Dynamic gene expression of GH/PRL-family hormone receptors in gill and kidney during freshwater-acclimation of Mozambique tilapia

In teleosts, prolactin (PRL) and growth hormone (GH) act at key osmoregulatory tissues to regulate hydromineral balance. This study was aimed at characterizing patterns of expression for genes encoding receptors for the GH/PRL-family of hormones in the gill and kidney of Mozambique tilapia (Oreochromis mossambicus) during freshwater (FW)-acclimation. Transfer of seawater (SW)-acclimated tilapia to FW elicited rapid and sustained increases in plasma levels and pituitary gene expression of PRL₁₇₇ and PRL₁₈₈; plasma hormone and pituitary mRNA levels of GH were unchanged. In the gill, PRL receptor 1 (PRLR1) mRNA increased markedly after transfer to FW by 6 h, while increases in GH receptor (GHR) mRNA were observed 48 h and 14 d after the transfer. By contrast, neither PRLR2 nor the somatolactin receptor (SLR) was responsive to FW transfer. Paralleling these endocrine responses were marked increases in branchial gene expression of a Na⁺/Cl^? cotransporter and a Na⁺/H⁺ exchanger, indicators of FW-type mitochondrion-rich cells (MRCs), at 24 and 48 h after FW transfer, respectively. Expression of Na⁺/K⁺/2Cl^? cotransporter, an indicator of SW-type MRCs, was sharply down-regulated by 6 h after transfer to FW. In kidney, PRLR1, PRLR2 and SLR mRNA levels were unchanged, while GHR mRNA was up-regulated from 6 h after FW transfer to all points thereafter. Collectively, these results suggest that the modulation of the gene expression for PRL and GH receptors in osmoregulatory tissues represents an important aspect of FW-acclimation of tilapia.     

Development of a novel ligand that activates JAK2/STAT5 signaling through a heterodimer of prolactin receptor and growth hormone receptor

The objective of this study was to determine if a functional heterodimer of prolactin receptor (PRLR) and growth hormone receptor (GHR) can be formed in humans. A novel ligand was designed that is composed of a GHR antagonist (B2036) and a PRLR antagonist (G129R) fused in tandem (B2036-G129R). Because both B2036 and G129R are binding site 2 inactive antagonists, the B2036-G129R fusion protein, in theory contains only two functional binding site 1s: one for GHR and one for PRLR. We examined the behavior of this chimeric ligand in cell lines known to express GHR, PRLR, or both receptors. The data presented show that B2036-G129R is inactive in IM-9 cells that express only GHR or Nb2 cells that express PRLR. In T-47D cells that coexpress PRLR and GHR, B2036-G129R activates JAK2/STAT5 signaling. These findings provide evidence that B2036-G129R is able to activate signal transduction through a heterodimer of PRLR and GHR in humans.     

The role of the bovine growth hormone receptor and prolactin receptor genes in milk, fat and protein production in Finnish Ayrshire dairy cattle

We herein report new evidence that the QTL effect on chromosome 20 in Finnish Ayrshire can be explained by variation in two distinct genes, growth hormone receptor (GHR) and prolactin receptor (PRLR). In a previous study in Holstein-Friesian dairy cattle an F279Y polymorphism in the transmembrane domain of GHR was found to be associated with an effect on milk yield and composition. The result of our multimarker regression analysis suggests that in Finnish Ayrshire two QTL segregate on the chromosomal region including GHR and PRLR. By sequencing the coding sequences of GHR and PRLR and the sequence of three GHR promoters from the pooled samples of individuals of known QTL genotype, we identified two substitutions that were associated with milk production traits: the previously reported F-to-Y substitution in the transmembrane domain of GHR and an S-to-N substitution in the signal peptide of PRLR. The results provide strong evidence that the effect of PRLR S18N polymorphism is distinct from the GHR F279Y effect. In particular, the GHR F279Y has the highest influence on protein percentage and fat percentage while PRLR S18N markedly influences protein and fat yield. Furthermore, an interaction between the two loci is suggested.     

