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.

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.

Development of a constitutively active mutant form of the prolactin receptor, a member of the cytokine receptor family.

The extracellular domain of the PRL receptor (PRL-R) is composed of two subdomains of approximately 100 amino acids, S1 and S2. To explore the functional significance of these subdomains in PRL binding and signal transduction, deletion mutants of S1 or/and S2 subdomains were constructed. We report here the inability of each of these mutant receptor forms to bind PRL after expression in COS-7 cells. We also studied the abilities of these different mutant receptors to respond to hormonal stimulation after transfection of each mutant complementary DNA into CHO-K1 cells along with a chimeric gene containing the promoter of a milk protein gene (beta-lactoglobulin) fused to chloramphenicol acetyltransferase coding sequence. Somewhat unexpectedly, a constitutively (PRL-independent) mutant form of the PRL-R was obtained after deletion of the S2 subdomain. Moreover, we analyzed, in CHO-K1 cells, the biological activity of chimeric receptors constructs in which each subdomain sequence was replaced by an unrelated, but coding, sequence of foreign protein, and we confirmed a specific requirement for the S1 sequence in the constitutive activity. In contrast, the S2 subdomain produced an inhibitory effect on S1 constitutive activity. Cotransfection experiments with the wild-type receptor and the constitutive mutant receptor provided evidence that the wild-type receptor was able to inhibit the constitutive activity of the deleted mutant. Furthermore, in the mouse mammary epithelial cell line HC11, the constitutive PRL-R form was able to induce transcription of the beta-casein gene in the absence of PRL. These results suggest a complex signal transduction process that implicates each extracellular PRL-R subdomain. Possible mechanisms for the constitutive effect are discussed.

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)

Short term effect of prolactin on intracellular calcium in Chinese hamster ovary cells stably transfected with prolactin receptor complementary deoxyribonucleic acid.

The mechanism of transduction of the PRL signal in target cells is poorly understood. We examined the effects of PRL on the intracellular free Ca2+ concentration in Chinese hamster ovary cells overexpressing functional PRL receptors. [Ca2+]i was determined by dual emission microspectrofluorimetry using indo-1 as the Ca2+ fluorescent probe. We demonstrate that at physiological concentrations (0.5-5 nM), PRL stimulates Ca2+ entry (type I) and/or induces a mobilization of calcium ions stored in intracellular compartments (type II). Two types of Ca2+ mobilization, distinguishable by their onset kinetics, were observed, a slow mobilization (type IIa; transition time to peak, approximately 10 sec) and a fast mobilization (type IIb; transition time to peak, < 2 sec). PRL responses were delayed (15-120 sec) compared to the well known activation by phosphatidylinositol 4,5 bisphosphate hydrolysis-coupled receptors. This suggests that inositol trisphosphate is not involved in PRL response or that phosphatidylinositol 4,5 bisphosphate hydrolysis is not directly coupled to the PRL receptor. The amplitude of the PRL-induced Ca2+ increases (300-1400 nM) would be sufficient to provoke several physiological responses, such as stimulation of secretion, cell proliferation, or gene activation. However, the relation between the increase in Ca2+ and activation of milk protein genes remains to be established.

20-alpha-Hydroxysteroid dehydrogenase from pseudopregnant rat ovary: obtention and characterization of a monoclonal antibody against the enzyme activity.

The enzyme 20-alpha-hydroxysteroid dehydrogenase (20-alpha-HSD) was purified from pseudopregnant rat ovaries and used as antigen for the development of a monoclonal antibody by the hybridoma technique. Spleen cells of BALB/c mice immunized with purified 20-alpha-HSD were fused with SP2/0 mouse myeloma cells. Among the colonies of hybrid cells, one (designated mAb-HSD 11) was found to be secreting antibodies (IgM) able to inhibit 20-alpha-HSD activity. The antibody-secreting hybridome was amplified by ascitic fluid production and the monoclonal antibody purified by Bakerbond ABx procedure. Purified mAb-HSD 11 was able to inhibit 20-alpha-HSD activity in a dose-dependent manner. Studies of Michaelis constants of 20-alpha-HSD indicate that this monoclonal antibody increases the Km for 20-alpha-dihydroprogesterone and decreases the Vmax.

Mutations of ovine and bovine placental lactogens change, in different ways, the biological activity mediated through homologous and heterologous lactogenic receptors.

