luciferase complementation (data not shown). As anticipated, GH triggered phosphorylation of both GHR-Nluc and PRLR-Cluc, and PRL only triggered phosphorylation of PRLR-Cluc (Figure 3B, left panel). Likewise, both GH and PRL triggered cellular STAT3 and STAT5 signaling (Figure 3B, right panel). Thus, both GHR-Nluc and PRLR-Cluc are amply expressed at the cell surface and normally allow ligand engagement and activation of signaling in these cells.

We next examined effects of GH or PRL treatment on GHR-Nluc/PRLR-Cluc complementation. Notably, and in sharp contrast to the augmentation of complementation by GH on GHR-Nluc/GHR-Cluc and PRLR-Nluc/PRLR-Cluc, GH treatment of γ2A-JAK2-GHR-Nluc/PRLR-Cluc cells yielded largely a dose-dependent decline in complementation over a 40-minute treatment duration, most notable after 15 minutes (Figure 3C). This suggests that a GHR-PRLR heteromer behaves quite differently in response to GH than does a GHR-GHR homodimer or a PRLR-PRLR homodimer. In principle, one possible arrangement of the heteromer is in the form of a GHR-PRLR heterodimer per se. This would imply that GH could bind to the heterodimer partners to somehow effect separation of their tails and resultant diminished complementation. However, another possible arrangement would be that the heteromer is comprised of GHR-Nluc/GHR-Nluc homodimers and PRLR-Cluc/PRLR-Cluc homodimers in a hetero-oligomeric complex. In this case, basal complementation would result from interaction of GHR-Nluc with PRLR-Cluc, each within nearby homodimers, and GH binding to each homodimer would result in the noncomplementing receptor tails within dimers moving closer together, but the complementing tails between dimers moving away from each other, thereby lessening the complementation signal. To test this, we asked what effect PRL treatment would have, reasoning that PRL cannot engage a GHR-PRLR heterodimer or a GHR-GHR homodimer, but rather only a PRLR-PRLR homodimer. Interestingly, PRL treatment of γ2A-JAK2-GHR-Nluc/PRLR-Cluc cells (Figure 3D) resulted in a dose-dependent decline in complementation, much like that seen in response to GH, favoring the hypothesis that the GHR-PRLR assemblage engaged by both ligands is one of heteromeric GHR-GHR and PRLR-PRLR dimers, rather than GHR-PRLR heterodimers. This heteromeric assemblage may be represented as a (GHR-GHR)x/(PRLR-PRLR)y multimer, where expression levels of each receptor might define x and y and thus the stoichiometry of the complex (eg, a dimer of dimers if x=y = 1, but a hexamer including 3 dimers if x = 1 and y = 2, for instance).

Effects of GHR and PRLR antagonists on GHR-PRLR hetero-complementation

G120R is a hGH mutant with a Gly-to-Arg substitution at residue 120. This substitution results in dramatically reduced site 2 binding affinity for interaction with both GHR and PRLR, but site 1 binding affinity remains intact; thus G120R functions as an antagonist against both GHR and PRLR (Table 1) (60). B2036 is another analog of hGH with a Gly-to-Lys change at residue 120 that reduces site 2 binding affinity, along with 8-amino acid substitutions at binding site 1 that specifically increase site 1 binding affinity to GHR, resulting in a GHR-specific antagonist (Table 1) (61). As above, G129R is a PRL mutant with a Gly-to-Arg substitution at residue 129 (site 2) that behaves as a PRLR-specific antagonist of both GH and PRL (Table 1) (58, 59). We tested the effects of each of these antagonists, G120R, B2036, and G129R, on GHR/PRLR hetero-complementation in γ2A-JAK2-GHR-Nluc/PRLR-Cluc cells. Notably, none of the antagonists, each of which has only one site capable of binding GHR (G120R and B2036) or PRLR (G120R and G129R), altered basal GHR-Nluc/PRLR-Cluc complementation (Figure 4A), confirming that single site binding is not sufficient to either reduce or increase proximity of the distal tails within heteromers.

Figure 4.

Figure 4.