Prolactin receptor signaling is essential for perinatal brown adipocyte function: a role for insulin-like growth factor-2

Background

The lactogenic hormones prolactin (PRL) and placental lactogens (PL) play central roles in reproduction and mammary development. Their actions are mediated via binding to PRL receptor (PRLR), highly expressed in brown adipose tissue (BAT), yet their impact on adipocyte function and metabolism remains unclear.

Methodology/Principal Findings

PRLR knockout (KO) newborn mice were phenotypically characterized in terms of thermoregulation and their BAT differentiation assayed for gene expression studies. Derived brown preadipocyte cell lines were established to evaluate the molecular mechanisms involved in PRL signaling on BAT function. Here, we report that newborn mice lacking PRLR have hypotrophic BAT depots that express low levels of adipocyte nuclear receptor PPARγ2, its coactivator PGC-1α, uncoupling protein 1 (UCP1) and the β3 adrenoceptor, reducing mouse viability during cold challenge. Immortalized PRLR KO preadipocytes fail to undergo differentiation into mature adipocytes, a defect reversed by reintroduction of PRLR. That the effects of the lactogens in BAT are at least partly mediated by Insulin-like Growth Factor-2 (IGF-2) is supported by: i) a striking reduction in BAT IGF-2 expression in PRLR KO mice and in PRLR-deficient preadipocytes; ii) induction of cellular IGF-2 expression by PRL through JAK2/STAT5 pathway activation; and iii) reversal of defective differentiation in PRLR KO cells by exogenous IGF-2.

Conclusions

Our findings demonstrate that the lactogens act in concert with IGF-2 to control brown adipocyte differentiation and growth. Given the prominent role of brown adipose tissue during the perinatal period, our results identified prolactin receptor signaling as a major player and a potential therapeutic target in protecting newborn mammals against hypothermia.     

Studies with a growth hormone antagonist and dual-fluorescent confocal microscopy demonstrate that the full-length human growth hormone receptor, but not the truncated isoform, is very rapidly internalized independent of Jak2-Stat5 signaling.

We have investigated trafficking of two negative regulators of growth hormone receptor (GHR) signaling: a human, truncated receptor, GHR1-279, and a GH antagonist, B2036. Fluorescent-labeled growth hormone (GH) was rapidly internalized by the full-length GHR, with >80% of the hormone internalized within 5 min of exposure to GH. In contrast, <5% of labeled GH was internalized by cells expressing truncated GHR1-279. Using another truncated receptor, GHR1-317 fused to enhanced green fluorescent protein (EGFP), we have exploited fluorescence energy transfer to monitor the trafficking of ligand-receptor complexes. The data confirmed that internalization of this truncated receptor is very inefficient. It was possible to visualize the truncated GHR1-317-EGFP packaged in the endoplasmic reticulum, its rapid movement in membrane bound vesicles to the Golgi apparatus, and subsequent transport to the cell membrane. The GH antagonist, B2036, blocked Jak2-Stat5-mediated GHR signaling but was internalized with a similar time course to native GH. The results: 1) demonstrate the rapid internalization of GH when studied under physiological conditions; 2) confirm the hypothesis that internalization of cytoplasmic domain truncated human GHRs is very inefficient, which explains their dominant negative action; and 3) show that the antagonist action of B2036 is independent of receptor internalization.     

Growth hormone and prolactin receptors in adipogenesis: STAT-5 activation, suppressors of cytokine signaling, and regulation of insulin-like growth factor I

