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M Edery

Publications and source records attributed to M Edery.

At least 55 records · Page 3Linked to original sources
PubMed1994

A limited cytoplasmic region of the prolactin receptor critical for signal transduction.

Prolactin receptors (PRL-R) are members of the cytokine receptor superfamily, which have in common, an absence of any known consensus sequence for signal transduction in their cytoplasmic domains. Four areas of high sequence homology have been identified in the cytoplasmic domains of PRL and growth hormone (GH) receptors, which may be important for signal transduction. The aim of this study was to investigate the role of these cytoplasmic regions in the functional activity of the PRL-R. Several mutant forms of PRL-R were constructed either by truncation or by deletion of the cDNA. Biological activities of these mutant receptors were assayed in CHO cells using a functional assay consisting in the co-transfection of PRL-R cDNA, along with a PRL responsive promoter fused to the coding sequence of the chloramphenicol acetyl transferase (CAT) gene. Progressive truncation of the cytoplasmic domain led to a progressive loss of ability to transactivate the CAT gene. Fully active PRL-R could be obtained when 217 of 358 aa of the cytoplasmic domain were present. Deletion of the first region of homology with the GH-R (residues 245-267) abolished the functional activity of PRL-R, whereas deletion of the second region of homology (residues 322-333) was without effect. These results indicate that a critical cytoplasmic region of 23 residues proximal to the transmembrane domain is essential for PRL signal transduction. There is strong homology within an 8-residue segment of this region with other members of the cytokine receptor superfamily, suggesting it contains a sequence necessary for signal transduction.

Amino Acid Sequence↗
PubMed1994

The GH receptor and signal transduction.

The primary structure of the growth hormone (GH) receptor in rabbits and humans determined by complementary DNA cloning revealed a single membrane-spanning protein of approximately 620 amino acids. A binding protein (bp) specific for GH has been identified in the serum of a number of species. In rabbits and man, a single 4.5-kb transcript has been identified that encodes the full-length receptor. In rats and mice, however, a smaller transcript produced by alternative splicing has been reported which is specific for the GHbp. Recently, the X-ray crystallographic structure of GH and its receptor have clearly shown the formation of an unusual homodimer, consisting of one molecule of GH and two molecules of hGHbp. Formation of the GH dimer is a necessary prerequisite for biological activity. The transcriptional activity of wild-type and mutant forms of GH receptor has been determined by co-transfecting the promoter of a GH-responsive gene, coupled to CAT along with the receptor cDNA. A 25-amino acid region near the transmembrane domain has been shown to be important for functional activity, although 8 amino acids (known as Box 1), rich in prolines, is essential. Alanine scanning mutagenesis has revealed that individual substitution of each residue is without effect, while the replacement of the last 2 or all 4 of the prolines abolishes activity. Finally, GH has been shown to induce rapid tyrosine phosphorylation of several proteins in cells expressing the receptor, one of which has recently been identified as the kinase JAK2 and another as MAP kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Receptor domains involved in signal transduction of prolactin and growth hormone.

Prolactin (PRL) and growth hormone (GH) receptors are members of a superfamily that include receptors for a number of cytokines. GH and its receptor form an unusual homodimer consisting of one molecule of GH and two molecules of receptor. A similar homodimer of the PRL receptor is probably required for biological effects to be seen. Using specific assays to measure the functional activity of PRL and GH receptors, a 25 amino acid juxtamembrane region has been identified as essential but not sufficient for normal action. More detailed studies have limited the region to eight amino acids, rich in prolines, that is highly conserved in many members of the receptor superfamily. Finally, GH and PRL have been shown to induce the rapid tyrosine phosphorylation of an associated kinase, Janus kinase 2, and of the receptor itself.

Animals↗
PubMed1993

Lack of hormone binding in COS-7 cells expressing a mutated growth hormone receptor found in Laron dwarfism.

