Ligand-independent dimerization of the human prolactin receptor isoforms: functional implications

Prolactin (PRL) contributes to the growth of normal and malignant breast tissues. PRL initiates signaling by engaging the PRL receptor (PRLr), a transmembrane (TM) receptor belonging to the cytokine receptor family. The accepted view has been that PRL activates the PRLr by inducing dimerization of the receptor, but recent reports show ligand-independent dimerization of other cytokine receptors. Using coimmunoprecipitation assays, we have confirmed ligand-independent dimerization of the PRLr in T47D breast cancer and HepG2 liver carcinoma cells. In addition, mammalian cells transfected with differentially epitope-tagged isoforms of the PRLr indicated that long, intermediate, and DeltaS1 PRLrs dimerized in a ligand-independent manner. To determine the domain(s) involved in PRLr ligand-independent dimerization, we generated PRLr constructs as follows: (1) the TM-ICD, which consisted of the TM domain and the intracellular domain (ICD) but lacked the extracellular domain (ECD), and (2) the ECD-TM, which consisted of the TM domain and the ECD but lacked the ICD. These constructs dimerized in a ligand-independent manner in mammalian cells, implicating a significant role for the TM domain in this process. These truncated PRLrs were functionally inert alone or in combination in cells lacking the PRLr. However, when introduced into cells containing endogenous PRLr, the ECD-TM inhibited human PRLr signaling, whereas the TM-ICD potentiated human PRLr signaling. These studies indicate that the ECD-TM and the TM-ICD are capable of modulating PRLr function. We also demonstrated an endogenous TM-ICD in T47D cells, suggesting that these findings are relevant to PRL-signaling pathways in breast cancer.     

Stoichiometric Structure-Function Analysis of the Prolactin Receptor Signaling Domain by Receptor Chimeras

The intracellular domain of the prolactin (PRL) receptor (PRLr) is required for PRL-induced signaling and proliferation. To identify and test the functional stoichiometry of those PRLr motifs required for transduction and growth, chimeras consisting of the extracellular domain of either the α or β subunit of human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GM-CSFr) and the intracellular domain of the rat PRLr were synthesized. Because the high-affinity binding of GM-CSF results from the specific pairing of one α- and one β-GM-CSFr, use of GM-CSFr/PRLr chimera enabled targeted dimerization of the PRLr intracellular domain. To that end, the extracellular domains of the α- and β-GM-CSFr were conjugated to one of the following mutations: (i) PRLr C-terminal truncations, termed α278, α294, α300, α322, or β322; (ii) PRLr tyrosine replacements, termed Y309F, Y382F, or Y309+382F; or, (iii) PRLr wild-type short, intermediate, or long isoforms. These chimeras were cotransfected into the cytokine-responsive Ba/F3 line, and their expression was confirmed by ligand binding and Northern and Western blot analyses. Data from these studies revealed that heterodimeric complexes of the wild type with C-terminal truncation mutants of the PRLr intracellular domain were incapable of ligand-induced signaling or proliferation. Replacement of any single tyrosine residue (Y309F or Y382F) in the dimerized PRLr complex resulted in a moderate reduction of receptor-associated Jak2 activation and proliferation. In contrast, trans replacement of these residues (i.e., αY309F and βY382F) markedly reduced ligand-driven Jak2 activation and proliferation, while cis replacement of both tyrosine residues in a single intracellular domain (i.e., αY309+382F) produced an inactive signaling complex. Analysis of these GM-CSFr–PRLr complexes revealed equivalent levels of Jak2 in association with the mutant receptor chains, suggesting that the tyrosine residues at 309 and 382 do not contribute to Jak association, but instead to its activation. Heterodimeric pairings of the intracellular domains from the known PRLr receptor isoforms (short-intermediate, short-long, and intermediate-long) also yielded inactive receptor complexes. These data demonstrate that the tyrosine residues at 309 and 382, as well as additional residues within the C terminus of the dimerized PRLr complex, contribute to PRL-driven signaling and proliferation. Furthermore, these findings indicate a functional requirement for the pairing of Y309 and Y382 in trans within the dimerized receptor complex.     