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Effect of antagonists on GHR-PRLR hetero-complementation. A, Neither the GHR-specific antagonist, B2036, the PRLR-specific antagonist, G129R, nor the general GHR and PRLR antagonist, G120R, affects basal GHR-PRLR hetero-complementation. γ2A-JAK2-GHR-Nluc/PRLR-Cluc cells were treated with vehicle, B2036, G129R, or G120R for 30 minutes, after which bioluminescence was determined. Data are displayed graphically as mean ± SE total flux (photons/s × 1000). No significant difference was detected (n = 9 per condition). B, Effect of G129R on PRL-induced GHR-PRLR hetero-complementation change. Serum-starved γ2A-JAK2-GHR-Nluc/PRLR-Cluc cells were preincubated with or without G129R and then treated with PRL (500 ng/ml). Bioluminescence was measured serially thereafter. See Materials and Methods for details. For PRL vs PRL+G129R, P < .05 at each time point. C, Effect of G120R on GH-induced GHR-PRLR hetero-complementation change. Serum-starved γ2A-JAK2-GHR-Nluc/PRLR-Cluc cells were preincubated with or without G120R and then treated with GH (500 ng/ml). Bioluminescence was measured serially thereafter. See Materials and Methods for details. For GH vs GH+G120R, P < .05 at each time point except 5 minutes. D, Comparison of G129R and B2036 effects on GH-induced GHR-PRLR hetero-complementation change. Serum-starved γ2A-JAK2-GHR-Nluc/PRLR-Cluc cells were preincubated ± G129R or B2036 and then treated with GH (500 ng/ml). Bioluminescence was measured serially thereafter. See Materials and Methods for details. The curve with dashed line and solid circles reflects GH's effect in the presence of G129R, presumably via GH's binding to GHR-GHR homodimers; The curve with solid line and empty circles reflects GH's effect in the presence of B2036, presumably via GH's binding to PRLR-PRLR homodimers. The curve with solid line and empty triangles was generated by mathematically combining the value at each time point of (GH+B2036) with that of (GH+G129R); thus, no error bars were applied. This combination tracing nearly overlapped with the experimental tracing reflecting GH-induced changes (dashed line and solid triangles), suggesting that GH's effect on hetero-complementation is the net effect of its independent binding to GHR-GHR homodimers and PRLR-PRLR homodimers. E, GH-induced complementation changes via each receptor are proportional to relative abundance of the receptors in different stable clone cells. Left panel, Cell lysates of 2 GHR-Nluc/PRLR-Cluc stable clones (γ2A-JAK2-GHR-Nluc/PRLR-Cluc cells and γ2A-JAK2-GHR-Nluc/PRLR-Cluc-2 cells) were resolved by SDS-PAGE and sequentially immunoblotted with anti-GHR (anti-GHRcyt-AL47) and with anti-PRLR (anti-PRLRcyt-AL84). γ2A-JAK2-GHR-Nluc/PRLR-Cluc-2 cells had a higher GHR to PRLR ratio than γ2A-JAK2-GHR-Nluc/PRLR-Cluc. Right panel, Stacked bar graphs showing the antagonists' effects on hetero-complementation in the 2 GHR-Nluc/PRLR-Cluc stable clones at time 40 minutes after GH (500 ng/ml) treatment. Black bar, GH's effect via PRLR-PRLR homodimers; the value was calculated as the complementation change from (GH+B2036) divided by that from GH. Gray bar, GH's effect via GHR-GHR homodimers; the value was calculated as the complementation change from (GH+G129R) divided by that from GH. Note that the sum of the 2 fractions is approximately 1 in each clone. Each bar combines data from 4 independent experiments and is displayed as fraction mean ± SE. Note that γ2A-JAK2-GHR-Nluc/PRLR-Cluc-2, with a greater GHR to PRLR ratio than γ2A-JAK2-GHR-Nluc/PRLR-Cluc, also had a greater proportion of complementation change via GHR-GHR homodimers (GH+G129R).

We also tested the effects of the antagonists on ligand-induced GHR-Nluc/PRLR-Cluc complementation changes. Although it did not affect basal hetero-complementation, G129R completely inhibited the PRL-induced decline of GHR-Nluc/PRLR-Cluc complementation (Figure 4B), consistent with the conclusion that the PRL-induced decrease is via its binding to the PRLR-PRLR homodimers within a (GHR-GHR)x/(PRLR-PRLR)y multimer.

In contrast to PRL, GH can engage both GHR-GHR and PRLR-PRLR. Consistent with this, GH's effect on GHR-Nluc/PRLR-Cluc complementation was entirely prevented by G120R, which blocks GH binding to both receptors (Figure 4C). Notably, selective inhibition of GH binding to GHR (with B2036) vs to PRLR (with G129R) altered GH-induced GHR-Nluc/PRLR-Cluc complementation changes in differing ways (Figure 4D). In principle, B2036's antagonism of GH's binding to GHR-GHR homodimers should not impair GH's binding to PRLR-PRLR homodimers; thus, the effects on hetero-complementation of (GH+B2036) are GH-induced changes via PRLR-PRLR homodimers (Figure 4D). Similarly, G129R's antagonism of GH's binding to PRLR-PRLR homodimers should not impair GH's binding to GHR-GHR homodimers; thus the effects on hetero-complementation of (GH+G129R) are GH-induced changes via GHR-GHR homodimers (Figure 4D). We reasoned that the GH-induced changes on hetero-complementation are the net changes from its independent binding to each receptor homodimer. To address this, we mathematically “combined” GH's effect via its binding to PRLR-PRLR homodimers (GH+B2036) with that via its binding to GHR-GHR (GH+G129R) and plotted a “combination tracing,” representing the net effects from GH's binding to each receptor homodimer (Figure 4D). Interestingly, the combined GH effect virtually overlapped with the experimental tracing arising from GH treatment alone (Figure 4D), strongly supporting the (GHR-GHR)x/(PRLR-PRLR)y multimer model.