A single point mutation in the growth hormone (GH) receptor gene generating a Phe-->Ser substitution in the extracellular binding domain of the receptor has been identified in one family with Laron type dwarfism. The mutation was introduced by site-directed mutagenesis into cDNAs encoding the full-length rabbit GH receptor and the extracellular domain or binding protein (BP) of the human and rabbit GH receptor, and also in cDNAs encoding the full length and the extracellular domain of the related rabbit prolactin (PRL) receptor. All constructs were transiently expressed in COS-7 cells. Both wild type and mutant full-length rabbit GH and PRL receptors, as well as GH and prolactin BPs (wild type and mutant), were detected by Western blot in cell membranes and concentrated culture media, respectively. Immunofluorescence studies showed that wild type and mutant full-length GH receptors had the same cell surface and intracellular distribution and were expressed with comparable intensities. In contrast, all mutant forms (full-length receptors or BPs), completely lost their modify the synthesis ligand. These results clearly demonstrate that this point mutation (patients with Laron syndrome) does not modify the synthesis or the intracellular pathway of receptor proteins, but rather abolishes ability of the receptor or BP to bind GH and is thus responsible for the extreme GH resistance in these patients.

Animals↗
PubMed1993

Roles of extracellular and cytoplasmic domains of the prolactin receptor in signal transduction to milk protein genes.

We have previously shown that the long form of the PRL receptor is able to activate milk protein gene transcription. In the present study, we have determined the respective contribution of the extracellular and the intracellular domains of this receptor to transcriptional activation of a milk protein gene by PRL. The membrane-anchored intracellular domain (pTMI) expressed alone was devoid of PRL binding activity, as expected, and did not constitutively stimulate expression of the target gene. The extracellular domain (pE), expressed alone as a soluble receptor form, binds PRL with 10-fold higher affinity than the full-length membrane receptor. This form was also unable to stimulate the expression of the reporter gene. However, expression of both mutants (pE + pTMI) in the same cell partially restored the ability of PRL to activate the beta-lactoglobulin promoter. Replacement of cysteine 184 by a serine in the extracellular domain of the receptor impairs this restoration of the biological response. However, introduction of the same mutation in the full-length receptor did not affect its functional activity. These results indicate that the membrane-anchored cytoplasmic domain of the PRL receptor has no constitutive activity, and that coexpression of individual extracellular and intracellular domains leads to restoration of receptor function. We propose that restoration may be the result of reconstitution of the holoreceptor through disulfide bonding, or it may be the result of interaction of the extracellular region with an external transducing molecule.

Animals↗
PubMed1993

Epidermal growth factor receptor levels in reproductive organs of female mice exposed neonatally to diethylstilbestrol.

Binding of epidermal growth factor (EGF) to membrane preparations of vagina, uterus, ovary, oviduct, and liver was examined in mice treated neonatally with diethylstilbestrol (DES) and compared with that in untreated mice. Binding in the vagina (12.5 +/- 0.73 fmol/mg protein) was somewhat higher than in the uterus (8.0 +/- 0.34 fmol/mg protein). Level of specific binding was of the order: liver (18.4 +/- 1.09 and 16.0 +/- 1.53 fmol/mg protein) > vagina (12.5 +/- 0.73 and 8.2 +/- 0.57 fmol/mg protein) > uterus (8.0 +/- 0.34 and 6.8 +/- 0.56 fmol/mg protein) > ovary (6.8 +/- 0.36 and 8.0 +/- 1.05 fmol/mg protein) > oviduct (2.1 +/- 0.32 and 1.7 +/- 0.05 fmol/mg protein) in control and neonatally DES-exposed mice, respectively. Thus, neonatal DES exposure significantly lowered the binding site level only in the vagina, without modifying the binding affinity (Kd = 5.4 x 10(-9) M in controls vs 4.6 x 10(-9) M in DES-exposed mice). Reduction of EGF receptor level in the vagina correlates with ovary-independent persistent proliferation and keratinization of the vagina induced by neonatal DES exposure. EGF receptors were immunohistochemically demonstrated in epithelial cells of vagina, uterus, and oviduct and in stromal cells in uterus and oviduct using a polyclonal antibody to human EGF receptor protein.

Animals↗
PubMed1992

The Nb2 form of prolactin receptor is able to activate a milk protein gene promoter.