The biological activities of ovine (o) and bovine (b) placental lactogens (PLs) and their mutated analogues were compared using several binding and in vitro bioassays. In almost all cases, the biological activities of these analogues mediated through rat (r) prolactin receptor (PRLR) showed little or no change, despite a remarkable decrease in their capacity to bind to the extracellular domain of rPRLR and despite compromised stability of the 2:1 complexes. These results indicate that mutations impairing the ability of oPL or bPL to form stable complexes with lactogenic receptors do not necessarily lead to a decrease in the biological activity, because the transient existence of the homodimeric complex is still sufficient to initiate the signal transduction. In contrast, oPL and bPL analogues completely, or almost completely, lost their ability to activate homologous PRLRs, and some of them even acted as site-2 antagonists. To explain the difference between the activity transduced through homologous and that transduced through heterologous PRLRs, we propose the novel term 'minimal time of homodimer persistence'. This concept assumes that in order to initiate the signal transduction, the associated kinase JAK2 has to be transphosphorylated and this requires a 'minimal time' of homodimer existence. In the case of homologous interaction between ruminant PLs and homologous PRLRs, this 'minimal time' is met, though the interaction with homologous PRLRs has a shorter half-life than that with heterologous PRLRs. Therefore oPL or bPL are active in cells possessing both homologous and heterologous PRLRs. Mutations of oPL or bPL lead to reduced affinity and, consequently, the 'time of homodimer persistence' is shortened. Although in the case of heterologous interaction the 'minimal time' is still sufficient to initiate the biological activity, in homologous interactions, which are already weaker than heterologous interactions, further destabilization of the complex shortens its persistence to below the 'minimal time', leading to full or partial loss of biological activity.

Cloning, preparation and characterization of biologically active recombinant caprine placental lactogen.

Caprine placental lactogen (cPL) cDNA was cloned by reverse transcription (RT)-PCR from total RNA of goat placenta. The PCR product encoding for the mature protein was gel purified, ligated to pGEM-T and finally subcloned into a pET8c prokaryotic expression vector. E. coli cells (BL-21) transformed with this vector overexpressed large amounts of cPL upon induction with Isopropyl-1-thio-beta-D-galactopyranoside. The expressed protein, found in the inclusion bodies, was refolded and purified to homogeneity on Q-Sepharose and SP-Sepharose columns, yielding two electrophoretically pure fractions (cPL-Q and cPL-S), composed of over 98% of monomeric protein of the expected molecular mass of approximately 23 kDa. Binding of cPL to the extracellular domain (ECD) of prolactin receptors (PRLR) from rat (r), rabbit (rb), and bovine (b), growth hormone receptors (GHR) from human (h) and rabbit, and binding to rabbit mammary gland membranes revealed similar binding profiles for cPL-Q, cPL-S and ovine (o)PL. Caprine PL was capable of forming 1:2 complexes with hGHR-ECD, rbGHR-ECD, rPRLR-ECD and rbPRLR-ECD whereas with bPRLR-ECD only a 1:1 complex was detected. The biological activity of both cPL fractions resulting from proper renaturation was further evidenced by their ability to stimulate proliferation of Nb2 cells, FDC-P1 cells transfected with rabbit or human GHRs and by stimulation of beta-casein synthesis in rabbit and ovine mammary gland acini cultures.

Involvement of both calcium influx and calcium mobilization in growth hormone-induced [Ca2+]i increases in Chinese hamster ovary cells.

This study reports rapid effects of growth hormone (GH) on the intracellular free calcium concentration ([Ca2+]i) in Chinese hamster ovary (CHO) cells stably expressing rabbit GH receptor. [Ca2+]i was measured by spectrofluorimetric methods in single cells and membrane Ca2+ currents by patch clamp techniques in the whole-cell configuration. In individual CHO cells, bathed in a standard saline solution containing 2 mM Ca2+, basal [Ca2+]i was 191 +/- 27 nM (mean +/- S.D.; n=83). Short term administration of GH (100 ng/ml, 30 s) induced a [Ca2+]i increase in 54% of cells tested (n = 398 of 743). Responses were clearly heterogeneous. Maximum calcium increase varied from 16 to 853 nM and time to peak varied from 4 to 320 s. On examination of the [Ca2+]i increases, it was possible to define two different types of calcium responses to GH. Experimental manipulations of extracellular and intracellular calcium concentrations demonstrated that GH-induced calcium increases involved both calcium influx and calcium mobilization. Calcium influx, a long lasting, small amplitude (63 +/- 34 nM) response, was observed in 121 out of 398 cells (30%) whereas calcium mobilization, a transient, large amplitude (263 +/- 175 nM) response, was observed in 277 out of 398 cells (70%). Moreover, patch clamp data show that influx did not involve the dihydropyridine-sensitive calcium channels.

Effect of PRL on MAPK activation: negative regulatory role of the C-terminal part of the PRL receptor.

Prolactin induces cell proliferation and cell differentiation through well-known MAPK Erk, and JAK2/STAT5 pathways depending on the cell line. The aim of the present study was to delineate the functional domains of the PRL receptor involved in PRL induced MAPK regulation. Using various PRL-R mutants of the cytoplasmic domain we found, that the membrane proximal domain is necessary for PRL induced MAPK activation and that the C-terminal part of the receptor exerts a negative regulatory role. A pharmacological approach, using different types of inhibitors, provided evidence that PRL induced MAPK activation requires both a MEK dependent pathway and a PI3K dependent pathway. The negative regulation induced by the carboxy-terminal part of the receptor involves a combination of tyrosine phosphatases and serine/threonine phosphatases as concluded from the actions of the phosphatase inhibitors: pervanadate, PAO and okadaic acid. The mechanism by which these phosphatases are recruited or are induced by the last 141 cytoplasmic residues of the receptor remains to be determined. Finally the negative regulatory role of the carboxy-terminal part of the receptor, first demonstrated in the present study, is discussed in terms of the regulation of different effects of PRL on growth and differentiation.

