Prolactin Receptor cDNA Research Articles

Molecular characterization, mRNA expression of prolactin receptor (PRLR) gene during pregnancy, nonpregnancy in the yak ( Bos grunniens)

Prolactin (PRL) plays central roles in a wide range of body functions in mammals, and the actions are mediated by the specific cell surface receptor, the prolactin receptor (PRLR). To better understand the role of PRL in the yak ( Bos grunniens), in the present study, we first cloned yak PRLR cDNA, and compared its mRNA expression in several tissues with cattle ( Bos taurus). By reverse transcriptase-polymerase chain reaction (RT-PCR) strategy, we obtained full-length of yak PRLR cDNA sequence comprised of an open reading frame of 1746 bp encoding a 581 amino acid protein, and contained a signal sequence and a transmembrane region. The intracellular domain had two pairs of cysteine residues and a WSXWS motif. The cytoplasmic domain comprised 323 residues and contained box 1 sequence. The yak PRLR shared 66.0–98.5% protein sequence identity with mammalian homologs. Real-time PCR analysis revealed that PRLR mRNA was higher in mammary tissue than in ovary and endometrium ( P < 0.01). During pregnancy, the ovary and mammary PRLR mRNA expression increased by 33- and 2.9-fold in yak, respectively, and increased by 46- and 3.8-fold in cattle, respectively. PRLR mRNA expression was higher ( P < 0.05) in mammary tissue and ovary of pregnant cow than that of pregnant yak. It is proposed that the increased ovarian and mammary PRLR mRNA expression during pregnancy may be associated with corpus luteum function for maintenance of pregnancy and mammary development for subsequent lactation.

The second prolactin receptor in Nile tilapia (Oreochromis niloticus): molecular characterization, tissue distribution and gene expression

Prolactin (PRL) is one of the most versatile hormones found in the pituitary of vertebrates and exerts its actions through binding to a specific PRL receptor (PRLR). Here we describe the cloning and characterization of a second prolactin receptor (ntPRLR2), isolated from the ovary of Nile tilapia (Oreochromis niloticus). The newly identified PRLR cDNA was 2011 bp in length and encoded 529 amino acids. It shared 31.6% identity in nucleotide sequence and 29.2% in deduced amino acid sequence with the first PRLR identified in Nile tilapia (ntPRLR1). Both of these ntPRLRs resemble the long form mammalian PRLRs. The nominated ntPRLR2 was further confirmed as a real prolactin receptor based on its competence to transactivate the beta-casein and c-fos promoters in the transiently ntPRLR2-transfected HEK293 cells. The ntPRLR2 gene also found to encode a 864-bp short form transcript in the ovary, which was confirmed by Northern blot analysis. A tissue distribution study by real-time PCR revealed that the mRNA of both receptors (ntPRLR1 and ntPRLR2) was widely expressed in different tissues, with an extremely high abundance in the osmoregulatory organs, including the gills, intestine and kidney. ntPRLR1 mRNA was more abundant than ntPRLR2 in the testis, while the reverse expression pattern was found in the ovary. In the ovary, ntPRLR2 mRNA demonstrated a distinct gonadal development-dependent expression profile, with significantly higher levels at a sexual mature stage than at sexual recrudescent and sexual regressed stages. When challenged with estradiol, ntPRLR2 mRNA expression was up-regulated by E2, whereas E2 had no significant effect on ntPRLR1.

Molecular cloning of prolactin receptor of the Peking duck

Prolactin receptor (PRLR) cDNA of Peking duck was cloned using reverse transcription-PCR methodology (GenBank accession no. EF502054). The duck PRLR (dPRLR) cDNA contains 2,439 bp including the ATG start codon and TAA stop that encodes 832 amino acid residues. The putative receptor protein had 2 strong hydrophobic stretches: one was the signal sequence in N-terminal region and the other was the single transmembrane region of 24 amino acid residues in the central portion. Excluding the signal sequence, the mature dPRLR contained 808 amino acid residues with a theoretical molecular mass of 91.5 kDa. The mature protein could be divided into 3 domains, extracellular domain (ECD), transmembrane domain, and intracellular domain. The ECD contained 2 homologous repeat units with approximately 67% amino acid sequence identity to each other. The membrane distal unit and proximal unit consisted of 204 and 212 amino acids, respectively. Each unit was similar to the singular ECD of the mammalian PRLR. The characteristic extracellular cysteines and the WSXWS motif of the cytokine receptor were conserved in both repeat units. The intracellular domain of dPRLR consisted of 368 amino acids at the C-terminus and contained 2 conserved regions-box 1 and box 2. The aforementioned characters of duck were all similar to chicken and mammalians, which implied that the dPRLR had same mechanism with other species. Sequence similarity analysis shows that the dPRLR shares approximately 45 to 97% amino identity and approximately 30 to 96% nucleic acid identity as compared with other species. Overall, the greatest sequence identity was found with goose PRLR. In a phylogenetic analysis, the duck PRLR was found to cluster with the PRLR of goose, turkey, chicken, and pigeon. Semiquantitative reverse transcription-PCR indicated that dPRLR mRNA was expressed in all 8 tissues. Expression in the kidney was greatest in varietals tissues.