We have recently cloned a cDNA encoding a mutant form of PRL receptor (PRL-R) from Nb2 cells, a PRL-dependent T lymphocyte-derived cell line. This cDNA is identical to the long form of the rat PRL-R, except for a deletion of 594 base pairs in the cytoplasmic domain, resulting in a mature receptor protein of 393 amino acids. Although a segment containing three cytoplasmic regions of moderate to high amino acid sequence identity with members of the PRL/GH receptor family is missing in this receptor form, the region of highest (70%) identity is retained. In the following studies, a homologous functional assay was developed to test the activity of three forms of receptor with respect to their ability to transmit a lactogenic signal. In this system, CHO cells were transiently transfected with a construct containing 2300 base pairs of the 5'-flanking sequence of the rat beta-casein gene fused to the chloramphenicol acetyltransferase (CAT) gene and an expression vector containing the various forms of rat PRL-R cDNA. The transfected cells were grown in serum-free medium in the absence or presence of PRL. In cells transfected with the long form of the PRL-R and beta-casein/CAT construct, a 7.2- +/- 0.9-fold induction (n = 3) of CAT activity was seen when cells were cultured in the presence of 400 ng/ml PRL and 1 micrograms/ml hydrocortisone. This level of stimulation was similar to that observed for the ovine beta-lactoglobulin/CAT construct in which a 5.7- +/- 1.2-fold (n = 3) effect was found.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Comparison of long and short forms of the prolactin receptor on prolactin-induced milk protein gene transcription.

The biological activities of long and short forms of the prolactin receptor have been compared. These two receptors expressed in mammalian cells were shown to bind prolactin with equal high affinity. The ability of these different forms to transduce the hormonal message was estimated by their capacity to stimulate transcription by using the promoter of a milk protein gene fused to the chloramphenicol acetyltransferase (CAT) coding sequence. Experiments were performed in serum-free conditions to avoid the effect of lactogenic factors present in serum. An approximately 17-fold induction of CAT activity was obtained in the presence of prolactin when the long form of the prolactin receptor was expressed, whereas no induction was observed when the short form was expressed. The present results clearly establish that only the long form of the prolactin receptor is involved in milk protein gene transcription.

Animals↗
PubMed1991

Effect of DMBA on the expression of prolactin receptors and IGF1 genes in rat mammary gland.

Prolactin receptor and IGF1 gene expression were measured in mammary glands from Sprague-Dawley rats at different times (10, 30; and 58 d) after administration of a single dose of 15 mg dimethylbenz(a)-anthracene (DMBA) per os at 55 d of age, and in DMBA-induced mammary tumors appearing in these rats at approximately 2 months after DMBA administration; The relative gene expression of prolactin receptor and insulin-like growth factor (IGF1) mRNAs was measured by hybridization to Northern blots prepared from pools of tissue. The probes used were 32P-labelled cDNAs specific to the extracellular domain of the receptor (E probe), common to all forms, and a probe specific to the intracellular position of the long form of the receptor (I probe), a human IGF1 probe, and chicken beta-actin probe, to correct for loss of tissue and different metabolic activity of the tissues. Hybridization with the prolactin receptor probes revealed bands at 2.5, 3; and 5.5 kb hybridizing with the long form of the receptor and a more intense band at 1.8 kb that corresponded to the short form of the receptor. There were no changes in the relative expression of prolactin receptor mRNAs in the mammary gland of control (oil-treated) or DMBA-treated rats, although there was a gradual diminution of expression with increasing age of the animals. In contrast, in DMBA-induced mammary tumors, there was a marked increase in the relative expression of prolactin receptor mRNAs with, however, no modification in the relative proportion of short and long forms.(ABSTRACT TRUNCATED AT 250 WORDS)

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1991

The prolactin/growth hormone receptor family.