Polyubiquitination of prolactin receptor stimulates its internalization, postinternalization sorting, and degradation via the lysosomal pathway

The ubiquitination of the receptor that mediates signaling induced by the polypeptide pituitary hormone prolactin (PRL) has been shown to lead to the degradation of this receptor and to the ensuing negative regulation of cellular responses to PRL. However, the mechanisms of PRL receptor (PRLr) proteolysis remain largely to be determined. Here we provide evidence that PRLr is internalized and primarily degraded via the lysosomal pathway. Ubiquitination of PRLr is essential for the rapid internalization of PRLr, which proceeds through a pathway dependent on clathrin and the assembly polypeptide 2 (AP2) adaptor complexes. Recruitment of AP2 to PRLr is stimulated by PRLr ubiquitination, which also is required for the targeting of already internalized PRLr to the lysosomal compartment. While mass spectrometry analysis revealed that both monoubiquitination and polyubiquitination (via both K48- and K63-linked chains) occur on PRLr, the results of experiments using forced expression of ubiquitin mutants indicate that PRLr polyubiquitination via K63-linked chains is important for efficient interaction of PRLr with AP2 as well as for efficient internalization, postinternalization sorting, and proteolytic turnover of PRLr. We discuss how specific ubiquitination may regulate early and late stages of endocytosis of PRLr and of related receptors to contribute to the negative regulation of the magnitude and duration of downstream signaling.     

Prolactin inhibits activity of pyruvate kinase M2 to stimulate cell proliferation

Mitogenic and prosurvival effects underlie the tumorigenic roles of prolactin (PRL) in the pathogenesis of breast cancer. PRL signaling is mediated through its receptor (PRLr). A proteomics screen identified the pyruvate kinase M2 (PKM2), a glycolytic enzyme known to play an important role in tumorigenesis, as a protein that constitutively interacts with PRLr. Treatment of cells with PRL inhibited pyruvate kinase activity and increased the lactate content in human cells in a manner that was dependent on the abundance of PRLr, activation of Janus kinase 2, and tyrosine phosphorylation of the intracellular domain of PRLr. Knockdown of PKM2 attenuated PRL-stimulated cell proliferation. The extent of this proliferation was rescued by the knock-in of the wild-type PKM2 but not of its mutant insensitive to PRL-mediated inhibition. We discuss a hypothesis that the inhibition of PKM2 by PRL contributes to the PRL-stimulated cell proliferation.     

Relative abundance of growth hormone receptor isoforms in rhesus monkey tissues and human transformed lymphocytes.

The growth hormone receptor (GHR) is expressed as one active, full-sequence isoform and one truncated, inactive one that lacks the intracellular signaling domain. The aim of this study was to investigate the variation in the tissue expression of the full and truncated mRNA and protein. Epstein-Barr virus-transformed human B lymphocyte lines were established from 9 normal individuals with a height standard deviation score (SDS) of - 0.1 +/- 1.1 (mean +/- SD). Tissues were also collected from 3 Rhesus monkeys, whose GHR has 94.1 % homology with the human molecule. mRNA quantitation was determined by Real Time Quantitative PCR. Growth hormone receptor expression in transformed lymphocytes was also studied by fluorescence-activated cell sorter analysis. Both isoforms were expressed in transformed lymphocytes, but individual variation in the relative mRNA expression was small (truncated isoform percentage of total receptor mRNA: 17.1 +/- 4.4, mean +/- SD). There was no correlation between donors' height SDS and the expression of either isoform or the ratio between them. Protein expression by FACS analysis showed wider variation among the subjects; however, the relative ratio was similar in all the subjects. In monkey tissues, the truncated receptor showed a tissue-specific distribution. In conclusion, the expression of both isoforms in transformed lymphocytes from normal subjects shows small differences at the RNA or protein levels, and does not correlate with height SDS. Growth hormone splice isoforms show tissue specificity, suggesting local regulation of splicing. Tissues with relatively high expression of the truncated isoform are likely to be more resistant to the effects of GH due to the dominant negative effect of this isoform. In addition, the differential tissue expression might influence the levels of growth hormone binding protein in the immediate milieu of each tissue.     