Overexpression of Activated Murine Notch1 and Notch3 in Transgenic Mice Blocks Mammary Gland Development and Induces Mammary Tumors

The mouse mammary tumor virus (MMTV) provirus was found to target the Notch1 gene, producing insertional mutations in mammary tumors of MMTV/neu transgenic (Tg) mice. In these mammary tumors, the Notch1 gene is truncated upstream of the transmembrane domain, and the resulting Notch1 intracellular domain (Notch1(intra)), deleted of most extracellular sequences, is overexpressed. Although Notch1(intra) transforms mammary epithelial cells in vitro, its role in mammary gland tumor formation in vivo was not studied. Therefore, we generated MMTV/Notch1(intra) Tg mice that overexpress murine Notch1(intra) in the mammary glands. We observed that MMTV/Notch1(intra) Tg females were unable to feed their pups because of impaired ductal and lobulo-alveolar mammary gland development. This was associated with decreased proliferation of ductal and alveolar epithelial cells during rapid expansion at puberty and in early pregnancy, as well as decreased production of beta-casein. Notch1(intra) repressed expression of the beta-casein gene promoter, as assessed in vitro with a beta-casein/luciferase reporter construct. The MMTV/Notch1(intra) Tg females developed mammary gland tumors, confirming the oncogenic potential of Notch1(intra) in vivo. Furthermore, MMTV/Notch3(intra) Tg mice exhibited a very similar phenotype. Thus, these Tg mice represent novel models for studying the role of Notch1 or Notch3 in the development and transformation of the mammary gland.

Association of polymorphisms for prolactin and prolactin receptor genes with broody traits in chickens

Prolactin (PRL) is generally accepted as crucial to the onset and maintenance of broodiness in avian species. The prolactin receptor (PRLR) plays an important role in the PRL signal transduction cascade. Two candidate genes, PRL and PRLR, were screened for polymorphisms in the chicken, and their genetic effects on broodiness were evaluated. Pedigreed hens (n = 155) of the Blue-shell chicken, a Chinese local breed, were observed for phenotypic broody traits including nesting days, broody days, repeats of broody cycles, and duration of broodiness. For polymorphism analysis, White Leghorns, Hy-Line brown egg layers, Avian broilers, and some other Chinese local breeds were included. Fifteen sets of primers were used to amplify the nucleotide sequences of the promotor of PRL and exons of PRLR. The PCR products were screened for polymorphisms using single-stranded conformational polymorphism protocol. Sequencing revealed a 24-bp insertion occurring in the promotor, −377 ˜ −354, of PRL (GenBank accession no. AB011434). A single nucleotide polymorphism (SNP), A9026G (GenBank accession no. AY237377), in exon 3 of PRLR was also detected, which led to a nucleotide transition in the 5′-untranslated region (5′-UTR) of PRLR cDNA. Two SNP, T14771C and G14820A (GenBank accession no. AY237376), were detected in exon 6 of the PRLR. The T14771C transition led to an amino acid variation, Leu340Ser, in PRLR, whereas the G14820A transition was a synonymous mutation. An association analysis showed that the genetic polymorphisms at PRLR3 and PRLR6 were not related to broodiness (P > 0.05), whereas the individuals without the insertion sequence at PRLpro2 were associated with broody traits (P < 0.05) and the incidence (>30%) of typical broody of genotypes +/− and −/− was higher (P < 0.01) than that of +/+. In addition, all White Leghorns were +/+ for PRLpro2, whereas local breeds with very strong broodiness were nearly all −/−. Homozygous insertion of the 24-bp sequence in the PRL promoter may decrease the expression of PRL, leading to nonbroodiness. The results suggested that PRLpro2 could be a genetic marker in breeding against broodiness in chickens.