PRL and GH are hormones with a wide spectrum of actions. Specific receptors are widely distributed in a number of classical target organs, but other tissues that are not known targets also contain measurable binding sites or receptor mRNA. The most likely explanation is that PRL and GH cause effects that have not yet been characterized in certain tissues. Cloning of the cDNAs encoding PRL and GH receptors has led to the discovery that the receptors, like the hormones themselves, form a gene family. Multiple receptor forms have been identified, including a short form, which for PRL is a membrane-bound receptor or for GH is a soluble BP, and a long form, which for both PRL and GH is a membrane-bound receptor. PRL and GH receptors, and the mRNAs encoding them, can be up- and down-regulated. GH induces an up-regulation of both GH and PRL receptors, whereas PRL stimulates an increase of only its own receptor. High concentrations of either hormone induce a homologous down-regulation of receptor expression. An assay has been developed to measure the functional activity of different forms of PRL receptor by cotransfecting a milk protein fusion gene specific to PRL coupled to a reporter-gene along with the cDNA of the PRL receptor. Although the short form represents the major form present in rat mammary gland, only the long form of receptor is able to stimulate milk protein gene transcription. For GH, increased expression of the receptor in some target cells is accompanied by a modest enhancement of the response to GH. No single second messenger mediating the action of either PRL or GH has been identified. Several potential components of the signal transduction pathways have been identified, but as yet none has clearly been shown to be able to mimic the effect of PRL or GH. Because of the wide range of biological actions associated with PRL, and the existence of various forms of PRL receptors, it is doubtful that one unifying mechanism of action will be found for this hormone. No human or animal model of a genetic defect of the PRL receptor has thus far been published. Mutations in the GH receptor gene have been demonstrated in Laron type dwarfism. Different exon deletions or point or nonsense mutations resulting in modifications in the extracellular, GH binding region of the GH receptor have been reported.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Prolactin receptor gene expression in rat mammary gland and liver during pregnancy and lactation.

The expression of two forms of PRL receptor messenger RNA was measured at different stages of pregnancy and lactation in mammary gland and liver from Sprague-Dawley rats, using 32P-labeled complementary DNA probes encoding the extracellular part of the receptor (E probe), common to the two forms and a probe encoding the intracellular part of the long form of the receptor (I probe), that only recognizes sequences specific to the long form of the receptor. Hybridizations were performed in Northern blots obtained from electrophoreses of poly (A+) enriched RNA preparations from mammary glands and livers of rats on days 0, 6, 12, 19, and 21 of pregnancy and 5, 10, 15, and 20 of lactation. The Northern blots were also hybridized with a chicken beta-actin probe, to correct for the amount of mRNA added and the different metabolic states of the tissues. Both tissues expressed the same forms of PRL receptor mRNAs, namely bands at 2.5, 3, and 5.5 kilobases encoding the long form of the receptor and a major band at 1.8 kilobases encoding the short form. The liver expressed all the receptor mRNA forms in much higher quantity than the mammary gland, independent of the reproductive state. In liver there was an increase of all the transcripts on day 19 of pregnancy, followed by an abrupt decline at the onset of lactation, to levels lower than those of virgin rats. In contrast, mammary gland PRL receptor mRNAs were low in virgin and pregnant animals, increased significantly at day 21 of pregnancy, and continued to increase throughout lactation. Treatment of day 19 pregnant rats with the antiprogesterone RU 486 induced, 24 h later, PRL receptor mRNAs in mammary gland but not in liver. There were no significant differences in the relative proportions of long to short forms of PRL receptor mRNAs at the different reproductive states, but the proportion of the long form was slightly greater in mammary gland than in liver. Membrane PRL receptor concentrations were also measured in the same tissues used for the mRNA study by binding to a 125I-labeled monoclonal antibody (U5), which specifically recognizes the PRL receptor at a site different from the hormone binding site. The quantity of receptor measured by U5 binding was approximately 3 times higher than that measured with 125I-labeled ovine PRL.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Growth of mouse endometrial luminal epithelial cells in vitro: functional integrity of the oestrogen receptor system and failure of oestrogen to induce proliferation.