Growth hormone GH stimulates lipolysis in mature adipocytes and primary preadipocytes but promotes adipogenesis in preadipocyte cell lines. The lactogenic hormones (prolactin PRL and placental lactogen) also stimulate adipogenesis in preadipocyte cell lines but have variable lipolytic and lipogenic effects in mature adipose tissue. We hypothesized that differences in expression of GH receptors GHR and PRL receptors PRLR during adipocyte development might explain some of the differential effects of the somatogens and lactogens on fat metabolism. To that end, we compared: (a) the expression of GHR and PRLR mRNAs in 3T3-L1 preadipocytes during the course of adipocyte differentiation; (b) the induction of STAT-5 activity by GH and PRL during adipogenesis; and (c) the acute effects of GH and PRL on the suppressors of cytokine signaling (SOCS-1-3 and cytokine-inducible SH2-domain-containing protein CIS) and IGF-I. In confluent, undifferentiated 3T3-L1 cells, the levels of GHR mRNA were approximately 250-fold higher than the levels of PRLR mRNA. Following induction of adipocyte differentiation the levels of PRLR mRNA rose 90-fold but GHR mRNA increased only 0.8-fold. Expression of both full-length (long) and truncated (short) isoforms of the PRLR increased during differentiation but the long isoform predominated at all time points. Mouse GH mGH stimulated increases in STAT-5a and 5b activity in undifferentiated as well as differentiating 3T3-L1 cells; mouse PRL mPRL had little or no effect on STAT-5 activity in undifferentiated cells but stimulated increases in STAT-5a and 5b activity in differentiating cells. mGH stimulated increases in SOCS-2 and SOCS-3 mRNAs in undifferentiated cells and SOCS-1-3 and CIS mRNAs in differentiating cells; mPRL induced CIS in differentiating cells but had no effect on SOCS-1-3. mPRL and mGH stimulated increases in IGF-I mRNA in differentiating cells but not in undifferentiated cells; the potency of mGH (3-6-fold increase, p < 0.01) exceeded that of mPRL (40-90% increase, p < 0.05). Our findings reveal disparities in the expression of PRLR and GHR during adipocyte development and differential effects of the hormones on STAT-5, the SOCS proteins, CIS, and IGF-I. These observations suggest that somatogens and lactogens regulate adipocyte development and fat metabolism through distinct but overlapping cellular mechanisms.     

Comparative FISH mapping on Z chromosomes of chicken and Japanese quail

Using direct R-banding fluorescence in situ hybridization, we assigned five functional genes-growth hormone receptor (GHR), prolactin receptor (PRLR), spleen tyrosine kinase (SYK), aldolase B (ALDOB), and muscle skeletal receptor tyrosine kinase (MUSK)-to the chicken Z chromosome. SYK and MUSK were newly localized to the chicken Z chromosome in this study. GHR and PRLR were situated close to each other on the short arm of the chicken Z chromosome, as are their counterparts on human chromosome 5. SYK, MUSK, and ALDOB, which have been mapped to human chromosome 9, were localized to the long arm of the chicken Z chromosome. Thus, the present results indicate the presence of conserved synteny between the chicken Z chromosome and human chromosomes 5 and 9. Using the same method, four of the genes (GHR, PRLR, ALDOB, and MUSK) were assigned to the Japanese quail Z chromosome. The locations of these four Z-linked genes were conserved between chicken and Japanese quail. The results support the notion that the avian Z chromosome and the mammalian X chromosome did not evolve from a common ancestral linkage group.     

The evolutionary radiation of Arvicolinae rodents (voles and lemmings): relative contribution of nuclear and mitochondrial DNA phylogenies

Background  

Mitochondrial and nuclear genes have generally been employed for different purposes in molecular systematics, the former to resolve relationships within recently evolved groups and the latter to investigate phylogenies at a deeper level. In the case of rapid and recent evolutionary radiations, mitochondrial genes like cytochrome b (CYB) are often inefficient for resolving phylogenetic relationships. One of the best examples is illustrated by Arvicolinae rodents (Rodentia; Muridae), the most impressive mammalian radiation of the Northern Hemisphere which produced voles, lemmings and muskrats. Here, we compare the relative contribution of a nuclear marker – the exon 10 of the growth hormone receptor (GHR) gene – to the one of the mitochondrial CYB for inferring phylogenetic relationships among the major lineages of arvicoline rodents.     

Structural basis of the signal transduction via transmembrane domain of the human growth hormone receptor

Background

Prior studies of the human growth hormone receptor (GHR) revealed a distinct role of spatial rearrangements of its dimeric transmembrane domain in signal transduction across membrane. Detailed structural information obtained in the present study allowed elucidating the bases of such rearrangement and provided novel insights into receptor functioning.