Positive and negative regulatory elements in the late lactation protein-A gene promoter from the tammar wallaby ( Macropus eugenii)

Little is known about the regulation of the marsupial-specific late lactation protein-A (LLP-A) gene, first expressed at mid-lactation in the mammary gland of the tammar wallaby. A genomic clone of LLP-A was sequenced and shown to include seven exons. The LLP-A promoter region of 1969 bp ligated to a secreted alkaline phosphatase (SEAP) gene reporter was co-transfected into CHO-K1 cells with prolactin (PRL) receptor cDNA. Transfected cells cultured with insulin, cortisol and PRL did not secrete SEAP into media. Similarly, this construct was not expressed in the mammary gland of eight lines of transgenic mice. In contrast, when the LLP-A promoter region was reduced to 850 bp, the expression of the SEAP reporter in CHO-K1 cells was constitutive and PRL-independent, despite the presence of two low affinity Stat5 binding sites. The 1969 bp promoter was analyzed using nine serial deletions ligated to the SEAP gene. The expression of these constructs was PRL-independent. Five putative inhibitory elements were identified between −1969 and −1796, −1404 and −1184, −1184 and −992, −992 and −757, and −591 and −425, and a putative enhancer or core transcription element between −425 and−239. These studies indicate that the complex temporal regulation of the LLP-A gene involves elements in its 5′-regulatory region.

Molecular cloning of bullfrog prolactin receptor cDNA: changes in prolactin receptor mRNA level during metamorphosis

In amphibian larvae, prolactin (PRL) is known to possess growth-promoting and anti-metamorphic activities. For further understanding of the role of PRL in larvae, bullfrog PRL receptor (bfPRLR) cDNA was obtained from the tail fin of premetamorphic tadpoles by use of the reverse transcription-polymerase chain reaction (RT-PCR) coupled with 5 ′- and 3 ′-rapid amplification of cDNA ends (RACE). The predicted bfPRLR was composed of 617 amino acids, contained a single transmembrane domain, and showed 33–57% sequence homologies with known sequences of vertebrate PRLRs. When bfPRLR was transiently expressed, specific binding of 125I-labeled bullfrog PRL (bfPRL) was observed. By Northern blot analysis, a 3-kb transcript was detected in the tail fin. By RT-PCR bfPRLR mRNA expression was detected mainly in the brain, kidney, skin, and tail throughout prometamorphic and middle climactic periods. The results of an RNase protection assay revealed that the bfPRLR mRNA level in the tail fin increased around the onset of climax (stage XX) and was maintained at a relatively high value at least until mid-climax (stage XXII). It also revealed that bfPRLR mRNA level in the kidney of larvae gradually rose as metamorphosis progressed. The results support the view that PRL in larval period acts not only on the larval organs but also on the organs that are necessary for the adult life to maintain or develop their structures and functions.

Expression of prolactin receptor mRNA in the abdominal gland of the newt Cynops ensicauda

To further the understanding that the structural development of the Cynops ensicauda abdominal gland and the synthesis of the pheromone silefrin in the gland are under the control of prolactin and androgen, we sought to demonstrate the presence of prolactin receptor (PRLR) mRNA in the gland. Firstly, PRLR cDNA was isolated from an abdominal gland cDNA library. A cDNA consisting of a 415-bp 5′-untranslated region, 1878-bp open reading frame and 175-bp 3′-untranslated region was obtained. The deduced amino acid sequence consisted of 626 amino acids with signal peptide and single transmembrane domain. By Northern blot analysis using partial C. ensicauda PRLR cDNA, two transcripts, of 3 and 10 kb, were detected for PRLR in the brain, liver, kidney, abdominal gland, oviduct and skin. RT-PCR coupled with Southern blot analysis showed that the PRLR gene was transcribed broadly in newt organs and revealed that PRLR mRNA levels in the abdominal gland were much higher in sexually developed newts than in the sexually undeveloped ones. By in situ hybridization, specific signals were detected in the epithelial cells of the abdominal gland of sexually developed newts, but much less in those of the sexually undeveloped ones.

Seasonal environmental changes modulate the prolactin receptor expression in an eurythermal fish.