To pursue this idea further, we isolated and tested a separate transfectant clone coexpressing GHR-Nluc and PRLR-Cluc. In this clone, designated γ2A-JAK2-GHR-Nluc/PRLR-Cluc-2 cells, the ratio of GHR-Nluc to PRLR-Cluc is greater than that in JAK2-GHR-Nluc/PRLR-Cluc cells by immunoblotting (Figure 4E, left panel). Intriguingly, consistent with its greater GHR-Nluc to PRLR-Cluc ratio, γ2A-JAK2-GHR-Nluc/PRLR-Cluc-2 has a greater proportion of GH's effect via GHR-GHR homodimers (GH+G129R) vs its effect via PRLR-PRLR homodimers (GH+B2036) than seen in γ2A-JAK2-GHR-Nluc/PRLR-Cluc (Figure 4E, right panel). Collectively, these data suggest a (GHR-GHR)x/(PRLR-PRLR)y multimer model, in which GHR-GHR and PRLR-PRLR homodimers are activated independently by their corresponding ligands and in proportion relative to the relationship between x and y. In contrast, these data do not support a scenario in which biofunctional GHR-PRLR heterodimers form and are activated per se by GH.

Effects of a novel GHR-PRLR agonist on GHR-PRLR hetero-complementation

B-G is a recombinant protein in which B2036 and G129R are fused in tandem with B2036 situated N-terminal to G129R (Figure 5A); this combined GHR-PRLR antagonist, by virtue of possessing single binding sites for each receptor (Table 1), can be an agonist in cells that express both GHR and PRLR, but not in cells expressing only GHR or PRLR (49). Indeed, B-G treatment of our γ2A-JAK2-GHR-Nluc/PRLR-Cluc cells resulted in acute tyrosine phosphorylation of both GHR and PRLR, as well as phosphorylation of STAT3 (Figure 5B), suggesting that both receptors are engaged by B-G. As expected, B-G treatment did not alter complementation in cells expressing GHR-Nluc/GHR-Cluc or PRLR-Nluc/PRLR-Cluc (data not shown). In contrast, B-G acutely and dose dependently augmented GHR/PRLR hetero-complementation in γ2A-JAK2-GHR-Nluc/PRLR-Cluc cells (Figure 5C). We emphasize that this B-G-induced pattern is dramatically different from that observed with either GH (Figure 3C) or PRL (Figure 3D) in the hetero-complementation setting. The augmentation of GHR-Nluc/PRLR-Cluc complementation from B-G was completely inhibited by either B2036 alone or G129R alone (Figure 5D), confirming that B-G engages both GHR and PRLR to affect GHR-PRLR heteromer complementation. In the context of the data presented in Figures 3 and 4, the data with B-G in Figure 5 may conform with the (GHR-GHR)x/(PRLR-PRLR)y multimer model, in that B-G binds to 1 GHR monomer within a GHR-GHR dimer and to 1 PRLR monomer within a nearby PRLR-PRLR dimer in the hetero-multimer. This B-G binding is envisioned to exert different effects on intracellular tail approximation (bringing GHR-Nluc within one GHR-Nluc-GHR-Nluc dimer into proximity with PRLR-Cluc within a PRLR-Cluc-PRLR-Cluc dimer to augment complementation) compared to GH or PRL (each bringing monomers within homodimers together and separating homodimers within the hetero-multimers).

Figure 5.

Figure 5.

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Effect of a novel GHR-PRLR agonist on GHR-PRLR hetero-complementation. A, Diagram of B-G: a novel GHR-PRLR agonist. B2036 and G129R are fused in tandem with a single binding site (site 1, in red) on B2036 for GHR and a single binding site (site 1, in yellow) on G129R for PRLR, respectively. Yellow “X” indicates the mutated site 2 of GH on B2036 or mutated site 2 of PRL on G129R. B, Agonist B-G induces phosphorylation of GHR and PRLR and triggers cellular signaling in γ2A-JAK2-GHR-Nluc/PRLR-Cluc cells. Treatment: ±B-G. Detergent cell extracts were immuno-precipitated with anti-GHR (anti-GHRcyt-AL47) (upper panel) or anti-PRLR (anti-PRLRcyt-AL84) (middle panel), resolved by SDS-PAGE and sequentially immunoblotted with anti-pY and anti-GHR (anti-GHRcyt-AL47) or anti-PRLR (anti-PRLRcyt-AL84). Lower panel, Immunoblot of cell extracts for phosphorylated and total STAT3. C, B-G induces concentration-dependent augmentation in hetero-complementation. After basal bioluminescence was determined in γ2A-JAK2-GHR-Nluc/PRLR-Cluc cells, B-G at indicated concentrations was added, and bioluminescence was serially determined. See Materials and Methods for details. For 0.5-μg/mL B-G vs 1-μg/mL B-G, P < .05 at each time point except 35 minutes. For 0.5-μg/mL B-G vs 5-μg/mL B-G or 10-μg/mL B-G, P < .05 at each time point. For 1-μg/mL B-G vs 5-μg/mL B-G or 10-μg/mL B-G, P < .05 at each time point except 35 and 40 minutes. For 5-μg/mL B-G vs 10-μg/mL B-G, P < .05 at 5 minutes. D, G129R or B2036 each completely antagonizes B-G-induced hetero-complementation changes. Serum-starved γ2A-JAK2-GHR-Nluc/PRLR-Cluc cells were preincubated ± G129R or B2036 and then treated ± B-G (1 μg/mL). Bioluminescence was measured serially thereafter. See Materials and Methods for details. For B-G vs B-G+G129R or B-G+B2036, P < .05 at each time point.