Differences in prolactin receptor (PRLR) in mouse and human fallopian tubes: evidence for multiple regulatory mechanisms controlling PRLR isoform expression in mice

The anterior pituitary-derived hormone prolactin (PRL) signals through the PRL receptor (PRLR) and is important for female reproductive function in mammals. In contrast to the extensive studies of PRLR expression and regulation in human and mouse ovary and uterus, the mechanisms controlling the regulation of PRLR isoform expression in the fallopian tube are poorly understood. Because dynamic interaction of hormonal signaling in gonadal tissue and the pituitary or in gonadal tissues themselves in mammals suggests endocrine or paracrine regulation of PRLR expression, we questioned whether differential regulation of PRLR isoforms by PRL ovarian-derived estrogen (E(2)) and progesterone (P(4)) exists in the fallopian tube and pituitary of prepubertal female mice. Western blot analysis showed distinct molecular separation of PRLR isoforms in mouse and human fallopian tubes, and cellular localization was found in mouse and human tubal epithelia but not in mouse tubal smooth muscle cells. These data support the concept of an isoform- and cell type-specific expression of PRLR in human and mouse fallopian tubes. Moreover, expression of the long form of PRLR decreased after PRL treatment and increased after blockage of endogenous PRL secretion by bromocriptine (an inhibitor of PRL secretion) in a time-dependent manner in mouse fallopian tube. The opposite regulation was observed in the pituitary. Treatment with exogenous E(2) or P(4) led to changes in PRLR expression in the fallopian tube similar to those of PRL treatment. However, E(2) and P(4) did not affect PRLR expression in the pituitary. Estrogen had no effect on the long form of PRLR expression, whereas P(4) regulated the long form of PRLR in the fallopian tube, as did PRL. Taken together, the data from our comparative study provide evidence that PRLR can be regulated by an interplay of two different mechanisms, PRL or ovarian steroid hormones independently or in combination in a tissue-specific manner. Furthermore, we found that ovarian steroid hormones selectively suppress the expression of PRLR isoforms in mouse fallopian tubes. These findings may contribute to our understanding of the mechanisms controlling PRLR isoform expression in the fallopian tube (in addition to ovary and uterus), with implications for female reproduction.     

Identification of fetal liver tyrosine kinase 3 (flt3) ligand domain required for receptor binding and function using naturally occurring ligand isoforms

We used a comparative approach to identify the fetal liver tyrosine kinase 3 (flt3) ligand structure required for binding and function. Two conserved bovine flt3 ligand isoforms, which differ in a defined region within the extracellular domain, were identified and shown to be uniformly transcribed in individuals with diverse MHC haplotypes. Notably, at the amino acid level, the extracellular domain of the bovine flt3 ligand isoform 1 is 81 and 72% identical with the extracellular domains of the human and murine flt3 ligands, respectively, whereas isoform-2 has a deletion within this domain. Bovine flt3 ligand isoform 1, but not 2, bound the human flt3 receptor and stimulated murine pro B cells transfected with the murine flt3 receptor. This retention of binding and function allowed definition of key residues by identifying sequences conserved among species. We have shown that a highly conserved, 18 aa sequence within the flt3 ligand extracellular domain is required for flt3 receptor binding and function. However, a peptide representing this sequence is insufficient for receptor binding as demonstrated by its failure to inhibit the bovine flt3 ligand isoform 1 binding to the human flt3 receptor. The requirement for flanking structure was confirmed by testing bovine flt3 ligand isoform 1 constructs truncated at specific residues outside the 18 aa sequence. Overall, the flt3 ligand structure required for function is markedly similar to that of the related hemopoietic growth factors, CSF-1 and steel factor. This definition of the required flt3 ligand structure will facilitate development of agonists to enhance dendritic cell recruitment for vaccines and immunotherapy.     

