Streptomyces matensis laminaripentaose hydrolase is an 'inverting' beta-1,3-glucanase.

The laminaripentaose-producing beta-1,3-glucanase of Streptomyces matensis is a member of the glycoside hydrolase family GH-64. We have constructed and purified a recombinant hexahistidine-tagged form of the enzyme for characterisation. The enzyme, which exists as a monomer in solution, hydrolyses beta-1,3-glucan by a mechanism leading to overall inversion of the anomeric configuration. This is the first determination of the mechanism prevailing in glycoside hydrolase family GH-64 and this is the first characterisation of an 'inverting' beta-1,3-glucanase.

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.

In vitro expression of long and short ovine prolactin receptors: activation of Jak2/STAT5 pathway is not sufficient to account for prolactin signal transduction to the ovine beta-lactoglobulin gene promoter.

The recent finding that sheep had long (l-oPRLR) and short (s-oPRLR) prolactin receptors provided new tools to further explore prolactin signaling to target genes. Here we used CHO cells transfected with l-oPRLR or s-oPRLR cDNAs to compare the activation of known key steps of prolactin signaling by the two receptors. We found that prolactin stimulated l-oPRLR tyrosine phosphorylation, although it lacked the last tyrosine residue found in other long prolactin receptors. In addition, l-oPRLR and s-oPRLR both responded to prolactin stimulation by (1) Janus kinase 2 (Jak2) tyrosine phosphorylation, (2) DNA-binding activation of signal transducer and activator of transcription 5 (STAT5), (3) stimulation of transcription from a promoter made of six repeats of STAT5-responsive sequence. However, although it contains STAT5-binding consensus sequences, the ovine beta-lactoglobulin promoter (-4000 to +40) was transactivated by l-oPRLR, but not by s-oPRLR. Taken together, our results indicate that activation of Jak2/STAT5 pathway alone is not sufficient to account for prolactin-induced transcription of this milk protein gene, and that sequences of its promoter, other than STAT5-specific sequences, account for the opposite transcriptional activation capabilities of l-oPRLR and s-oPRLR.

Prolactin signal transduction to milk protein genes: carboxy-terminal part of the prolactin receptor and its tyrosine phosphorylation are not obligatory for JAK2 and STAT5 activation.

In this study, we have developed several Chinese Hamster ovary (CHO) cell clones stably expressing various deletion mutant forms of the rabbit prolactin receptor (rbPRL-R) to better define the domains of the receptor involved in JAK2 kinase interaction, STAT5 activation, and to assess the role of tyrosine phosphorylation of the PRL-R in signal transduction. We observed that the box 1 region of the receptor was critical for productive interaction with JAK2 and its tyrosine phosphorylation after PRL stimulation. However, this region appeared to require the presence of additional cytoplasmic domain region(s), such as box 2, to exert its complete effect. In addition, we found that a mutant form lacking the 141 C-terminal residues lost the capacity to be tyrosine phosphorylated in response to PRL but remained able to activate JAK2 kinase and STAT5 transcription factor, indicating that it contained the minimal sequence required for STAT5 activation. The absence of tyrosine phosphorylation of this C-terminal rbPRL-R mutant upon PRL stimulation indicated that the phosphorylation of the PRL-R normally occured in the last 141 animo acids (aa) containing three tyrosines and was not absolutely necessary for induction of these early events in PRL signal transduction. Transfectant cell lines expressing wild type (WT) PRL-R and this C-terminal mutant form were able to induce CAT activity upon PRL stimulation when transiently transfected with the ovine-beta-lactoglobulin promoter, containing STAT5 recognition sites, fused to the CAT reporter gene. The comparison between transcriptional activity of these two receptor forms leads to the conclusion that the C-terminal region of the rbPRL-R, containing the physiological sites for tyrosine phosphorylation, is probably responsible for an amplification of the PRL signal to milk protein genes.

Preparation and characterization of recombinant prolactin receptor extracellular domain from rat.

Complementary (c)DNA of the extracellular domain of rat prolactin receptor (rPRLR-ECD) was cloned in the prokaryotic expression vector pTrc99A, and expressed in Escherichia coli following induction with isopropyl-b-D-thiogalactopyranoside. The expressed rPRLR-ECD protein, contained within the refractile body pellet was solubilized in 4.5 M urea, refolded and purified on a Q-Sepharose column by stepwise elution with NaCl. Only approximately 10% of the expressed protein refolded as a monomeric fraction, yielding 5-6 mg/l of induced culture. The purified protein was over 98% homogeneous, as shown by SDS-PAGE in the presence or absence of reducing agent, and by chromatography on a Superdex column. Its molecular mass, determined by SDS-PAGE in the absence of reducing agent, was 28 kDa, and by gel filtration, 25.6 kDa. Binding experiments indicated high affinity for bovine placental lactogen (bPL) and human growth hormone (hGH) as compared to ovine (o) or rat PRLs. Gel filtration was used to determine the stoichiometry of rPRLR-ECD's interaction with these hormones. At a 5 microM initial concentration of the hormones, formation of 2:1 (ECD:ligand) complexes was detected with bPL, hGH and oPRL whereas only 1:1 complex was formed with rPRL. Dilution (25-fold) of these complexes did not affect the stoichiometry with bPL, whereas with hGH a clear tendency towards dissociation of the initial 2:1 complex to 1:1 complex was observed. This tendency was even stronger in the case of oPRL. Although all four hormones exhibited nearly identical activities in the Nb2-11C lymphoma cell bioassay, the ability of the purified rat or rabbit PRLR-ECD to inhibit hormonal mitogenic activity generally reflected their affinity for the respective hormones. In view of these and former results, we suggest that unlike in the GH:GHR-ECD interaction, the inability of lactogenic hormones to form a 1:2 complex with soluble recombinant PRLR-ECDs does not necessarily predicts lack of biological activity.

