In vivo modulation of epidermal growth factor receptor phosphorylation in mice expressing different gangliosides

We studied in this work the in vivo phosphorylation of the epidermal growth factor receptor (EGFr) in skin from knockout mice lacking different ganglioside glycosyltransferases. Results show an enhancement of EGFr phosphorylation, after EGF stimulation, in skin from Sial-T2 knockout and Sial-T2/GalNAc-T double knockout mice as compared with wild-type and Sial-T1 knockout mice. Qualitative analysis of ganglioside composition in mice skin suggest that the increase of EGFr phosphorylation observed in skin from Sial-T2 knockout and Sial-T2/GalNAc-T double knockout mice in response to EGF might not be primary attributed to the expression of GD3 or a-series gangliosides in mice skin. These studies provide, for the first time, an approach for studying the molecular mechanisms involved in the in vivo regulation of EGFr function by gangliosides.     

Genetic regulation of mouse glycosylphosphatidylinositol-phospholipase D

Glycosylphosphatidylinositol phospholipase D (GPI-PLD) has been proposed to be responsible for cleaving membrane-associated glycosylphosphatidyl inositol (GPI) molecules to generate inositol phosphoglycan (IPGs), which have growth factor-mimetic properties. We have cloned the mouse liver GPI-PLD cDNA, which has a sequence that differs from that previously isolated from a mouse glucagonoma cell library. Using a highly specific and very sensitive RNase protection assay, we found that the GPI-PLD expressed in adult/post-natal brain, antrum and insulin-producing cells is identical to that isolated from liver. The expression of mouse GPI-PLD in liver shows a complex genetic regulation with a mouse strain-specific variation. In addition, GPI-PLD mRNA levels were higher in 4-week old animals compared to older animals, and the GPI-PLD mRNA levels increased in mice that developed insulin dependent type 1 diabetes spontaneously. This suggests that the expression of liver GPI-PLD in mice is highly regulated.     

Identification of a gene induced by glucocorticoids in murine T-cells: a potential G protein-coupled receptor.

Using cDNA cloning techniques we previously identified a set of genes induced by glucocorticoids and cAMP in murine T-lymphocytes. We report here the sequence of one of these cDNA clones (clone 4.2), renamed here as glucocorticoid-induced receptor (GIR), which encodes a potential new member of the family of receptors that couple to G-proteins. Several different forms of cDNA for this gene were isolated and shown to correspond to multiple mRNA species in lymphoid cells using an RNase protection assay. The cDNA clone corresponding to the most abundant form of GIR mRNA encodes a precursor protein of 423 amino acids, with a putative signal peptide of 17 amino acids. A hydropathy plot reveals the presence of seven hydrophobic regions, with significant similarities to other G-protein-coupled receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and sequencing of the rat preproepidermal growth factor cDNA: comparison with mouse and human sequences.

We have cloned and sequenced the cDNA corresponding to the rat preproepidermal growth factor (ppEGF) mRNA. The cDNA contained 4,801 nucleotides, similar to that reported for the mouse (4,749 nucleotides) and the human mRNAs (4,871 nucleotides). The predicted protein sequence would contain 1,133 amino acids, smaller than that reported for the mouse (1,217 amino acids) and the human sequences (1,207 amino acids). The results of the sequencing of several cDNA clones suggested the existence of more than one structural gene for ppEGF. In addition, there was an occurrence of alternative splicing events, resulting in deletions of entire exons from the mature mRNA. These alternative splicing events do not create frameshift mutations but cause a deletion of one or more of the "EGF-like" repeat units from the ppEGF. There is approximately the same homology between the rat and mouse amino acid sequences both in the EGF region and in the other regions of the ppEGF protein. We conclude that, because of this conservation of homology, there may be an important function performed by these other regions of the ppEGF besides their function as a precursor for the EGF protein.     