Normal endometrial luminal epithelial cells isolated from ovariectomized approximately 40-day-old BALB/cCrgl mice were purified by Percoll density gradient centrifugation and grown as primary cultures in collagen gel matrix and serum-free medium. Cells increased threefold in number during the 9-day culture period. Deletion of insulin, epidermal growth factor or bovine serum albumin resulted in decreased growth. Addition of any single factor to the unsupplemented medium had no effect. Relatively high levels of cytosolic oestrogen receptors and progestin receptors were demonstrable in the cultures. Addition of oestrogen did not enhance epithelial cell proliferation. On the contrary, all doses of oestrogen (180 fmol/l to 218 nmol/l) were inhibitory. Continuous exposure to oestradiol-17 beta (1.8 nmol/l) for 9 days in serum-free medium resulted in a decrease in cytosolic oestrogen receptors with an associated nuclear accumulation of oestrogen receptors. A corresponding increase in cytosolic progestin receptors was also observed, indicating that no qualitative modification of the oestrogen receptor system had occurred. Thus, as previously reported for vaginal epithelial cells, oestrogen, despite its stimulation of specific product synthesis (progestin receptors), did not increase proliferation of endometrial luminal epithelial cells in this culture system.

Animals↗
PubMed1991

The growth hormone/prolactin receptor gene family.

The identification of cDNAs encoding receptors for growth hormone and prolactin have allowed the identification of the primary structure of these receptors. The expression of these receptors can be measured in various tissues by estimation of mRNA levels using cDNA probes and measurements of protein levels by radioimmunoassay or immunoblot analysis using monoclonal or polyclonal antibodies to the receptors. Site-directed mutagenesis of ligand-binding domains will lead to an understanding of the residues important for hormone binding. In addition, mutagenesis of the cytoplasmic domains of the receptors performed together with functional tests should help identify regions of the receptor involved in the process of signal transduction. Finally, the identification of the enlarged receptor family, including receptors for cytokines, should shed some light on additional functions of growth hormone prolactin, especially in regulating immune functions.

Animals↗
PubMed1990

Prolactin stimulates milk protein promoter in CHO cells cotransfected with prolactin receptor cDNA.

A functional biological system was developed by cotransfecting mammalian cell lines with the cDNA of the prolactin receptor (PRL-R) and a fusion gene containing the promoter of the milk protein, ovine beta-lactoglobulin linked to the coding sequence of the chloramphenicol acetyltransferase (CAT) gene. Surprisingly, this system is effective even if a non-mammary cell line is used, since Chinese hamster ovary (CHO) cells transfected both transiently and stably with PRL-R cDNA respond to PRL, as observed by stimulation of the reporter gene. This newly developed system should help precisely define the functional domains of both the PRL-R molecule and of the regulatory elements of a PRL target gene.

Animals↗
PubMed1990

Expression of two forms of prolactin receptor in rat ovary and liver.

The screening of a size-selected cDNA library from the ovary revealed the existence of a second form of PRL receptor in the rat. The polypeptide sequence deduced from cDNAs has a much longer cytoplasmic domain (357 amino acids) than the form previously identified in the liver (57 amino acids). Nucleotide sequence analysis and comparison with rabbit, mouse, and human PRL receptor cDNAs suggests that the two forms of rat PRL receptor result from alternative splicing of a primary transcript. Complementary DNAs encoding the long form of the receptor were also found in a library prepared from estradiol-treated rat liver, although they represent a minor fraction of total PRL receptor cDNAs obtained from this tissue. DNA polymerase chain reaction amplification of cDNA confirmed the presence of the two receptor forms in both the ovary and liver. Northern analysis, using probes that specifically hybridize with either form of mRNA, indicates a major transcript of 1.8 kilobases (kb) in estradiol-treated liver, which encodes the receptor with a short cytoplasmic domain, while the long form of the receptor is encoded by mRNAs of 2.5 and 3 kb. In the ovary, a complex pattern of hybridization to multiple mRNAs (1.8-5.5 kb) is obtained with the probe specific to the long form, and essentially only a 5.5-kb mRNA is obtained with the probe specific to the short form. The predicted size of the mature form of the long PRL receptor (PRL-R2) is 591 amino acid residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