Protein Kinase C�� Modulates Ligand-induced Cell Surface Death Receptor Accumulation: A MECHANISTIC BASIS FOR ENZASTAURIN-DEATH LIGAND SYNERGY*

Although treatment with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) is known to protect a subset of cells from induction of apoptosis by death ligands such as Fas ligand and tumor necrosis factor-α-related apoptosis-inducing ligand, the mechanism of this protection is unknown. This study demonstrated that protection in short term apoptosis assays and long term proliferation assays was maximal when Jurkat or HL-60 human leukemia cells were treated with 2–5 nm PMA. Immunoblotting demonstrated that multiple PKC isoforms, including PKCα, PKCβ, PKCϵ, and PKCθ, translocated from the cytosol to a membrane-bound fraction at these PMA concentrations. When the ability of short hairpin RNA (shRNA) constructs that specifically down-regulated each of these isoforms was examined, PKCβ shRNA uniquely reversed PMA-induced protection against cell death. The PKCβ-selective small molecule inhibitor enzastaurin had a similar effect. Although mass spectrometry suggested that Fas is phosphorylated on a number of serines and threonines, mutation of these sites individually or collectively had no effect on Fas-mediated death signaling or PMA protection. Further experiments demonstrated that PMA diminished ligand-induced cell surface accumulation of Fas and DR5, and PKCβ shRNA or enzastaurin reversed this effect. Moreover, enzastaurin sensitized a variety of human tumor cell lines and clinical acute myelogenous leukemia isolates, which express abundant PKCβ, to tumor necrosis factor-α related apoptosis-inducing ligand-induced death in the absence of PMA. Collectively, these results identify a specific PKC isoform that modulates death receptor-mediated cytotoxicity as well as a small molecule inhibitor that mitigates the inhibitory effects of PKC activation on ligand-induced death receptor trafficking and cell death.     

The c-Kit/D816V mutation eliminates the differences in signal transduction and biological responses between two isoforms of c-Kit

Activating mutations of codon 816 of the Kit gene have been implicated in malignant cell growth of acute myeloid leukemia (AML), systemic mastocytosis and germ cell tumors. Substitution of aspartic acid with valine (D816V) renders the receptor independent of ligand for activation and signaling. Wild-type c-Kit is a tyrosine kinase receptor that requires its ligand, stem cell factor (SCF), for activation. Several isoforms of c-Kit exist as a result of alternative mRNA splicing, of which two are characterized by the presence or absence of four amino acids (GNNK? and GNNK+, respectively) in the extracellular domain. The two isoforms show differences in signal transduction and biological activities and the shorter isoform seems to be highly expressed than the longer isoform in human malignancies. In this study we analysed the signal transduction downstream of the oncogenic c-Kit mutant D816V in an isoform specific context, using the hematopoietic cell line Ba/F3 stably transfected with the different versions of isoform and mutant receptor. Our data show that in contrast to the differences shown in the activation of wild-type c-Kit isoforms, both isoforms of c-Kit/D816V are constitutively phosphorylated to the same extent. By the use of Western blot analysis we investigated the activation of different signaling proteins and found that both D816V/GNNK? and D816V/GNNK+ constitutively phosphorylated Gab2, Shc, SHP-2 and Cbl to almost the same extent as c-Kit/GNNK?. In addition, both isoforms of c-Kit/D816V induced SCF-independent cell survival and proliferation equally well. This is in contrast to wild-type c-Kit, where c-Kit/GNNK? induced better cell survival and stronger proliferation than c-Kit/GNNK+, and both required stimulation with SCF. Taken together, these findings reveal that the differences in downstream signal transduction and biological responses between the two GNNK isoforms are eliminated by the D816V mutant.     

Combining restriction digestion and touchdown PCR permits detection of trace isoforms of histamine H3 receptor

The conserved sequences of the mouse histamine H3 receptor at the potential alternative splice junctions suggest that the splice isoforms found in guinea pig, rat, human, and hamster may also be present in the mouse. However, the trace amount isoforms are hard to be detected by the regular PCR approach. In this paper, we report a method in which the unspliced long isoform is cut by restriction endonuclease so that the short isoforms can be amplified to detectable levels to confirm the existence of the splice isoforms of H3 receptor mRNA in the mouse. This method is applicable to the detection of trace amounts of splice isoforms that coexist with the long, more abundant isoforms.     