The Fer tyrosine kinase regulates an axon retraction response to Semaphorin 3A in dorsal root ganglion neurons

Background  

Fps/Fes and Fer are the only two members of a distinct subclass of cytoplasmic protein tyrosine kinases. Fps/Fes was previously implicated in Semaphorin 3A (Sema3A)-induced growth cone collapse signaling in neurons from the dorsal root ganglion (DRG) through interaction with and phosphorylation of the Sema3A receptor component PlexinA1, and members of the collapsin response mediator protein (CRMP) family of microtubule regulators. However, the potential role of the closely related Fer kinase has not been examined.     

Functional heterodimerization of prolactin and growth hormone receptors by ovine placental lactogen

Although homo- or heterodimerization are common mechanisms for activation of cytokine receptors, cross-talk between two distinct receptors in this superfamily has been never shown. Here we show a physiologically relevant example indicating that such an interaction does occurs, thus raising the hypothesis that heterodimerization between distinct cytokine receptors may be a novel mechanism contributing to the diversity of cytokine signaling. These findings were documented using both surface plasmon resonance and gel filtration experiments and show that ovine placental lactogen (PL) heterodimerizes the extracellular domains (ECDs) of ruminant growth hormone receptor (GHR) and prolactin receptor (PRLR). We also show that PL or PL analogues that exhibit little or no activity in cells transfected with PRLRs and no activity in cells transfected with ovine GHRs exhibit largely enhanced activity in cells cotransfected with both PRLRs and GHRs. Furthermore, chimeric receptors consisting of cytosolic and transmembrane part of ovine GHR or ovine PRLR and ECDs of human granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) alpha or beta were constructed. Upon transfection into Chinese hamster ovary cells along with reporter luciferase gene and stimulation by GM-CSF, a significant increase in luciferase activity occurred when GM-CSFR-alpha-PRLR and GM-CSFR-beta-GHR or GM-CSFR-alpha-GHR and GM-CSRR-beta-PRLR were cotransfected. In conclusion, we show that ovine PL is capable of functional heterodimerization of GHR and PRLR and that when their cytosolic parts, coupled to the ECD of GM-CSF receptors, are heterodimerized by GM-CSF, they are capable of transducing biological signal.     

Structural Characterization of the Stem-Stem Dimerization Interface between Prolactin Receptor Chains Complexed with the Natural Hormone

The most promising approach to targeting the tumor-growth-promoting actions of prolactin (PRL) mediated by its autocrine/paracrine pathway has been the development of specific PRL receptor (PRLR) antagonists. However, the optimization of such antagonists requires a thorough understanding of the activation mechanism of PRLR. We have thus conducted a systematic X-ray crystallographic study in order to visualize the successive steps of PRLR activation by PRL. We report here the structure at 3.35 Å resolution of the 1:2 complex between natural PRL and two PRLR chains (PRLR1 and PRLR2), corresponding to the final activated state of PRLR. Further than our previously published structure involving an affinity-matured PRL variant, this structure allowed to visualize for the first time the loop L5 spanning PRLR2 residues Thr133-Phe140, revealing its central implication for the three intermolecular interfaces of the complex. We equally succeeded in obtaining a comprehensive picture of the PRLR-PRLR dimerization interface, also called stem-stem interface. Site-directed mutagenesis was conducted to probe the energetic importance of stem-stem contacts highlighted by the structure. Surprisingly, in spite of significant structural differences between the PRL/PRLR₂ complex and the 1:2 growth hormone/growth hormone receptor complex, our mutational data suggest that hot-spot residues that stabilize the receptor dimerization interface are equivalent in the two complexes. This study provides a new overall picture of the structural features of PRLR involved in stabilizing its complex with PRL.     