Eurythermal fish have evolved compensatory responses to the cyclical seasonal changes of the environment. The complex adaptive mechanisms include the transduction of the physical parameters variations into molecular signals. Studies in carp have indicated that prolactin and growth hormone expression is associated with acclimatization, suggesting that the pituitary gland is a relevant physiological node in the generation of the homeostatic rearrangement that occurs in this adaptive process. Here, we report the cloning and characterization of a full-length carp prolactin receptor cDNA, which codes for the long form of the protein resembling that found in mammalian prolactin receptors. We identified up to three receptor transcript isoforms in different tissues of the teleost and assessed cell- and temporal-specific transcription and protein expression in carp undergoing seasonal acclimatization. The distinctive pattern of expression that carp prolactin receptor (cPRLr) depicts upon seasonal acclimatization supports the hypothesis that prolactin and its receptor are clearly involved in the new homeostatic stage that the eurythermal fish needs to survive during the cyclical changes of its habitat.

103. Cloning and expression of the ovarian prolactin receptor (PRLR) in the tammar wallaby (Macropus eugenii)

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Expression of prolactin receptor (PRL-R) mRNA in the caudal gland of Hokkaido sika deer (Cervus nippon yesoensis Heude, 1884)

We applied the in situ hybridization technique to the Hokkaido sika deer (Cervus nippon yesoensis) caudal glands using probes designed from the red deer (Cervus elaphus) prolactin receptor cDNA. Prolactin receptor mRNA expression was consistently detected in the secretory epithelial cells collected from fawns and lactating hinds. This suggests that the presence of developed caudal glands during late pregnancy and lactation may be caused by increased blood levels of prolactin. During late pregnancy and lacation, visual range is limited for deer in forests and grasslands because of the flourishing leaves and sprigs. Therefore, we suggest that the scent emitted from the caudal gland in a particular season coordinates with and reinforces the visual alarm effect of the rump patch and tail.

Molecular characterization of the prolactin receptor in two fish species, tilapiaOreochromis niloticusand rainbow trout,Oncorhynchus mykiss: A comparative approach

We present recent information on the molecular characterization of the prolactin receptor (PRL-R) in two teleost species, tilapia (Oreochromis niloticus) and rainbow trout (Oncorhynchus mykiss), in the perspective of improved understanding of the physiological differences in the control of osmoregulatory function between these two fish species. Although our interest will mainly focus on osmoregulatory organs, we will also discuss evidence of the presence of PRL-R in other tissues such as gonads and hematopoietic organs. The first fish PRL-R was characterized in tilapia. This receptor is similar to that of the long form of mammalian PRL-R, but the most conserved region (extracellular domain) has only 53% identity with mammalian PRL-R. A rainbow trout PRL-R cDNA has been also isolated and appeared very similar in structure to tilapia PRL-R. Expression of the PRL-R gene was studied by Northern blotting for various tissues from tilapia and trout, and a unique transcript size of 3.2-3.4 kb was observed in all tissues studied (including male and female gonads, skin, brain, spleen, head, kidney, and circulating lymphocytes). Osmoregulatory organs (gills, kidney, intestine) were the richest tissues. Using in situ hybridization, PRL-R transcripts were localized in gill chloride cells, both in trout and tilapia. Analysis of PRL-R transcript levels in gills, kidney, and intestine indicated the maintenance of a high level of expression during adaptation to a hyperosmotic environment. These results support PRL being a pleiotropic hormone in fish and suggest the presence of a unique PRL-R form in tilapia and in trout. Finally, characterization of hormone receptor binding has been carried out in both species using a radioreceptor assay (in tilapia) or surface plasmon resonance (SPR) technology (in trout). These studies indicated the presence of a stable hormone-receptor complex in tilapia, while PRL binds to its receptor through an unstable homodimeric complex in trout. Thus, the characteristics of PRL binding on its receptor appear to be significantly different in tilapia and trout. Whether such differences may lead to different signal transduction mechanisms and osmoregulatory actions of PRL in these two euryhaline species merits further investigation.Key words: prolactin receptor, genetic expression, hormone-receptor interaction, surface plasmon resonance, fish osmoregulation.