Discussion

GHR and PRLR are canonical hormone receptor members of the cytokine receptor superfamily that have both ordered and disordered elements of similarity in the ECDs and ICDs, respectively (24, 62). In humans, both receptors bind and can be activated by GH, but PRL binds and activates only PRLR (35–37). GHR is expressed in most cells, albeit at varying levels. PRLR is highly expressed mainly in reproductive tissues and breast epithelium. The different expression levels of these receptors among tissues and in ontogeny bespeak their important regulatory roles in growth, metabolism, and reproductive biology. In addition, both receptors have been implicated in oncogenesis and/or cancer behavior. We have been interested in understanding how GHR and PRLR, when coexpressed in cells, function and/or interact relative to each other (42, 43). In these recent studies, we have been intrigued by the tendency of GHR and PRLR to physically associate, as determined by specific coimmunoprecipitation, in cells that naturally express both receptors and by the influence of PRLR, presumably by virtue of this interaction, on steady state GHR levels. Although substantial information exists on ligand-independent dimerization of GHR with GHR and PRLR with PRLR (25–32), the nature of the GHR-PRLR association (eg, stoichiometry, interaction determinants, organizing principles) is largely unknown. We reason that better understanding of these aspects of GHR-PRLR association will be highly relevant for our understanding of both the physiology and pathophysiology of GH and PRL action, particularly in humans.

We have begun to approach this issue by using our luciferase complementation assay, exploiting its sensitivity and specificity, building on our previous study of GHR-GHR complementation (44), and employing specific and nonspecific GHR and PRLR antagonists. We first explored PRLR-PRLR complementation and found abundant and specific ligand-independent PRLR-Nluc/PRLR-Cluc complementation that was augmented in response to either GH or PRL, akin to GHR-GHR complementation augmentation by GH. We interpret this to indicate that PRLR-PRLR homodimers are engaged by GH or PRL and that such engagement enhances closeness of the distal tails of the PRLR monomers within the dimer (Figure 6A). In contrast, we interpret our findings concerning coexpression of GHR and PRLR to indicate that the basal (ligand-independent) arrangement is as a (GHR-GHR)x/(PRLR-PRLR)y multimer (Figure 6B), in which x and y are determined by relative GHR and PRLR abundance. In this model, GHR and PRLR are envisioned to interact between homodimers via surfaces, most likely in the extracellular and/or transmembrane domains, other than those surfaces involved in homomeric association within dimers. This conclusion is based on the responses to GH (Figure 6C) and PRL (Figure 6D), in which both ligands lessened, rather than augmented, GHR/PRLR complementation in a fashion that use of GHR and PRLR antagonists revealed was related to GH and PRL binding to homodimeric (GHR-GHR and PRLR-PRLR) assemblages within the GHR-PRLR heteromer. Further supporting this model is the effect of B-G (Figure 6E) that augments GHR-PRLR complementation. We take this result to indicate that B-G, by virtue of the extended reach of this tandem molecule and that it has within it 1 GHR and 1 PRLR binding site, is able to engage GHR and PRLR monomers that are each situated within a homodimer component of the hetero-multimer.

Figure 6.

Figure 6.

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Model of (GHR-GHR)x/(PRLR-PRLR)y multimer. A, PRLRs exist as preformed homodimers that are engaged by either GH or PRL. Both GH and PRL engagement acutely increase PRLR-Nluc-PRLR-Cluc complementation (demonstrated by the yellow dendritic shape with the upward arrow) that could be envisioned by enhanced closeness of the distal tails of the PRLR monomers within the dimer. PRLRs are in orange. GH is in green. PRL is in purple. 1 and 2 in yellow indicate the 2 asymmetric binding sites of GH or PRL for each PRLR monomer within the dimer. B, GHRs and PRLRs form a hetero-assemblage as (GHR-GHR)x/(PRLR-PRLR)y multimer. For illustrative purposes, this diagram shows a situation of x = 1 and y = 1. GHR-GHR homodimer is in gray and PRLR-PRLR homodimer is in orange. Left panel shows the view from the side; right panel shows the view from the top. C, GH reduces GHR-Nluc/PRLR-Cluc heteromer complementation (demonstrated by the yellow dendritic shape with the downward arrow). GH binds to GHR-GHR homodimers and PRLR-PRLR homodimers within a (GHR-GHR)x/(PRLR-PRLR)y multimer. GH brings tails of GHR monomers within GHR-GHR homodimers together and of PRLR monomers within PRLR-PRLR homodimers together, respectively, but overall yields a net separation of GHR tails from PRLR tails within the heteromer. D, PRL reduces GHR-Nluc/PRLR-Cluc heteromer complementation (demonstrated by the yellow dendritic shape with the downward arrow). PRL binds to PRLR-PRLR homodimers within a (GHR-GHR)x/(PRLR-PRLR)y multimer, but not to GHR-GHR homodimers. PRL brings tails of PRLR monomers within PRLR-PRLR homodimers together but separates PRLR tails from GHR tails within the heteromer. E, B-G augments GHR-Nluc/PRLR-Cluc heteromer complementation (demonstrated by the yellow dendritic shape with the upward arrow). B-G binds to 1 GHR monomer (via site 1 of B2036, in red) within a GHR-GHR dimer and to 1 PRLR monomer (via site 1 of G129R, in yellow) within a nearby PRLR-PRLR dimer in the hetero-multimer. This B-G binding brings 1 GHR tail within 1 GHR-GHR dimer into proximity with 1 PRLR tail within a PRLR-PRLR dimer. Two possible binding combinations are shown in the 2 panels (left and right).