The rabbit mammary gland prolactin receptor is tyrosine-phosphorylated in response to prolactin in vivo and in vitro.

We report the first in vivo study demonstrating tyrosine phosphorylation of mammary gland proteins including the prolactin receptor, in response to the injection of prolactin. Immunoblotting of mammary gland membrane extracts revealed that subunits of 200, 130, 115, 100, 90, 70, and 45 kDa display increased tyrosine phosphorylation within 5 min of prolactin administration. The 100-kDa component was identified as the full-length prolactin receptor by a variety of means including immunoprecipitation and immunoblotting with monoclonal (U5, 917, 110, and 82) and polyclonal (46) antibodies to the prolactin receptor. Maximal receptor phosphorylation was seen within 1 min of hormone injection, and to obtain a strong response it was necessary to deprive rabbits of their endogenous prolactin for 36 h. Rapid tyrosine phosphorylation of the full-length receptor was verified by its demonstration in Chinese hamster ovary cells stably transfected with rabbit prolactin receptor cDNA. Both in vivo and in vitro, the phosphorylation signal was transient, being markedly reduced within 10 min of exposure to prolactin. Tyrosine-phosphorylated receptor was shown to be associated with JAK 2 by immunoblotting of receptor immunoprecipitated from transfected Chinese hamster ovary cells with polyclonal 46. A 48-kDa ATP-binding protein was also shown to be associated with the mammary gland receptor by U5 or polyclonal 46 immunoprecipitation of receptor complexes following covalent labeling with [alpha-32P]azido-ATP. Our demonstration of prolactin receptor tyrosine phosphorylation raises the possibility of signaling pathways regulated by receptor/SH2 protein interaction, which would facilitate prolactin specific responses. The fact that a period of hormone deprivation is needed for significant hormone triggered receptor phosphorylation indicates that the mammary gland receptor exists in a largely desensitized state in vivo, analogous to the related growth hormone receptor.

cDNA cloning and genomic analysis of a new multigene family sharing common phylogenetic and expression profiles with the laminin receptor gene.

We have isolated a human laminin receptor (LR) cDNA which bears a different sequence in its 5' end with regard to the corresponding region in the regular LR mRNA. This different sequence hybridizes to a 1 kb mRNA. We have cloned a 740 bp cDNA for this transcript (cDNA 48-1). Search on sequence data bases revealed no sequence homology with known messengers or proteins. Using cDNA 48-1 in a simplified version of the protocol with which we had previously characterized the LR gene as a member of a retrogene family in mammals, we show in the present paper that the gene of this new transcript exhibits phylogenic, expression and amplification features that strickingly recall those of the LR gene.

Laminin expression by two clones isolated from the colon carcinoma cell line LoVo that differ in metastatic potential and basement-membrane organization.

In the present report we describe the characteristics of 2 clones, E2 and C5, isolated from the human colon adenocarcinoma cell line LoVo. When grafted to immunosuppressed newborn rats, these clones formed tumors that varied with regard to differentiation rate, basement-membrane organization and lung metastatic potential. Production and distribution of laminin by E2, C5 and related tumors was studied by immunohistochemistry with an anti-laminin monoclonal antibody 4C12 (MAb 4C12). In lowly metastatic E2-derived tumors, strong regular stainings were observed which were strictly peri-tumoral and corresponded to the basal lamina. Since the antibody interacted with human laminin (the graft) but not with rat laminin (the host), this result indicated that basement-membrane laminin was supplied mainly by tumor-cell synthesis. In highly metastatic C5-derived tumors, the staining obtained with MAb 4C12 was peri-cellular and unorganized. Laminin synthesis by E2 and C5 cells in sub-cultures or soon after dissociation from explanted tumors was studied by metabolic labelling with 35S-methionine under steady-state conditions followed by immunoprecipitation and SDS-PAGE. High-molecular-weight laminin comprised by disulfide-linked A and B chains, i.e., heterotrimeric laminin, was found in cell lysates and in the secretion medium of cell lines and tumor cells. In addition, B1B2 dimers and free B chains were observed in cell lysates. Quantitatively, laminin expression by E2 and C5 clones or tumor cells was not significantly different. These findings suggest that basement-membrane defects in invasive clone LoVo C5 were not due to laminin under-expression.