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.

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.

Endocytosis and degradation of prolactin and its receptor in Chinese hamster ovary cells stably transfected with prolactin receptor cDNA.

Molecular cloning of the prolactin (PRL) receptor cDNA has revealed different forms of the receptor: among them, the longest form encodes a transmembrane protein of 592-598 amino acids and was originally found in rabbit mammary gland as well as in human and rat tissues. It contains a cytoplasmic domain of 358 amino acids. In CHO cells transfected with the PRL receptor cDNA, PRL is able to induce the specific expression of a reporter gene provided with the promoter of the milk protein gene beta-lactoglobulin. The cDNA encoding this long receptor form has been expressed permanently after stable transfection of Chinese hamster ovary (CHO) cells. In these cells, we have determined the fate of the bound hormone and of the receptor. At 37 degrees C, transfected cells were able to endocytose 125I-labeled human growth hormone (hGH) or ovine prolactin (oPRL) at an initial rate of about 1 fmol/h at 100 pM labeled hormone and 10(6) cells/well. Lowering the temperature to 15 degrees C slowed the endocytosis of [125I]hGH by a factor of 5. These results were confirmed by electron microscopy with oPRL labeled with colloidal gold. At 37 degrees C, the receptor underwent rapid insertion to the cell surface and constitutive endocytosis (half-life 80 min). This rate of endocytosis was enhanced in the presence of 10 nM oPRL (half-life 8 min), leading to down-regulation of the receptor by exhaustion of the intracellular receptor pool. After down-regulation, the cell surface was replenished with newly synthesized PRL receptor with a half-time of 8-10 min. If cycloheximide was added, almost no receptors could be found on the cell surface. These results indicate that in transfected cells the PRL receptor behaved largely as in classical target cells. A "conveyor belt" endocytosis behavior was found, with degradation of the endocytosed receptors, and occupation by the hormone enhancing this process. Moreover, since the PRL receptor belongs to a family of receptors in which companion protein(s) seem to play important roles, transfected CHO cells appear to provide the expressed receptors with the necessary element(s) to function as in normal PRL target cells.

Prolactin receptor and signal transduction to milk protein genes.

After cloning of the mammary gland prolactin (PRL) receptor cDNA, a functional assay was established using co-transfection of PRL receptor cDNA together with a milk protein promoter/chloramphenicol acetyl transferase (CAT) construct in Chinese hamster ovary (CHO) cells. Different mutants of the PRL receptor were tested in this CAT assay to delimit the domains in the receptor necessary for signal transduction to milk protein genes. In CHO cells stably transfected with PRL receptor cDNA, high numbers of PRL receptor are expressed. By metabolic labeling and immunoprecipitation, expressed PRL receptor was identified as a single species of 100 kDa. Using these cells, we analyzed the effects of PRL on intracellular free Ca++ concentration. PRL stimulates Ca++ entry and induces secondary Ca++ mobilization. The entry of Ca++ is a result of an increase in K+ conductance that hyperpolarizes the membranes. We have also analyzed tyrosine phosphorylation induced by PRL. In CHO cells stably transfected with PRL receptor cDNA, PRL induced a very rapid and transient tyrosine phosphorylation of a 100-kDa protein which is most probably the PRL receptor. The same finding was obtained in mammary membranes after PRL injection to lactating rabbits. Whereas tyrosine kinase inhibitors genistein and lavendustin were without effect, PRL stimulation of milk protein gene promoters was partially inhibited by 2 microM herbimycin in CHO cells co-transfected with PRL receptor cDNA and the beta lactoglobulin CAT construct. Taken together these observations indicate that the cytoplasmic domain of the PRL receptor interacts with one or several tyrosine kinases, which may represent early postreceptor events necessary for PRL signal transduction to milk protein genes.

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.

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.

Introduction of a proximal Stat5 site in the murine alpha-lactalbumin promoter induces prolactin dependency in vitro and improves expression frequency in vivo.

In order to establish a possible correlation between in vitro prolactin induction and the transcriptional activity of mammary gene promoters in transgenic mice, a functional Stat5-binding site was created by means of site-directed mutagenesis at position -70 on a 560 bp murine alpha-lactalbumin promotor linked to a CAT reporter gene. Surprisingly, the wild-type promoter was constitutively active in vitro and could not be induced by prolactin. Introducing the proximal Stat5 site abolished this constitutive activity and resulted in prolactin dependence in both CHO-K1- and HC11-transfected cells. In transgenic mice, both the frequency of lines expressing the transgene and the prevalence of mid to late pregnancy expression were increased.