Although derived only from biochemical and signaling data and luciferase complementation data, we see our model as robust in its internal consistency. Further, it provides a template for further mutagenesis studies that aim to map GHR and PRLR regions involved in these interactions. Companion structural studies will add clarity to this model. However, a major virtue of our luciferase complementation studies is that they are performed in situ in living cells, whereas structural methods often rely on overexpression of purified recombinant proteins and cell-free analysis and may thus not adequately reflect intracellular events.

We and others have investigated GHR regions involved in ligand-independent homodimerzation. Our previous data implicate the “dimerization interface” in ECD subdomain 2 as key for prehomodimerization (30), whereas others stress the transmembrane domain (TMD)'s role (29). It is intriguing to consider whether regions dictating homodimerization might be important in dictating the higher order nature of heteromeric complexes of GHR and PRLR. Because GHR and PRLR TMDs differ substantially, it might be hypothesized that the TMD contributes more strongly to homodimer formation than to hetero-multimer formation; however, it is conceivable that the nonhomodimerizing “face” of the TMD (or of ECD subdomain 2) or a region(s) contained in ECD subdomain 1 could mediate hetero-multimerization. We also realize that the ICD may have a role in multimer formation, either intrinsically or via association with an intracellular protein(s), such as JAK2. We think that ICD involvement is less likely, however, based on our findings that neither JAK2 nor the receptor ICD is required for either GHR-GHR complementation (44) or PRLR-PRLR complementation (data not shown) and that both receptor ICDs are thought to be intrinsically disordered (62), unlike their ECDs and TMDs. We are also mindful that higher order homo-multimers may also form, either in situations in which only GHR or PRLR is expressed or even when both receptors are coexpressed. Indeed, Sedek et al (63) used blue native electrophoresis to demonstrate the likely existence of (presumably) homo-multimeric GHR complexes in cell extracts. Assuming their existence in intact cells, whether or not such homo-multimeric (GHR-GHR or PRLR-PRLR) complexes may signal differently than the hetero-multimeric (GHR-GHR/PRLR-PRLR) complexes suggested by our current studies is a question that warrants further investigation. Such studies should be facilitated by our observations using the luciferase complementation system.

Although GHR-PRLR association is not required for either GH or PRL signaling, we note that our previous studies suggest that interactions between the receptors may regulate important aspects of their availability and function in human breast cancer cells (42, 43). Further, these receptors are commonly expressed in human breast tissue and in breast cancers and both hormones likely have important roles in breast cancer pathogenesis and/or behavior, either via autocrine/paracrine or endocrine means (9, 64–67). We envision that relative GHR to PRLR ratios within particular breast cancers, for example, might dictate the degree to which homodimer, homo-multimer, and hetero-multimer formation is favored and that understanding the determinants of such assemblages might be useful if future therapies are individualized to target a cancer's GHR/PRLR expression profile. This might be thought of as akin to tailored breast cancer therapies currently employed that are based on estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2/neu) status. In the case of GHR and PRLR, specific peptide or antibody antagonists may have differential efficacy depending on the relative complement of the receptors present and their tendency to associate. Thus, basic studies that dissect homodimer, homo-multimer, and hetero-multimer interaction domains may have translational impact. Our findings represent a point of departure for such future studies that utilize complementation methods, as well as biophysical and biochemical analysis to further this dissection.

Acknowledgments

We thank the expert assistance of Sharon Samuel and helpful discussion with members of the Frank laboratory. Parts of this work were presented at the 97th Annual The Endocrine Society Meeting in San Diego, CA, 2015.

This work was supported by National Institutes of Health Grants DK58259 and DK46395 (to S.J.F.) and a Veterans Affairs Merit Review award (S.J.F.).

Disclosure Summary: The authors have nothing to disclose.

Footnotes

Abbreviations:
B-G
B2036-G129R
Cluc
C-terminal luciferase
ECD
extracellular domain
EPOR
erythropoietin receptor
ER
estrogen receptor
GHR
GH receptor
hGH
human GH
hPRL
human PRL
ICD
intracellular domain
JAK2
Janus Kinase 2
luc
luciferase
Nluc
N-terminal luciferase
p-
phospho-
PRL
prolactin
PRLR
PRL receptor
STAT
signal transducer and activator of transcription
TMD
transmembrane domain.

