Protein tyrosine phosphatase alpha (PTPalpha) and contactin form a novel neuronal receptor complex linked to the intracellular tyrosine kinase fyn.

Glycosyl phosphatidylinositol (GPI)-linked receptors and receptor protein tyrosine phosphatases (RPTPs), both play key roles in nervous system development, although the molecular mechanisms are largely unknown. Despite lacking a transmembrane domain, GPI receptors can recruit intracellular src family tyrosine kinases to receptor complexes. Few ligands for the extracellular regions of RPTPs are known, relegating most to the status of orphan receptors. We demonstrate that PTPalpha, an RPTP that dephosphorylates and activates src family kinases, forms a novel membrane-spanning complex with the neuronal GPI-anchored receptor contactin. PTPalpha and contactin associate in a lateral (cis) complex mediated through the extracellular region of PTPalpha. This complex is stable to isolation from brain lysates or transfected cells through immunoprecipitation and to antibody-induced coclustering of PTPalpha and contactin within cells. This is the first demonstration of a receptor PTP in a cis configuration with another cell surface receptor, suggesting an additional mode for regulation of a PTP. The transmembrane and catalytic nature of PTPalpha indicate that it likely forms the transducing element of the complex, and we postulate that the role of contactin is to assemble a phosphorylation-competent system at the cell surface, conferring a dynamic signal transduction capability to the recognition element.

T1 is a c-Fos- and FosB-responsive gene which is induced by growth factors through multiple signal transduction pathways.

Stimulation of quiescent cells with growth factors triggers changes in gene expression through multiple signal transduction pathways. One of these changes in Swiss 3T3 cells is the strong accumulation of T1 mRNA which encodes a secreted glycoprotein of the immunoglobulin superfamily. Proliferating cells continued to express T1 mRNA at a lower level, whereas growth arrest induced either by serum deprivation or by contact inhibition was paralleled by the disappearance of the T1 mRNA. T1 mRNA synthesis in response to serum and platelet-derived growth factor stimulation is mediated through protein kinase C-dependent and protein kinase C-independent pathways. Activation of protein kinase A also led to T1 gene expression. Ongoing protein synthesis is a prerequisite for T1 gene induction by growth factors which defines T1 as a delayed early serum-responsive gene. The ability of the immediate early transcription factors c-Fos and FosB to directly induce the T1 gene was demonstrated in a conditional expression system in the absence of protein synthesis. Furthermore, all known inducers of the T1 gene also lead to c-fos gene activation. Thus we show that the T1 gene is regulated by signals which are transduced through multiple pathways and provide evidence that the Fos proteins play an important role in the integration of these pathways.

Growth factor-mediated induction of the delayed early gene T1 depends on a 12-O-tetradecanoylphorbol 13-acetate-responsive element located 3.6 kb upstream of the transcription initiation site.

The T1 gene is a delayed early serum-responsive gene which encodes a secreted glycoprotein of the immunoglobulin superfamily. We have addressed the question of what promoter elements are needed to allow for growth factor-mediated T1 gene expression. By deletion analysis we have identified a 448-bp DNA region 3.5-4.0 kb upstream of the transcription start site which can confer serum inducibility onto a foreign minimal promoter. Within this sequence there is a 12-O-tetradecanoylphorbol 13-acetate (TPA)-responsive element (TRE) which is essential for T1 promoter induction in response to the forced expression of the transcription factor AP-1 in NIH 3T3 fibroblasts and F9 teratocarcinoma cells. This TRE is crucial for growth factor-mediated T1 gene expression. A point mutation within this TRE attenuated serum inducibility. Two E boxes are positioned 6 and 40 bp downstream of the TRE. Point mutations within these sequence motifs reduced basal T1 promoter activity and serum inducibility. Additional, as-yet-unidentified, promoter elements within the 448-bp serum-responsive region are required for T1 gene activation in response to growth stimulation.

Kinetic analysis of two closely related receptor-like protein-tyrosine-phosphatases, PTP alpha and PTP epsilon.

Among transmembrane protein-tyrosine-phosphatases, the membrane distal catalytic domain (D2) of protein-tyrosine-phosphatase alpha (PTP alpha) is unusual in having low but detectable activity in the absence of the membrane proximal catalytic domain (D1). To investigate the catalytic properties of PTP alpha D2 in association with D1, kinetic parameters of activity were established for PTP alpha D1D2 proteins containing an inactivating point mutation in D1 and/or D2. In this context, D2 activity was unchanged by the presence (N-terminal or C-terminal) or absence of inactive D1, and the presence or absence of inactive D2 affected the velocity but not the Km of D1 catalysis. While D1 appears to be the major catalytic contributor to PTP alpha activity, D2 possesses a significantly higher substrate-specific activity relative to wild-type D1D2 than the D2 domains of other protein-tyrosine-phosphatases. Also, PTP alpha D2 is an active phosphatase with comparable or better efficiency, on the basis of k(cat)/Km criteria, to some of the dual specificity phosphatases. Kinetic parameters of a closely related receptor-like protein-tyrosine-phosphatase, PTP epsilon, were determined. PTP epsilon D1 is the major, if not the only, catalytic moiety of PTP epsilon, and has much higher turnover numbers than D1 of PTP alpha. The PTP epsilon D2 activity is insignificant compared to that of PTP epsilon-D1D2, with lower turnover numbers than PTP alpha D2. Thus, the intrinsic activity of PTP alpha D2 is high compared to other D2 domains and, more outstandingly, its activity relative to D1 appears unique. These are also apparent upon in vitro assay of full-length PTP alpha catalytic mutants expressed in mammalian cells. Together. these results suggest potential catalytic and regulatory roles for PTP alpha D2, and that PTP alpha may be an optimal model transmembrane protein-tyrosine-phosphatase for investigating the former within the cell.