Unmodified prolactin (PRL) and S179D PRL-initiated bioluminescence resonance energy transfer between homo- and hetero-pairs of long and short human PRL receptors in living human cells

We have used bioluminescence resonance energy transfer (BRET) to examine the interaction between human prolactins (PRLs) and the long (LF) and two short isoforms (SF1a and SF1b) of the human PRL receptor in living cells. cDNA sequences encoding the LF, SF1a, and SF1b were subcloned into codon-humanized vectors containing cDNAs for either Renilla reniformis luciferase (Rluc) or a green fluorescent protein (GFP(2)) with a 12- or 13-amino acid linker connecting the parts of the fusion proteins. Transfection into human embryonic kidney 293 cells demonstrated maintained function of Rluc and GFP(2) when linked to the receptors, and confocal microscopy demonstrated the localization of tagged receptors in the plasma membrane by 48 h after transfection. All three tagged receptors transduced a signal, with the LF and SF1a stimulating, and SF1b inhibiting, promoter activity of an approximately 2.4-kb beta-casein-luc construct. Both unmodified PRL (U-PRL) and the molecular mimic of phosphorylated PRL, S179D PRL, induced BRET with all combinations of long and short receptor isoforms except SF1a plus SF1b. No BRET was observed with the site two-inactive mutant, G129R PRL. This is the first demonstration, 1) that species homologous PRL promotes both homo- and hetero-interaction of most long and short PRLR pairs in living cells, 2) that both U-PRL and S179D PRL are active in this regard, and 3) that there is some aspect of SF1a-SF1b structure that prevents this particular hetero-receptor pairing. In addition, we conclude that preferential pairing of different receptor isoforms is not the explanation for the different signaling initiated by U-PRL and S179D PRL.     

Prolactin upregulates its receptors and inhibits lipolysis and leptin release in male rat adipose tissue

Prolactin (PRL) is recognized as a metabolic regulator during lactation, but little information exists on its actions in male adipose tissue. We examined whether PRL affects the expression of its receptors (PRLR), lipolysis, and adipokine secretion in male rats. Both long and short PRLR isoforms were induced 40-50-fold during differentiation of epididymal preadipocytes, with a 10-fold higher expression of the long isoform. PRL upregulated both isoforms before and after differentiation. PRL suppressed lipolysis in epididymal explants and mature adipocytes in a dose- and time-dependent manner, which was reversed by a Jak2 inhibitor. PRL also inhibited leptin, but not adiponectin, release. We conclude that PRL inhibits lipolysis and leptin release by acting directly on adipocytes via interaction with either of its receptors and activation of a Jak2-dependent signaling pathway(s). This is the first demonstration of substantial effects of PRL on male adipocytes.     

Activation and association of the Tec tyrosine kinase with the human prolactin receptor: mapping of a Tec/Vav1-receptor binding site

Stimulation of the PRL receptor (PRLr) results in the activation of the guanine nucleotide exchange factor (GEF) p95Vav1 with corresponding alterations in cytoarchitecture and cell motility. To better understand the mechanisms involved in the regulation of Vav1 activity, the role of the tyrosine kinase p70Tec was examined. Coimmunoprecipitation and in vitro kinase assays revealed that ligand stimulation of the PRLr resulted in the rapid activation of Tec and its concomitant association with the PRLR: When coexpressed in COS-1 cells, both Vav1 and Tec were found to associate with the PRLr in the presence of ligand. In the absence of receptor, a constitutive complex between Vav1 and Tec was noted. Both Vav1 and Tec, however, were capable of independent engagement of a bipartite intracellular domain of the PRLR: Deletion mapping studies confined this interaction to residues 323 to 527 of the intracellular domain of the PRLR: Furthermore, Tec enhanced the GEF activity of Vav1 as evidenced by an increase in GTP-bound Rac1. These data would suggest a pivotal function for the formation of a Tec/Vav1/PRLr complex during PRL-driven signal transduction, given the role of Vav1 in the control of cell proliferation and the regulation of Rho family-mediated cytoskeletal alterations.