Effect of recombinant growth hormone on expression of growth hormone receptor, insulin-like growth factor mRNA and serum level of leptin in growing pigs

Growth hormone (GH) plays an important role in regulation of animal growth, metabolism and lactation[1]. Numerous studies have shown that exogenous somatotropin (ST) can increase average daily weight gain, improve feed efficiency, stimulate protein deposition and muscle growth and decrease lipid accretion rate[1]. The original somatomedin hypothesis suggested that the effect of GH on postnatal growth was mediated by insulin-like growth hormone factor 1 (IGF-I) which was thought to be deriv…     

Single-step, multiple retroviral transduction of human T cells

Background

Retroviral transduction of human peripheral blood T cells has considerable potential in the development of gene therapy strategies for immunological disorders. New vectors and experimental procedures have been developed for efficient transduction of several genes into human T cells.

Methods

Bicistronic retroviral vectors encoding distinct cell markers were used for the simultaneous multiple transduction of a human T‐cell line (MT‐2), as well as of human peripheral blood T cells from normal donors. Transduction efficiencies were evaluated by flow cytometry and double‐ and triple‐transduced cells were isolated by fluorescence cell sorting.

Results

Four new bicistronic retroviral vectors were developed that express different gene markers under the control of the internal ribosome entry site (IRES) of the encephalomyocarditis virus. These markers are, respectively, enhanced green fluorescent protein (EGFP), β‐galactosidase, and truncated versions of human nerve growth factor receptor (ΔNGFR) and human growth hormone receptor (ΔGHR). A single 1 h spinoculation infection, performed in the presence of polybrene and using transiently produced amphotropic retroviral particles, was sufficient to obtain transduction efficiencies consistently greater than 50% on human peripheral blood T lymphocytes which had been previously stimulated for 3 days with immobilized anti‐CD3. The transient production of viral particles encoding EGFP, ΔNGFR, and ΔGHR markers in the same viral supernatant has allowed up to three different genes to be introduced simultaneously into human T cells.

Conclusions

This study describes new experimental conditions for efficient single‐step multiple transduction of human primary T lymphocytes. The procedure could be of interest for the development of gene therapy approaches. Copyright © 2002 John Wiley & Sons, Ltd.

     

Tyrosine residues of the erythropoietin receptor are dispensable for erythroid differentiation of human CD34+ progenitors

To study the role of the cytoplasmic domain and particularly the tyrosine residues of the erythropoietin receptor (EpoR) in erythroid differentiation of human primary stem cells, we infected cord blood-derived CD34+ cells with retroviruses encoding chimeric receptors containing the extracellular domain of the prolactin receptor (PRLR) and the cytoplasmic domain of either the normal EpoR or a truncated EpoR devoid of tyrosine residues. Erythroid differentiation of the infected progenitors could thus be studied after stimulation by PRL. The complete PRLR was used to assess its ability to substitute for EpoR in erythroid differentiation. Typical erythroid day-14 colonies were observed from CD34+ cells grown in PRL when infected with any of the three viral constructs. These results demonstrate that: (i) the activation of the virally transduced PRLR leads to erythroid colony formation showing that erythroid terminal differentiation can be induced by a non-erythroid receptor in human progenitors; (ii) a chimeric receptor PRLR/EpoR is able to transduce a signal leading to terminal erythroid differentiation of human CD34+ cells; (iii) in contrast to results previously reported in murine models, tyrosine residues of the EpoR are not required for growth and terminal differentiation of human erythroid progenitors.     

Growth hormone receptor is a target for presenilin-dependent gamma-secretase cleavage