References

  • 1. Huising MO, Kruiswijk CP, Flik G. Phylogeny and evolution of class-I helical cytokines. J Endocrinol. 2006;189:1–25. [DOI] [PubMed] [Google Scholar]
  • 2. Teilum K, Hoch JC, Goffin V, Kinet S, Martial JA, Kragelund BB. Solution structure of human prolactin. J Mol Biol. 2005;351:810–823. [DOI] [PubMed] [Google Scholar]
  • 3. Isaksson OG, Edén S, Jansson JO. Mode of action of pituitary growth hormone on target cells. Annu Rev Physiol. 1985;47:483–499. [DOI] [PubMed] [Google Scholar]
  • 4. Kaplan S. Hormonal regulation of growth and metabolic effects of growth hormone. In: Kostyo J, Goodman HM, eds. Handbook of Physiology. New York, NY: Oxford University Press; 1999:129–143. [Google Scholar]
  • 5. Møller N, Jørgensen JO. Effects of growth hormone on glucose, lipid, and protein metabolism in human subjects. Endocr Rev. 2009;30:152–177. [DOI] [PubMed] [Google Scholar]
  • 6. Waters MJ, Hoang HN, Fairlie DP, Pelekanos RA, Brown RJ. New insights into growth hormone action. J Mol Endocrinol. 2006;36:1–7. [DOI] [PubMed] [Google Scholar]
  • 7. Goffin V, Binart N, Touraine P, Kelly PA. Prolactin: the new biology of an old hormone. Annu Rev Physiol. 2002;64:47–67. [DOI] [PubMed] [Google Scholar]
  • 8. Rui H. Prolactin. In: Oppenheim JJ, Feldman M, eds. Cytokine Reference On-Line. London, UK: Academic Press, Harcourt; 2000:267–283. [Google Scholar]
  • 9. Clevenger CV, Furth PA, Hankinson SE, Schuler LA. The role of prolactin in mammary carcinoma. Endocr Rev. 2003;24:1–27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Goffin V, Touraine P, Pichard C, Bernichtein S, Kelly PA. Should prolactin be reconsidered as a therapeutic target in human breast cancer? Mol Cell Endocrinol. 1999;151:79–87. [DOI] [PubMed] [Google Scholar]
  • 11. Vonderhaar BK. Prolactin involvement in breast cancer. Endocr Relat Cancer. 1999;6:389–404. [DOI] [PubMed] [Google Scholar]
  • 12. Arendt LM, Schuler LA. Transgenic models to study actions of prolactin in mammary neoplasia. J Mammary Gland Biol Neoplasia. 2008;13:29–40. [DOI] [PubMed] [Google Scholar]
  • 13. Arendt LM, Rugowski DE, Grafwallner-Huseth TA, Garcia-Barchino MJ, Rui H, Schuler LA. Prolactin-induced mouse mammary carcinomas model estrogen resistant luminal breast cancer. Breast Cancer Res. 2011;13:R11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. Wennbo H, Gebre-Medhin M, Gritli-Linde A, Ohlsson C, Isaksson OG, Törnell J. Activation of the prolactin receptor but not the growth hormone receptor is important for induction of mammary tumors in transgenic mice. J Clin Invest. 1997;100:2744–2751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15. Llovera M, Pichard C, Bernichtein S, et al. Human prolactin (hPRL) antagonists inhibit hPRL-activated signaling pathways involved in breast cancer cell proliferation. Oncogene. 2000;19:4695–4705. [DOI] [PubMed] [Google Scholar]
  • 16. Levina VV, Nolen B, Su Y, et al. Biological significance of prolactin in gynecologic cancers. Cancer Res. 2009;69:5226–5233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Tan D, Chen KE, Khoo T, Walker AM. Prolactin increases survival and migration of ovarian cancer cells: importance of prolactin receptor type and therapeutic potential of S179D and G129R receptor antagonists. Cancer Lett. 2011;310:101–108. [DOI] [PubMed] [Google Scholar]
  • 18. Wen Y, Zand B, Ozpolat B, et al. Antagonism of tumoral prolactin receptor promotes autophagy-related cell death. Cell Rep. 2014;7:488–500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. Zhang C, Cherifi I, Nygaard M, et al. Residue 146 regulates prolactin receptor folding, basal activity and ligand-responsiveness: potential implications in breast tumorigenesis. Mol Cell Endocrinol. 2015;401:173–188. [DOI] [PubMed] [Google Scholar]
  • 20. Swanson SM, Unterman TG. The growth hormone-deficient Spontaneous Dwarf rat is resistant to chemically induced mammary carcinogenesis. Carcinogenesis. 2002;23:977–982. [DOI] [PubMed] [Google Scholar]
  • 21. Shen Q, Lantvit DD, Lin Q, et al. Advanced rat mammary cancers are growth hormone dependent. Endocrinology. 2007;148:4536–4544. [DOI] [PubMed] [Google Scholar]
  • 22. Ramsey MM, Ingram RL, Cashion AB, et al. Growth hormone-deficient dwarf animals are resistant to dimethylbenzanthracine (DMBA)-induced mammary carcinogenesis. Endocrinology. 2002;143:4139–4142. [DOI] [PubMed] [Google Scholar]
  • 23. Steuerman R, Shevah O, Laron Z. Congenital IGF1 deficiency tends to confer protection against post-natal development of malignancies. Eur J Endocrinol. 2011;164:485–489. [DOI] [PubMed] [Google Scholar]
  • 24. Boutin JM, Edery M, Shirota M, et al. Identification of a cDNA encoding a long form of prolactin receptor in human hepatoma and breast cancer cells. Mol Endocrinol. 1989;3:1455–1461. [DOI] [PubMed] [Google Scholar]