The Pyruvate Dehydrogenase Complex: Cloning of the Rat Somatic El α Subunit and Its Coordinate Expression with the mRNAs for the E1β E2, and E3 Catalytic Subunits in Developing Rat Brain

Abstract: We report the isolation of cDNA clones encoding the somatic form of the E1α subunit of the pyruvate dehydrogenase complex of rat. The deduced amino acid sequence has 99.5, 98, and 97% identity, respectively, with the orthologous proteins of mouse, human, and pig and 98.5% identity with a rat E1α sequence reported previously. The cDNAs isolated in this and earlier studies predict different E1α subunit mRNA sizes and amino acid sequences. These differences have been investigated by PCR, northern blot hybridization, and RNase protection. We have used our E1α cDNA, in conjunction with cDNA probes to the E1β, E2, and E3 catalytic subunits of rat pyruvate dehydrogenase complex and also to rat citrate synthase, to perform RNase protection assays of developing rat whole brain RNA. The results show a 2.5-fold increase in the concentration of each of the subunit mRNAs and a 1.2-fold increase in citrate synthase mRNA from late foetal stage to 5 days post partum. Thereafter, the mRNA levels remained constant. These data indicate that the respective six-and threefold increases in the amounts of pyruvate dehydrogenase complex and citrate synthase found to occur in rat brain between birth and adulthood are mediated principally by translational and/or posttranslational mechanisms.     

Molecular cloning and expression of the human and mouse homologues of the Drosophila dachshund gene

Recent genetic analysis of the Drosophila dachshund (dac) gene has established that dac encodes a novel nuclear protein that is involved in both eye and leg development. In the Drosophila eye, dac expression appears to be controlled by the product of the eyeless/Pax6 gene. In order to analyze the Pax6 pathway in vertebrates we have isolated and characterized the cDNA and genomic clones corresponding to the human and mouse homologues of Drosophila dac. A full-length human cDNA encoding dachshund (DACH) encodes the 706 amino acids protein with predicted molecular weight of 73 kDa. A 109 amino acid domain located at the N-terminus of the DACH showed significant sequence and secondary structure homologies to the ski/sno oncogene products. Northern blot analysis found human DACH predominantly in adult kidney, heart, and placenta, with less expression detected in the brain, lung, skeletal muscle and pancreas. A panel of human cell lines was studied and most notably a large proportion of neuroblastomas expressed DACH mRNA. Mouse Dach encodes a protein of 751 amino acids with predicted molecular weight of 78 kDa that is 95% identical to the human DACH. RNase protection analysis showed the highest Dach mRNA expression in the adult mouse kidney and lung, whereas lower expression was detected in the brain and testis. RT/PCR analysis readily detected Dach mRNA in the adult mouse cornea and retina. Dach mRNA expression in the mouse E11.5 embryo was observed primarily in the fore and hind limbs, as well as in the somites. Received: 9 February 1999 / Accepted: 19 April 1999     

Affinity purification, peptide analysis, and cDNA sequence of the mouse interferon gamma receptor

The receptor for mouse interferon gamma (IFN-gamma) was purified from detergent-solubilized plasma membranes of EL-4, a thymoma cell line which expresses a high number of receptors on its cell surface. The purification was carried out by immunoaffinity chromatography using an anti-receptor monoclonal antibody. The purified receptor was subjected to NH2-terminal sequence analysis as well as sequencing of endopeptidase-generated peptides. One of the peptides was found to be identical to a portion of the published amino acid sequence of the human IFN-gamma receptor deduced from cDNA. This information was utilized to construct a mixed-sequence oligodeoxynucleotide probe which permitted the isolation of a full-length cDNA clone coding for the mouse IFN-gamma receptor. The mouse IFN-gamma receptor cDNA is comprised of 105 base pairs of the 5'-untranslated region, an open reading frame coding for a 477-amino acid serine-rich protein having calculated Mr 52,276, and a 3'-untranslated region of 539 base pairs. The receptor is first synthesized as a pre-protein from which a 25-amino acid signal peptide is cleaved. The receptor contains a hydrophobic transmembrane portion near the center of the molecule. Northern blot analysis of various cell lines showed that each contained a single 2.0-kilobase mRNA. A direct correlation between the amount of IFN-gamma receptor mRNA and the level of receptor expressed on the cell surface was observed. The mouse and human IFN-gamma receptors are structurally similar, showing 51% over-all homology in amino acid sequence. Mouse IFN-gamma receptor cDNA when inserted in a mammalian shuttle vector and transfected into COS-7 monkey cells was able to direct the expression of specific binding activity for mouse IFN-gamma.     