Growth hormone receptor (GHR) is a cytokine receptor superfamily member that binds growth hormone (GH) via its extracellular domain and signals via interaction of its cytoplasmic domain with JAK2 and other signaling molecules. GHR is a target for inducible metalloprotease-mediated cleavage in its perimembranous extracellular domain, a process that liberates the extracellular domain as the soluble GH-binding protein and leaves behind a cell-associated GHR remnant protein containing the transmembrane and cytoplasmic domains. GHR metalloproteolysis can be catalyzed by tumor necrosis factor-alpha-converting enzyme (ADAM-17) and is associated with down-modulation of GH signaling. We now study the fate of the GHR remnant protein. By anti-GHR cytoplasmic domain immunoblotting, we observed that the remnant induced in response to phorbol ester or platelet-derived growth factor has a reliable pattern of appearance and disappearance in both mouse preadipocytes endogenously expressing GHR and transfected fibroblasts expressing rabbit GHR. Lactacystin, a specific proteasome inhibitor, did not appreciably change the time course of remnant appearance or clearance but allowed detection of the GHR stub, a receptor fragment slightly smaller than the remnant but containing the C terminus of the remnant (receptor cytoplasmic domain). In contrast, MG132, another (less specific) proteasome inhibitor, strongly inhibited remnant clearance and prevented stub appearance. Inhibitors of gamma-secretase, an aspartyl protease, also prevented the appearance of the stub, even in the presence of lactacystin, and concomitantly inhibited remnant clearance in the same fashion as MG132. In addition, mouse embryonic fibroblasts derived from presenilin 1 and 2 (PS1/2) knockouts recapitulated the gamma-secretase inhibitor studies, as compared with their littermate controls (PS1/2 wild type). Confocal microscopy indicated that the GHR cytoplasmic domain became localized to the nucleus in a fashion dependent on PS1/2 activity. These data indicate that the GHR is subject to sequential proteolysis by metalloprotease and gamma-secretase activities and may suggest GH-independent roles for the GHR.     

Leucine Supplementation Improves Acquired Growth Hormone Resistance in Rats with Protein-Energy Malnutrition

Background

Protein-energy malnutrition (PEM) can lead to growth hormone (GH) resistance. Leucine supplementation diets have been shown to increase protein synthesis in muscles. Our study aimed at investigating if long-term leucine supplementation could modulate GH-insulin-like growth factor (IGF)-1 system function and mammalian target of rapamycin (mTOR)-related signal transduction in skeletal muscles in a rat model of severe malnutrition.

Methodology/Principal Findings

Male Sprague-Dawley rats (n = 50; weight, 302 ± 5 g) were divided into 5 treatment groups, including 2 control groups (a normal control group that was fed chow and ad libitum water [CON, n = 10] and a malnourished control group [MC, n = 10] that was fed a 50% chow diet). After undergoing a weight loss stage for 4 weeks, rats received either the chow diet (MC-CON, n = 10), the chow diet supplemented with low-dose leucine (MC-L, n = 10), or the chow diet supplemented with high-dose leucine (MC-H, n = 10) for 2 weeks. The muscle masses of the gastrocnemius, soleus, and extensor digitorum longus were significantly reduced in the MC group. Re-feeding increased muscle mass, especially in the MC-L and MC-H groups. In the MC group, serum IGF-1, IGF-binding protein (IGFBP)-3, and hepatic growth hormone receptor (GHR) levels were significantly decreased and phosphorylation of the downstream anabolic signaling effectors protein kinase B (Akt), mTOR, and ribosomal protein S6 kinase 1 (S6K1) were significantly lower than in other groups. However, serum IGF-1 and IGF binding protein (IGFBP)-3 concentrations and hepatic growth hormone receptor (GHR) levels were significantly higher in the MC-L and MC-H groups than in the MC-CON group, and serum IGFBP-1 levels was significantly reduced in the MC-L and MC-H groups. These changes were consistent with those observed for hepatic mRNA expression levels. Phosphorylation of the downstream anabolic signaling effectors Akt, mTOR, and S6K1 were also significantly higher in the MC-L and MC-H groups than in the MC-CON group.

Conclusion/Significance

Our data are the first to demonstrate that long-term supplementation with leucine improved acquired growth hormone resistance in rats with protein-energy malnutrition. Leucine might promote skeletal muscle protein synthesis by regulating downstream anabolic signaling transduction.     

The receptors for prolactin and growth hormone are localized in the same region of human chromosome 5

Prolactin receptor (PRLR) and growth hormone receptor (GHR) are encoded by members of a gene family containing regions of identical sequences. To determine their chromosomal locations, cDNA probes for these genes were used. Analysis of hybridization to several somatic cell hybrids, together with hybridization in situ to metaphase chromosomes, resulted in the assignment of the loci for both receptors to human chromosome 5 in the region p13----p14. Thus, these proteins may be encoded by a cluster of related genes that evolved from a common ancestral gene, in a manner consonant with that of their ligands.