  • 25. Qazi AM, Tsai-Morris CH, Dufau ML. Ligand-independent homo- and heterodimerization of human prolactin receptor variants: inhibitory action of the short forms by heterodimerization. Mol Endocrinol. 2006;20:1912–1923. [DOI] [PubMed] [Google Scholar]
  • 26. de Vos AM, Ultsch M, Kossiakoff AA. Human growth hormone and extracellular domain of its receptor: crystal structure of the complex. Science. 1992;255:306–312. [DOI] [PubMed] [Google Scholar]
  • 27. Cunningham BC, Ultsch M, De Vos AM, Mulkerrin MG, Clauser KR, Wells JA. Dimerization of the extracellular domain of the human growth hormone receptor by a single hormone molecule. Science. 1991;254:821–825. [DOI] [PubMed] [Google Scholar]
  • 28. Gent J, van Kerkhof P, Roza M, Bu G, Strous GJ. Ligand-independent growth hormone receptor dimerization occurs in the endoplasmic reticulum and is required for ubiquitin system-dependent endocytosis. Proc Natl Acad Sci USA. 2002;99:9858–9863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29. Brown RJ, Adams JJ, Pelekanos RA, et al. Model for growth hormone receptor activation based on subunit rotation within a receptor dimer. Nat Struct Mol Biol. 2005;12:814–821. [DOI] [PubMed] [Google Scholar]
  • 30. Yang N, Wang X, Jiang J, Frank SJ. Role of the growth hormone (GH) receptor transmembrane domain in receptor predimerization and GH-induced activation. Mol Endocrinol. 2007;21:1642–1655. [DOI] [PubMed] [Google Scholar]
  • 31. Gadd SL, Clevenger CV. Ligand-independent dimerization of the human prolactin receptor isoforms: functional implications. Mol Endocrinol. 2006;20:2734–2746. [DOI] [PubMed] [Google Scholar]
  • 32. Brooks AJ, Waters MJ. The growth hormone receptor: mechanism of activation and clinical implications. Nat Rev Endocrinol. 2010;6:515–525. [DOI] [PubMed] [Google Scholar]
  • 33. Clevenger CV, Kline JB. Prolactin receptor signal transduction. Lupus. 2001;10:706–718. [DOI] [PubMed] [Google Scholar]
  • 34. Herrington J, Carter-Su C. Signaling pathways activated by the growth hormone receptor. Trends Endocrinol Metab. 2001;12:252–257. [DOI] [PubMed] [Google Scholar]
  • 35. Cunningham BC, Bass S, Fuh G, Wells JA. Zinc mediation of the binding of human growth hormone to the human prolactin receptor. Science. 1990;250:1709–1712. [DOI] [PubMed] [Google Scholar]
  • 36. Somers W, Ultsch M, De Vos AM, Kossiakoff AA. The x-ray structure of a growth hormone-prolactin receptor complex. Nature. 1994;372:478–481. [DOI] [PubMed] [Google Scholar]
  • 37. Fu YK, Arkins S, Fuh G, et al. Growth hormone augments superoxide anion secretion of human neutrophils by binding to the prolactin receptor. J Clin Invest. 1992;89:451–457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38. Hughes JP, Friesen HG. The nature and regulation of the receptors for pituitary growth hormone. Annu Rev Physiol. 1985;47:469–482. [DOI] [PubMed] [Google Scholar]
  • 39. Keeler C, Jablonski EM, Albert YB, et al. The kinetics of binding human prolactin, but not growth hormone, to the prolactin receptor vary over a physiologic pH range. Biochemistry. 2007;46:2398–2410. [DOI] [PubMed] [Google Scholar]
  • 40. Biener E, Martin C, Daniel N, et al. Ovine placental lactogen-induced heterodimerization of ovine growth hormone and prolactin receptors in living cells is demonstrated by fluorescence resonance energy transfer microscopy and leads to prolonged phosphorylation of signal transducer and activator of transcription (STAT)1 and STAT3. Endocrinology. 2003;144:3532–3540. [DOI] [PubMed] [Google Scholar]
  • 41. Herman A, Bignon C, Daniel N, Grosclaude J, Gertler A, Djiane J. Functional heterodimerization of prolactin and growth hormone receptors by ovine placental lactogen. J Biol Chem. 2000;275:6295–6301. [DOI] [PubMed] [Google Scholar]
  • 42. Xu J, Zhang Y, Berry PA, et al. Growth hormone signaling in human T47D breast cancer cells: potential role for a growth hormone receptor-prolactin receptor complex. Mol Endocrinol. 2011;25:597–610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43. Xu J, Sun D, Jiang J, et al. The role of prolactin receptor in GH signaling in breast cancer cells. Mol Endocrinol. 2013;27:266–279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44. Liu Y, Berry PA, Zhang Y, et al. Dynamic analysis of GH receptor conformational changes by split luciferase complementation. Mol Endocrinol. 2014;28:1807–1819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45. Luker KE, Smith MC, Luker GD, et al. Kinetics of regulated protein-protein interactions revealed with firefly luciferase complementation imaging in cells and living animals. Proc Natl Acad Sci USA. 2004;101:12288–12293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46. Villalobos V, Naik S, Piwnica-Worms D. Detection of protein-protein interactions in live cells and animals with split firefly luciferase protein fragment complementation. Methods Mol Biol. 2008;439:339–352. [DOI] [PubMed] [Google Scholar]