Genomic analysis of the 67-kDa laminin receptor in normal and pathological tissues: circumstantial evidence for retroposon features.

We have cloned two cDNAs for the human 67-kDa laminin receptor (LR). In the present report we show that these clones hybridize to many restriction fragments in Southern experiments in human. This particular pattern is accounted for by the presence of up to 16 and 21 copies of the laminin receptor gene per haploid genome in human and mouse, respectively. In contrast, a single gene copy is found in chicken. Chromosomal localization reveals four main loci: LAMRP1, laminin receptor pseudogene 1 (Chr 3); LAMRP2, laminin receptor pseudogene 2 (Chr 12); LAMRP3, laminin receptor pseudogene 3 (Chr 14); LAMRP4, laminin receptor pseudogene 4 (Chr X). Comparison of our experimental results to the known features of processed retropseudogenes enabled us to conclude that the LR gene belongs to a retroposon family in mammals.

Modulable Southern and Northern blot hybridization apparatus for routine screening of gene amplification and expression.

An apparatus for Northern and Southern blot hybridization is described. It allows from one to twenty-four blots to be processed at the same time, with different probes. All the pre-, post- and hybridization steps are performed without handling the filters and the experimenter is totally protected from beta radiations. The development of such modulable materials has become necessary since Southern and Northern techniques are becoming routine assays in hospitals, particularly in the field of oncology, in prognosis and for hereditary diseases, as an antenatal diagnosis procedure.

Laminin biosynthesis in the extracellular matrix-producing cell line PFHR9 studied with monoclonal and polyclonal antibodies.

The biosynthesis of the basement-membrane glycoprotein laminin in the mouse teratocarcinoma cell line PFHR9 was studied by immunoelectron microscopy and pulse-chase experiments using monoclonal and polyclonal antibodies. By immunoelectron microscopy, most of the protein was found to be aggregated on the outer cell surface. Cytoplasmic stainings were rare and were located next to the intracellular side of the plasma membrane. Sequential immunoprecipitations of cell extracts with a monoclonal antibody (4C12) sensitive to the laminin native conformation and with a polyclonal antibody enables laminin, the B1 subunit and a 410 kDa molecule to be distinguished. Most of the laminin is of the A(B1B2) type, and the 410 kDa molecule appears to be a B1B2 heterodimer. The assembly of laminin from subunits is completed in less than 1 h, and B chains are incorporated via the formation of the B heterodimers. The B2 and A chains are not found as free forms, so their levels appear to be the rate-limiting factors for the assembly of the dimers and laminin respectively. The formation of an uncross-linked A(B1B2) complex as a short-lived intermediate in the biosynthetic process is possible. Together with immunoelectron microscopy, the present study suggests that the protein is rapidly exported after assembly to accumulate on the outer side of the cell membrane. The biosynthesis of laminin in the PFHR9 cell line appears to be similar to that in other matrix-producing cell lines.

[Laminin: biosynthesis, structure and functions]. / Laminine: biosynthèse, structure et fonctions.

Biosynthesized by epithelial, endodermal and Swann type cells, laminin (Lam) is a cross shaped multifunctional glycoprotein formed by the multimeric assembly of subunits which result from the activation of several genes. In vivo, depending on its location, because of its adhesive properties and multivalent affinities, Lam is in association as a part of supramolecular complexes together with compounds of the plasma, the basement membrane and the cell coat. In the basement membrane (MB) Lam has structural and functional roles. It may also be adsorbed on the cell coat or secreted. It is interacting with epithelial cells by the way of a plasma membrane receptor and has a role to play in cellular differentiation and proliferation. Lam is a molecular link of epithelial cells to MB. These features implicate the molecule in organogenesis, embryogenesis and post-traumatic healing. As a structural component of MB and as an attachment factor, Lam is involved: 1 in tumoral invasion which allows metastatic spreading, 2 in homing because metastatic cell display an increased receptivity to the molecule. The study of Lam expression, Lam receptivity and their factors of control should lead to a better understanding of the biochemical and molecular basis of differentiation, embryogenesis, organogenesis and metastasis.

Quantitative in situ hybridization of 3H-labeled complementary deoxyribonucleic acid (cDNA) to the messenger ribonucleic acid of thyroglobulin in human thyroid tissues.

Thyroglobulin (Tgb) mRNA content was studied in human thyroid tissues using liquid hybridization and in situ hybridization. Liquid hybridization revealed no differences in mRNA content, except in the case of colloid adenoma in which a lower amount of Tgb mRNA was found. Conditions for quantitative in situ hybridization of [3H]DNA complementary to the mRNA of Tgb are described. In situ hybridization allowed correlation of the morpho-functional state of the follicles and their content of Tgb mRNA.