Sequence and functional characterisation of the marmoset monkey ( Callithrix jacchus) prolactin receptor: comparative homology with the human long-form prolactin receptor

This study demonstrates the cloning and in-vitro characterisation of the marmoset monkey ( Callithrix jacchus) prolactin receptor cDNA. The marmoset prolactin receptor cDNA was generated by reverse transcription-polymerase chain reaction using adrenal RNA and primers designed from prolactin receptor conserved regions. Sequence analysis predicts a mature protein of 598 amino acids exclusive of the 24 amino acid signal peptide. The marmoset prolactin receptor cDNA shares 93 and 61% base pair, and 89 and 61% amino acid sequence homologies with the long form human and rat prolactin receptor cDNA, respectively. The marmoset prolactin receptor cDNA sequence retains all the receptor sequences that have been shown previously to be essential for ligand binding, structural integrity and signal transduction. Transfection of human 293 fibroblast cells with the marmoset prolactin receptor cDNA (three independent experiments) confirmed the expression of a receptor that has high binding affinity to human growth hormone ( K a=3.6±0.07 nM −1 and B max=7.55±2.06×10 −11 M) and human prolactin ( K a=3.1±0.12 nM −1 and B max=2.87±0.66×10 −11 M). Functionality of the receptor was assessed by co-transfection of 293 fibroblast cells with marmoset prolactin receptor cDNA and the Jak2 cDNA, or marmoset prolactin receptor and a Stat5 responsive element linked to the luciferase coding sequence. Incubation of the cells with 18 nM ovine prolactin resulted in rapid phosphorylation of Jak2 as ascertained by Western blotting. In addition, the marmoset prolactin receptor cDNA led to 9.06±0.47-fold induction of luciferase gene activity. This was comparable with the induction observed following transfection with the human prolactin receptor cDNA (8.55±0.5-fold). In-vivo prolactin receptor expression in the marmoset monkey was assessed by ribonuclease protection assay and detected in a number of tissues including female reproductive organs. These data confirm the cloning and functionality of the marmoset prolactin receptor cDNA. The marmoset prolactin receptor shares a high sequence homology with the long-form human prolactin receptor, and both receptors bind hormones with comparable affinity and confer a similar intracellular response. The marmoset monkey may provide a useful tool to investigate the role of prolactin in primate reproduction.

Role of protein kinases in the prolactin-induced intracellular calcium rise in Chinese hamster ovary cells expressing the prolactin receptor.

There is still only limited understanding of the early steps of prolactin signal transduction in target cells. It has been shown that prolactin actions are associated with cell protein phosphorylation, Ca2+ increases, and so on. However, the link between the activation of kinases and calcium influx or intracellular Ca2+ mobilization has not yet been clearly established. Chinese hamster ovary (CHO) cells, stably transfected with the long form of rabbit mammary gland prolactin receptor (PRL-R) cDNA were used for PRL-R signal transduction studies. Spectrofluorimetric techniques were used to measure intracellular calcium ([Ca2+]i) in cell populations with Indo1 as a calcium fluorescent probe. We demonstrate that, although protein kinase C activation (PMA or DiC8) caused a calcium influx in CHO cells, prolactin-induced PKC activation was not responsible for the early effect of prolactin on [Ca2+]i. Activation of protein kinase A (PKA) or protein kinase G did not modify [Ca2+]i and inhibition of PKA pathway did not affect the prolactin response. In the same way, phosphatidylinositol-3 kinaseinhibition had no effect on the prolactin-induced Ca2+ increase. On the other hand, tyrosine kinase inhibitors (herbimycin A, lavendustin A, and genistein) completely blocked the effect of prolactin on [Ca2+]i (influx and release). W7, a calmodulin-antagonist, and a specific inhibitor of calmodulin kinases (KN-62), only blocked prolactin-induced Ca2+ influx but had no significant effect on Ca2+ release. Using pharmacological agents, we present new data concerning the involvement of protein phosphorylations in the early effects of prolactin on ionic channels in CHO cells expressing the long form of PRL-R. Our results suggest that, at least in the very early steps of prolactin signal transduction, serine-threonine phosphorylation does not participate in the prolactin-induced calcium increase. On the other hand, tyrosine phosphorylation is a crucial, very early step, since it controls K+ channel activation, calcium influx, and intracellular calcium mobilization. Calmodulin acts later, since its inhibition only blocks the prolactin-induced Ca2+ influx.

Cloning of a cDNA for Xenopus prolactin receptor and its metamorphic expression profile.