Cooperative interactions of AP-1 and basic helix-loop-helix transcription factors regulate T1 gene expression.

The T1 gene is a murine, delayed early serum-responsive gene that encodes glycoproteins of the interleukin-1 receptor (IL-1R) family. Transcription of the T1 gene leads to production of two mRNAs that encode a transmembrane protein, which is highly similar to the type-1 IL-1R, and a secreted protein, which consists solely of the extracellular part. Fibroblasts, in contrast to mast cells, express predominantly the shorter form of the protein, and several mitogens cause strong, transient induction of the T1 gene in these cells. Here we describe the identification of a 148 bp enhancer element that is positioned 3.6 kb upstream of the transcription initiation site. A TPA-responsive element (TRE) and three identical E-boxes are located within this sequence. Introduced point mutations confirmed the necessity of these sites for full T1 promoter activity. The TRE and the distal E-box are absolutely indispensable for promoter function, whereas the two proximal E-boxes contribute less to promoter strength. In vitro the three E-boxes are bound by different protein complexes.

Amelioration of murine colitis by feeding a solution of lysed Escherichia coli.

BACKGROUND: Immune reactivity towards the bacterial intestinal flora plays an important part in the pathogenesis of inflammatory bowel disease. Disease activity can be positively influenced by the administration of living probiotic bacteria. We investigated the effect of soluble bacterial antigens extracted from Escherichia coli (strain Laves) on the disease activity of murine colitis. METHODS: C3H.IL-10-/- and BALB/c mice with dextran sulphate sodium-induced colitis were treated with either a bacterial lysate from E. coli or with a placebo. Mice were monitored and inflammation was assessed by histological scoring, analysis of fecal IL-1beta and measurement of cytokine production by ELISA. T cell proliferation was quantified by 3H-thymidine incorporation. RESULTS: Clinically and histologically, bacterial-lysate-treated mice revealed significantly (P < 0.05) fewer signs of colitis than placebo-treated mice. Fecal IL-1beta and mucosal TNF-alpha and IFN-gamma concentrations were significantly lower (P < 0.05) in verum-treated mice than in the placebo group. Furthermore, lymphocyte proliferation after stimulation with lipopolysaccharide or caecal bacterial antigen was significantly (P < 0.05) reduced in verum-treated mice. CONCLUSION: The use of E. coli lysate is effective in the amelioration of murine colitis. This effect may be due to a decreased Th1 reaction and to an induction of tolerance against bacterial antigens.

Double-blind randomised placebo-controlled phase III study of an E. coli extract plus 5-fluorouracil versus 5-fluorouracil in patients with advanced colorectal cancer.

The primary aim of this study was to evaluate the toxicity (mucositis, diarrhea and leucopenia) of a therapy with 5-fluorouracil (CAS 51-21-8; 5-FU) plus an E. coli extract (LC-Extract, Laves coli extract, Colibiogen inject, cell-free soluble fraction from lysed E. coli, Laves strain) in comparison with 5-FU plus placebo. Secondary endpoints included general toxicity, response rate according to WHO, survival time and quality of life. 164 patients with advanced colorectal cancer were enrolled in this randomised, placebo-controlled, double-blind, multicenter phase III study. The treatment consisted of 0.167 ml/kg/d LC-Extract or placebo followed by 500-750 mg/m2/d 5-FU on five consecutive days, repeated every three weeks for up to six treatment cycles. 158 (77 verum, 81 placebo) patients were evaluable for toxicity, 144 (72 verum, 72 placebo) evaluable for response. The therapy with LC-Extract was well tolerated. Adverse events that occurred during the study were mainly judged as 5-FU- or tumor-related. Toxicity from treatment with 600 mg/m2/d 5-FU in both treatment groups was very low. After treatment with 750 mg/m2/d 5-FU patients in the placebo-group experienced a higher CTC toxicity than in the LC-Extract groups. Remission rate and survival time showed a slight trend in favour of LC-Extract. These results suggest a positive benefit-risk ratio of the additional application of LC-Extract to 5-FU in the treatment of advanced colorectal cancer especially for administration of high doses of 5-FU.

The axonally secreted cell adhesion molecule, axonin-1. Primary structure, immunoglobulin-like and fibronectin-type-III-like domains and glycosyl-phosphatidylinositol anchorage.

Axonin-1 is an axon-associated cell adhesion molecule (AxCAM) of the chicken, which promotes neurite outgrowth by interaction with the AxCAM L1(G4) of the neuritic membrane. Here we report the cloning and sequence determination of a cDNA encoding axonin-1. Peptides generated by enzymatic cleavage showed similarity to the AxCAM F11. Degenerated polymerase chain reaction (PCR) primers were designed and an axonin-1 fragment was amplified from mRNA of embryonic retina. Screening of a cDNA library from embryonic brain resulted in the isolation of a 4.0-kb cDNA insert with an open reading frame of 3108 nucleotides. The deduced polypeptide of 1036 amino acids includes a putative hydrophobic N-terminal signal sequence of 23 or 25 amino acids and a C-terminal hydrophobic sequence of 29 amino acids which is suggestive of sequences serving as signal for the attachment of a glycosyl-phosphatidylinositol (glycosyl-PtdIns) anchor. The putative mature form of axonin-1 comprises six immunoglobulin-like repeats, followed by four fibronectin-type III repeats. Axonin-1 exhibits 75% amino acid identity with the AxCAM TAG-1 of the rat, suggesting that it is the chicken homologue of TAG-1. Like TAG-1, axonin-1 is glycosyl-PtdIns-anchored to the neuronal membrane; in contrast to TAG-1, it does not exhibit an Arg-Gly-Asp sequence.