CSF-1 activates MAPK-dependent and p53-independent pathways to induce growth arrest of hormone-dependent human breast cancer cells.

The CSF-1 receptor (CSF-1R) is expressed in >50% of human breast cancers. To investigate the consequence of CSF-1R expression, hormone-dependent human breast cancer cell lines, MCF-7 and T-47D, were transfected with CSF-1R. Unexpectedly, CSF-1 substantially inhibited estradiol (E2) and insulin-dependent proliferation of MCF-7 transfectants (MCF-7fms) and prevented cyclin E/cdk2 and cyclin A/cdk2 activation, consistent with a G1 arrest. In contrast, CSF-1 increased DNA synthesis in T-47D transfectants (T-47Dfms) alone and with E2 or insulin. In response to CSF-1, there was a marked and sustained upregulation of the cyclin-dependent kinase inhibitor, p21Waf1/Cip1, in MCF-7fms but not T-47Dfms. CSF-1 also markedly upregulated cyclin D1 in MCF-7fms. The coordinate increase in cyclin D1 and p21 had the effect of decreasing the specific but not absolute activity of cyclin D1/cdk4. p53 was not involved since CSF-1 induction of p21 was unaffected by dominant-negative p53 expression. ERK activation by CSF-1 was robust and sustained in MCF-7fms and to a much lesser extent in T-47Dfms. Using pharmacological and transient transfection approaches, we showed that ERK activation was necessary and sufficient for p21 induction in MCF-7fms. Moreover, activated MEK inhibited E2-stimulated cdk2 activity. Our findings indicate that the consequence of CSF-1R-mediated signals in human breast cancer cells is dependent on the genetic background of the particular tumor.

Interaction of the release factors with the Escherichia coli ribosome: structurally and functionally-important domains.

There are two major domains of interaction between the Escherichia coli release factors (RF-1 and RF-2) and each subunit of the ribosome. RF-2 has a binding domain on the shoulder and lower head region of the small subunit at the small lobe distant from the decoding site. This is in close proximity to one of the domains on the large subunit which includes the body dimer of L7/L12 and L11. The other domains of interaction, at the decoding site on the small subunit, and at the peptidyltransferase centre of the large subunit of the ribosome, are some distance from the first two, although the evidence for direct contact with the ribosome is less comprehensive. The release factors may therefore have two distinct structural domains, and in support of this concept RF-1 and RF-2 can both be cleaved into two fragments by papain. Region-specific antibodies, and antibodies against defined peptide within the RF sequences have given an indication that a significant part of an interacting RF molecule is in close proximity to the ribosome surface, confirming an observation by immunoelectron microscopy which suggested that the RF penetrates deeply into the cleft between the two subunits. A region of highly conserved primary sequence between the two release factors from E coli is also conserved in those from B subtilis suggesting it forms an important structural or functional domain. Antibodies against peptides from the N-terminal end of this region strongly inhibit binding of the RF to the ribosome.

Structure and expression of the mouse growth hormone receptor/growth hormone binding protein gene.

The mouse growth hormone receptor/growth hormone-binding protein (GHR/BP) gene produces several distinct mRNA forms through alternative splicing, including mRNAs encoding the membrane-bound growth hormone receptor (GHR) and the soluble growth hormone-binding protein (GHBP). Transcripts are also heterogeneous in their 5' regions due to alternative selection of two major 5' untranslated region (5'UTR) sequences, designated L1 and L2. Here we report the cloning of all mouse GHR/BP coding exons as well as the exon encoding 5'UTR L2, the most widely expressed 5'UTR. The mouse GHR/GHBP gene contains 11 coding exons, 9 of which are homologous in size and sequence to human GHR exons 2-10. The two mouse exons that do not have homologs in the human gene are designated exons 4B and 8A. Exon 4B, located between exons 4 and 5, encodes an 8-amino acid segment of the ligand binding domain that is unique to mouse GHR and GHBP. Analysis by reverse transcriptase-polymerase chain reaction indicated that exon 4B is constitutively present in mouse GHR and GHBP mRNA. Exon 8A encodes the GHBP hydrophilic tail and 3'UTR sequence. 5'UTR L2 is encoded by a single exon located at least 27 kb upstream of exon 2 and at least 12 kb upstream of the exon encoding 5'UTR L1. The transcription start sites of UTR L2 were mapped and the 5' flanking region sequenced. The exon and proximal promoter region are GC rich, and share a high level of conservation with the equivalent exons in the sheep, bovine and human GHR genes. A CCAAT motif and several putative Sp1 motifs are present, and there is no TATA box. Homology between the mouse sequence and other species is limited to a region of 450 bp upstream of the exon due to the insertion of a fragment of a LINE-1 element upstream of the mouse L2 exon. Ribonuclease protection assays were used to confirm that 5'UTR L2 is widely expressed in multiple tissues and is the predominant form of transcript except in the liver during pregnancy, in which 5'UTR L1 is the major form.