A pituitary hormone, prolactin (PRL) shows various effects on cellular metabolism in amphibians, such as stimulation of larval tissue growth and inhibition of metamorphic changes. All these effects are mediated by its cell surface receptor. However, lack of information on PRL receptor (PRL-R) gene expression has made the physiological importance of the PRL/PRL-R system obscure in amphibian metamorphosis. Hence, a Xenopus PRL-R cDNA was cloned, its structure was characterized, and specific binding of PRL to Xenopus PRL-R expressed in COS-7 cells was confirmed. In adult tissues, high level expression was found in the lung, heart, brain, thymus and skin, and low level in the oviduct, kidney and spinal cord. The developmental expression pattern showed that PRL-R messenger ribonucleic acid (mRNA) was expressed in the brain and tail from premetamorphosis and the level increased toward late metamorphosis, suggesting that PRL may inhibit the metamorphic changes in those organs. The level of brain PRL-R mRNA reached a peak just at the start of the metamorphic climax stages and then decreased, whereas in the tail, mRNA expression peaked at late metamorphosis. In the kidney, mRNA expression increased and reached a maximum level at the end of metamorphosis. The results obtained were discussed in relation to metamorphosis.

Evidence for multiple prolactin receptor transcripts in the turkey

Multiple prolactin receptor (PRL-R) mRNA transcript isoforms have been identified in mammals, but there are conflicting reports concerning the number of avian PRL-R isoforms. We hypothesized that multiple turkey PRL-R transcript isoforms exist and that PRL-R mRNA abundance may be related to reproductive status. Two turkey PRL-R cDNA fragments were generated using reverse transcriptase polymerase chain reaction (RT-PCR) that displayed a high degree of similarity to mammalian and avian PRL-R. Northern blot analysis of poly A+ mRNA hybridized to a turkey PRL-R riboprobe revealed a 3.1-kb band in the liver, oviduct, and testes. Additional 1.5- and 10.7-kb transcripts were found in the liver and testes, respectively. Hybridization of the same Northern blot to a chicken PRL-R probe verified the presence of a 3.1-kb transcript in all three tissues. A Northern blot was used to examine turkey PRL-R transcript isoform expression in laying hens. A 3.1-kb band was found in the pineal, infundibulum, magnum, isthmus, kidney, and intestine. In addition, 10.7- and 7.3-kb bands were detected in the pineal, magnum, isthmus, and intestine. Turkey PRL-R transcript isoforms were also examined throughout the reproductive cycle. The 10.7-, 7.3-, and 3.1-kb isoforms were detected in the oviduct, intestine, and pineal during each reproductive state. Turkey PRL-R mRNA levels were also compared during the reproductive cycle. Turkey PRL-R mRNA levels were greatest in laying hen pineal glands (P<0.05) and in incubating hen oviducts. This study provides the first evidence for multiple PRL-R mRNA transcript isoforms in turkeys.

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.

Molecular characterization of the prolactin receptor in two fish species, tilapia &lt;i&gt;Oreochromis niloticus&lt;/i&gt; and rainbow trout, &lt;i&gt;Oncorhynchus mykiss&lt;/i&gt;: A comparative approach

We present recent information on the molecular characterization of the prolactin receptor (PRL-R) in two teleost species, tilapia (Oreochromis niloticus) and rainbow trout (Oncorhynchus mykiss), in the perspective of improved understanding of the physiological differences in the control of osmoregulatory function between these two fish species. Although our interest will mainly focus on osmoregulatory organs, we will also discuss evidence of the presence of PRL-R in other tissues such as gonads and hematopoietic organs. The first fish PRL-R was characterized in tilapia. This receptor is similar to that of the long form of mammalian PRL-R, but the most conserved region (extracellular domain) has only 53% identity with mammalian PRL-R. A rainbow trout PRL-R cDNA has been also isolated and appeared very similar in structure to tilapia PRL-R. Expression of the PRL-R gene was studied by Northern blotting for various tissues from tilapia and trout, and a unique transcript size of 3.2-3.4 kb was observed in all tissues studied (including male and female gonads, skin, brain, spleen, head, kidney, and circulating lymphocytes). Osmoregulatory organs (gills, kidney, intestine) were the richest tissues. Using in situ hybridization, PRL-R transcripts were localized in gill chloride cells, both in trout and tilapia. Analysis of PRL-R transcript levels in gills, kidney, and intestine indicated the maintenance of a high level of expression during adaptation to a hyperosmotic environment. These results support PRL being a pleiotropic hormone in fish and suggest the presence of a unique PRL-R form in tilapia and in trout. Finally, characterization of hormone receptor binding has been carried out in both species using a radioreceptor assay (in tilapia) or surface plasmon resonance (SPR) technology (in trout). These studies indicated the presence of a stable hormone-receptor complex in tilapia, while PRL binds to its receptor through an unstable homodimeric complex in trout. Thus, the characteristics of PRL binding on its receptor appear to be significantly different in tilapia and trout. Whether such differences may lead to different signal transduction mechanisms and osmoregulatory actions of PRL in these two euryhaline species merits further investigation.