  • 47. Paulmurugan R, Gambhir SS. Firefly luciferase enzyme fragment complementation for imaging in cells and living animals. Anal Chem. 2005;77:1295–1302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48. Yang N, Langenheim JF, Wang X, Jiang J, Chen WY, Frank SJ. Activation of growth hormone receptors by growth hormone and growth hormone antagonist dimers: insights into receptor triggering. Mol Endocrinol. 2008;22:978–988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49. Langenheim JF, Chen WY. Development of a novel ligand that activates JAK2/STAT5 signaling through a heterodimer of prolactin receptor and growth hormone receptor. J Recept Signal Transduct Res. 2009;29:107–112. [DOI] [PubMed] [Google Scholar]
  • 50. Zhang Y, Guan R, Jiang J, et al. Growth hormone (GH)-induced dimerization inhibits phorbol ester-stimulated GH receptor proteolysis. J Biol Chem. 2001;276:24565–24573. [DOI] [PubMed] [Google Scholar]
  • 51. Jiang J, Liang L, Kim SO, Zhang Y, Mandler R, Frank SJ. Growth hormone-dependent tyrosine phosphorylation of a GH receptor-associated high molecular weight protein immunologically related to JAK2. Biochem Biophys Res Commun. 1998;253:774–779. [DOI] [PubMed] [Google Scholar]
  • 52. Chen H, Kleinberger JW, Takane KK, et al. Augmented Stat5 signaling bypasses multiple impediments to lactogen-mediated proliferation in human β-cells. Diabetes. 2015;64:3784–3797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53. Kohlhuber F, Rogers NC, Watling D, et al. A JAK1/JAK2 chimera can sustain α and γ interferon responses. Mol Cell Biol. 1997;17:695–706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54. Loesch K, Deng L, Cowan JW, et al. Janus kinase 2 influences growth hormone receptor metalloproteolysis. Endocrinology. 2006;147:2839–2849. [DOI] [PubMed] [Google Scholar]
  • 55. Deng L, He K, Wang X, et al. Determinants of growth hormone receptor down-regulation. Mol Endocrinol. 2007;21:1537–1551. [DOI] [PubMed] [Google Scholar]
  • 56. Deng L, Jiang J, Frank SJ. Growth hormone-induced JAK2 signaling and GH receptor down-regulation: role of GH receptor intracellular domain tyrosine residues. Endocrinology. 2012;153:2311–2322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57. Jiang J, Wan Y, Wang X, et al. Inhibitory GH receptor extracellular domain monoclonal antibodies: three-dimensional epitope mapping. Endocrinology. 2011;152:4777–4788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58. Chen WY, Ramamoorthy P, Chen N, Sticca R, Wagner TE. A human prolactin antagonist, hPRL-G129R, inhibits breast cancer cell proliferation through induction of apoptosis. Clin Cancer Res. 1999;5:3583–3593. [PubMed] [Google Scholar]
  • 59. Goffin V, Bernichtein S, Touraine P, Kelly PA. Development and potential clinical uses of human prolactin receptor antagonists. Endocr Rev. 2005;26:400–422. [DOI] [PubMed] [Google Scholar]
  • 60. Chen WY, Chen NY, Yun J, Wagner TE, Kopchick JJ. In vitro and in vivo studies of antagonistic effects of human growth hormone analogs. J Biol Chem. 1994;269:15892–15897. [PubMed] [Google Scholar]
  • 61. Goffin V, Bernichtein S, Carrière O, Bennett WF, Kopchick JJ, Kelly PA. The human growth hormone antagonist B2036 does not interact with the prolactin receptor. Endocrinology. 1999;140:3853–3856. [DOI] [PubMed] [Google Scholar]
  • 62. Haxholm GW, Nikolajsen LF, Olsen JG, et al. Intrinsically disordered cytoplasmic domains of two cytokine receptors mediate conserved interactions with membranes. Biochem J. 2015;468:495–506. [DOI] [PubMed] [Google Scholar]
  • 63. Sedek M, van der Velden LM, Strous GJ. Multimeric growth hormone receptor complexes serve as signaling platforms. J Biol Chem. 2014;289:65–73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 64. Raccurt M, Lobie PE, Moudilou E, et al. High stromal and epithelial human gh gene expression is associated with proliferative disorders of the mammary gland. J Endocrinol. 2002;175:307–318. [DOI] [PubMed] [Google Scholar]
  • 65. Wu ZS, Yang K, Wan Y, et al. Tumor expression of human growth hormone and human prolactin predict a worse survival outcome in patients with mammary or endometrial carcinoma. J Clin Endocrinol Metab. 2011;96:E1619–E1629. [DOI] [PubMed] [Google Scholar]
  • 66. Goffin V, Touraine P. The prolactin receptor as a therapeutic target in human diseases: browsing new potential indications. Expert Opin Ther Targets. 2015;19:1229–1244. [DOI] [PubMed] [Google Scholar]
  • 67. Mertani HC, Garcia-Caballero T, Lambert A, et al. Cellular expression of growth hormone and prolactin receptors in human breast disorders. Int J Cancer. 1998;79:202–211. [DOI] [PubMed] [Google Scholar]

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