Alternative 5'-untranslated regions of mouse GH receptor/binding protein messenger RNA are derived from sequences adjacent to the major L2 promoter.

Heterogeneity of 5' untranslated region (5'UTR) sequences is a common feature of growth hormone receptor/binding protein (GHR/BP) mRNA from a number of species. Two major 5'UTR sequences (designated L1 and L2 in the mouse) have been cloned from rodents, ruminants and primates, and are known to correspond to transcripts generated from independently regulated promoters. A variable number of other 5'UTRs with diverse sequences have been cloned from rat, human and bovine tissues. To characterize alternative 5'UTR usage in mouse GHR/BP mRNA, we carried out 5' rapid amplification of cDNA ends using RNA from non-pregnant mouse liver and adipose tissue. Three novel 5'UTR sequences were obtained. Sequencing of genomic DNA revealed that exons corresponding to these three sequences are clustered within 1 kb downstream of the exon encoding 5'UTR L2, and the associated L2 promoter. The novel 5'UTRs are present at very low levels relative to the total pool of GHR/BP mRNA in liver, fat, kidney, and mammary gland as determined by ribonuclease protection assays. On the basis of these data, we propose that these 5'UTR sequences may result from the use of cryptic transcription start sites and splice donor sites under the influence of the adjacent L2 promoter/enhancer region.

Prolactin receptor expression in the epithelia and stroma of the rat mammary gland.

The importance of prolactin (PRL) in regulating growth and differentiation of the mammary gland is well known. However, it is not well established whether PRL acts solely on the mammary epithelia or if it can also directly affect the mammary stroma. To determine where PRL could exert its effects within the mammary gland, we investigated the levels of expression and the localization of the PRL receptor (PRLR) in the epithelia and stroma of the rat mammary gland at different physiological stages. For these studies, we isolated parenchymal-free 'cleared' fat pads and intact mammary glands from virgin, 18-day-pregnant and 6-day-lactating rats. In addition, intact mammary tissues were enzymatically digested to obtain epithelial cells, free of stroma. The mammary tissues, intact gland, stroma and isolated epithelia, were then used for immunocytochemistry, protein extraction and isolation of total RNA. PRLR protein was detected in tissues using specific polyclonal antisera (PRLR-l) by immunocytochemistry and Western blot analysis. Messenger RNA for PRLR was measured by ribonuclease protection assay. Immunocytochemistry and Western blots with the PRLR-1 antisera detected PRLR in wild-type rat and mouse tissues, whereas the receptor protein was absent in tissues from PRLR gene-deficient mice. PRLR was found to be present both in the epithelia and stroma of mammary glands from virgin, pregnant and lactating rats, as determined by immunocytochemistry and Western blotting. Western blots revealed the predominance of three bands migrating at 88, 90 and 92 kDa in each of the rat mammary samples. These represent the long form of the PRLR. During pregnancy and lactation, PRLR protein increased in the epithelial compartment of the mammary gland but did not change within the stromal compartment at any physiological stage examined. We also found PRLR mRNA in both the epithelia and stroma of the mammary gland. Again, the stroma contained lower levels of PRLR mRNA compared with the epithelia at all physiological stages examined. Also, the PRLR mRNA levels within the stroma did not change significantly during pregnancy or lactation, whereas PRLR mRNA within the epithelia increased twofold during pregnancy and fourfold during lactation when compared with virgin rats. We conclude from this study that PRLR is expressed both in the stromal and epithelial compartment of the mammary gland. This finding suggests PRL may have a direct affect on the mammary stroma and by that route affect mammary gland development.

A single proteolytic cleavage in release factor 2 stabilizes ribosome binding and abolishes peptidyl-tRNA hydrolysis activity.

The structural and functional organization of Escherichia coli polypeptide chain release factors 1 and 2 (RF-1 and RF-2) was investigated by limited proteolysis with trypsin and chymotrypsin. A protease-sensitive site was found in a similar position in both factors at the beginning of a highly conserved region in the C-terminal part of the proteins. Chymotrypsin cleavage of RF-2 yielded a nicked form with the fragments associated. This nicked factor lost in vitro peptidyl-tRNA hydrolysis activity (a peptidyltransferase function) but had enhanced in vitro codon-ribosome binding activity (a decoding site function). It inhibited codon-dependent f[3H]Met-tRNA hydrolysis activity of intact RF-1 and RF-2, presumably as a result of an increased affinity for ribosomes. These data are consistent with a model whereby the release factor acts like a tRNA analog spanning the decoding and peptidyltransferase centers on the ribosome. The proteolytic sensitivity of the RFs most likely reflects an exposed surface loop. We propose that this loop interacts with the ribosomal peptidyltransferase site and that the stabilization of factor:ribosome binding upon cleavage could be explained by conformational coupling between domains on the factor for codon-ribosome binding at the decoding site and interaction with peptidyltransferase.