雌激素拮抗剂对胎盘EGF受体及其基因表达的影响

中期孕鼠在他莫昔芬作用下,其颌下腺,血清中ＥＧＦ含量下降,胎盘中ＥＧＦ受体结合位点数下降以及它的ｍＲＮＡ表达受到抑制,再次证实了他莫昔芬抑制雌激素诱导ＥＧＦ受体ｍＲＮＡ的表达。从而使ＥＧＦ受体结合位点数减少,因此,他莫昔芬对孕鼠胚胎生长发育有不可忽视的影响。     

Mouse keratin 19: complete amino acid sequence and gene expression during development

The complete amino acid sequence of the mouse keratin 19 (K19) was determined from a partial sequence of cDNA isolated from a mouse (day 10.5) embryo library and an amplified genomic fragment. Analysis of the sequence reveals strong evolutionary conservation with other K19s. Examination of the expression of the gene encoding K19 (K19) during development using an RNase protection assay reveals it is expressed in extra-embryonic tissues by day 8.5 and in the embryo proper by at least day 9.5. Furthermore, the K19 gene is induced in differentiating F9 embryonal carcinoma cells. These results indicate that K19 is another keratin, in addition to the K8-K18 pair, which is synthesized early during mouse development. Finally, Southern analysis of the K19 gene reveals that it is found as a unique copy in the mouse genome, in contrast to what is found in humans, which have at least one processed pseudogene.     

Small interfering RNAs suppress the expression of endogenous and GFP-fused epidermal growth factor receptor (erbB1) and induce apoptosis in erbB1-overexpressing cells

Deregulated and excessive expression of epidermal growth factor receptor (EGFR or erbB1), a transmembrane receptor tyrosine kinase specific for the epidermal growth factor (EGF), is a feature and/or cause of a wide range of human cancers, and thus inhibition of its expression is potentially therapeutic. In RNA interference (RNAi), duplexes of 21-nucleotide RNAs (small interfering RNA, siRNA) corresponding to mRNA sequences of particular genes are used to efficiently inhibit the expression of the target proteins in mammalian cells. Here we show that by using RNAi the expression of endogenous erbB1 can be specifically and extensively (90%) suppressed in A431 human epidermoid carcinoma cells. As a consequence, EGF-induced tyrosine phosphorylation was inhibited and cell proliferation was reduced due to induction of apoptosis. We established an inverse correlation between the level of expressed erbB1 and EGF sensitivity on a cell-by-cell basis using flow cytometry. A431 cells expressing endogenous erbB1 were transfected with erbB1 fused C-terminally to enhanced green fluorescent protein (EGFP). Selective inhibition of the expression of the fusion protein was achieved with an siRNA specific for the EGFP mRNA, whereas the erbB1-specific siRNAs inhibited the expression of both molecules. siRNA-mediated inhibition of erbB1 and other erbB tyrosine kinases may constitute a useful therapeutic approach in the treatment of human cancer.     

Molecular cloning and characterization of a novel rat activin receptor.

We report the isolation of a full-length rat cDNA for a new activin receptor. The deduced amino acid sequence of this receptor shows 67 percent overall identity with that of a previously identified mouse activin receptor. As predicted for the mouse activin receptor, the amino acid sequence of the rat receptor is consistent with a polypeptide containing an extracellular ligand binding domain, a hydrophobic transmembrane domain, and a serine/threonine kinase intracellular domain. In an expression assay, this new receptor was found to bind I125 radiolabeled activin.