The prolactin receptor from the brushtail possum ( Trichosurus vulpecula): cDNA cloning, expression and functional analysis

A full length, prolactin receptor cDNA clone has been isolated from the brushtail possum ( Trichosurus vulpecula). This clone encodes a 625 amino acid protein which shares 60–70 and 54% sequence identity with prolactin receptor (long form) sequences from mammalian and avian species, respectively. Sequence similarity was highest in the extra-cellular, hormone-binding domain and in specific regions of the intracellular domain which regulates prolactin receptor signalling in cells. Prolactin receptor mRNA was detected in a wide range of possum tissues and in the mammary gland the PRL-R gene was differentially expressed during lactation with peak mRNA levels being detected during the first 6 days of lactation and after day 115 throughout late lactation. This pattern of PRL-R mRNA expression in the mammary gland is similar to that observed for circulating prolactin in the lactating possum. In CHO cells transiently transfected with the possum prolactin receptor, expression of a β-lactoglobulin promoter/reporter gene construct was increased 3-fold by adding prolactin. The possum prolactin receptor is therefore capable of binding ovine prolactin and activating the Jak2/Stat5 signalling cascade. This provides evidence for the highly conserved nature of the prolactin signalling pathway in mammalian evolution.

Distinct Cytoplasmic Regions of the Prolactin Receptor Are Required for Prolactin-induced Calcium Entry

Two cytoplasmic regions of the prolactin (PRL) receptor are well documented for their participation in PRL signal transduction, the membrane proximal box 1 and the COOH-terminal region. In order to study the role of these regions in PRL-induced Ca2+ increase, we use Chinese hamster ovary (CHO) cells stably transfected with mutated PRL receptor cDNA. These cells express the long form of PRL receptor deleted from box 1 (CHO Delta1 cells) or the 141 amino acids of the COOH-terminal region (CHO H3 cells). The patch-clamp technique in "whole-cell" configuration and microfluorimetric techniques were used singly or in combination. Data obtained for these cells were compared with those we have recently published using CHO cells expressing the wild-type long form of the PRL receptor (CHO TSE32). In contrast to CHO TSE32 cells, exposure of CHO Delta1 or H3 cells to PRL (0.05-50 nM) did not modify [Ca2+]i. We have previously shown that the PRL-induced calcium influx via voltage-insensitive, Ca2+ channels was due to the activation of tyrosine kinase-dependent K+ channels that hyperpolarize the CHO TSE32 cell membrane (hyperpolarization-driven Ca2+ influx). Therefore, two events are involved in PRL-induced Ca2+ changes (i) JAK2-activation of K+ channels and (ii) intracellular messenger-opening of Ca2+ channels. In CHO Delta1 cells, PRL (0.05-50 nM) neither hyperpolarized the membrane potential nor stimulated the JAK2-dependent K+ current, confirming the pivotal role played by box 1/JAK2 in the PRL-induced activation of K+ channels. However, when these cells were voltage-clamped below the resting membrane potential, application of 5 nM PRL resulted in an increase in Ca2+ influx. Therefore, box 1/JAK2 was not involved in the opening of these Ca2+ channels. In CHO H3 cells, 5 nM PRL activated the K+ current and hyperpolarized the membrane potential without any effect on [Ca2+]i. Moreover, PRL was also ineffective on CHO H3 cells voltage-clamped below the resting membrane potential. Therefore, the COOH-terminal region is involved in the production of the intracellular messenger that opens voltage-independent Ca2+ channels. We conclude from these findings that box 1 and COOH-terminal regions are both needed for PRL-induced Ca2+ changes.