The leader peptides of attenuation-regulated chloramphenicol resistance genes inhibit translational termination.

Placing a translation stop codon at the ribosomal pause site in the leader of the attenuation-regulated cat-86 gene activates cat expression in the absence of the inducer, chloramphenicol. Genetic experiments have shown that this phenomenon depends on the amino acid sequence of the leader-encoded peptide and could readily be explained if the peptide was an inhibitor of translation termination. Here we demonstrate that the cat-86 leader pentapeptide is an in vitro inhibitor of translation termination in addition to its previously described antipeptidyltransferase activity.

Functional domains in the Escherichia coli release factors. Activities of hybrids between RF-1 and RF-2.

Chimeras between Escherichia coli release factors RF-1 and RF-2 have been constructed to study the role of the release factors in termination, in particular whether each possesses specific domains for recognition of the stop codon, and for facilitating peptidyl-tRNA hydrolysis. One hybrid factor showed normal codon-recognition activity but was defective in its ability to facilitate hydrolysis. Overexpression of this protein was toxic to the cell. Conversely, another hybrid factor showed complete loss of codon recognition but retained some hydrolysis activity. These two functional activities of the release factors were not localised in domains within either the amino-terminal or carboxy-terminal halves of the primary sequence as previously predicted. Evidence from the activities of the hybrid proteins and from earlier studies suggests that a combination of residues from the beginning and middle of the sequence, including a region of very high sequence conservation, contribute to the hydrolysis domain, whereas residues from both the amino-terminal and carboxy-terminal halves of the molecule are important for the codon recognition domain.

Escherichia coli release factor 3: resolving the paradox of a typical G protein structure and atypical function with guanine nucleotides.

Escherichia coli release factor 3 (RF3) is a G protein involved in the termination of protein synthesis that stimulates the activity of the stop signal decoding release factors RF1 and RF2. Paradoxically for a G protein, both GDP and GTP have been reported to modulate negatively the activity of nucleotide-free RF3 in vitro. Using a direct ribosome binding assay, we found that RF3xGDPCP, a GTP analogue form of RF3, has a 10-fold higher affinity for ribosomes than the GDP form of the protein, and that RF3xGDPCP binds to the ribosome efficiently in the absence of the decoding release factors. These effects show that RF3 binds to the ribosome as a classical translational G protein, and suggest that the paradoxical inhibitory effect of GTP on RF3 activity in vitro is most likely due to untimely and unproductive ribosome-mediated GTP hydrolysis. Nucleotide-free RF3 has an intermediate activity and its binding to the ribosome exhibits positive cooperativity with RF2. This cooperativity is absent, however, in the presence of GDPCP. The observed activities of nucleotide-free RF3 suggest that it mimics a transition state of RF3 in which the protein interacts with the decoding release factor while it enhances the efficiency of the termination reaction.

Translational termination efficiency in both bacteria and mammals is regulated by the base following the stop codon.

The translational stop signal and polypeptide release factor (RF) complexed with Escherichia coli ribosomes have been shown to be in close physical contact by site-directed photochemical cross-linking experiments. The RF has a protease-sensitive site in a highly conserved exposed loop that is proposed to interact with the peptidyltransferase center of the ribosome. Loss of peptidyl-tRNA hydrolysis activity and enhanced codon-ribosome binding by the cleaved RF is consistent with a model whereby the RF spans the decoding and peptidyltransferase centers of the ribosome with domains of the RF linked by conformational coupling. The cross-link between the stop signal and RF at the ribosomal decoding site is influenced by the base following the termination codon. This base determines the efficiency with which the stop signal is decoded by the RF in both mammalian and bacterial systems in vivo. The wide range of efficiencies correlates with the frequency with which the signals occur at natural termination sites, with rarely used weak signals often found at recoding sites and strong signals found in highly expressed genes. Stop signals are found at some recoding sites in viruses where -1 frame-shifting occurs, but the generally accepted mechanism of simultaneous slippage from the A and P sites does not explain their presence here. The HIV-1 gag-pol-1 frame shifting site has been used to show that stop signals significantly influence frame-shifting efficiency on prokaryotic ribosomes by a RF-mediated mechanism. These data can be explained by an E/P site simultaneous slippage mechanism whereby the stop codon actually enters the ribosomal A site and can influence the event.