beta-NGF-endopeptidase: structure and activity of a kallikrein encoded by the gene mGK-22

Mouse nerve growth factor (NGF) is cleaved at a histidine-methionine bond to release an NH2-terminal octapeptide (NGF1-8). The enzyme responsible, beta-NGF-endopeptidase, is structurally and functionally similar to gamma-NGF and epidermal growth factor-binding protein (EGF-BP) and cleaves mouse low molecular weight kininogen to produce bradykinin-like activity. These data have suggested that, like gamma-NGF and EGF-BP, beta-NGF-endopeptidase is a mouse glandular kallikrein. Evidence for a physiological role for NGF1-8 encouraged studies to further characterize the structure and function of this enzyme. Purified beta-NGF-endopeptidase migrated as a single band on isoelectric focusing and reducing SDS-polyacrylamide gels. As was expected, it removed NGF1-8 from NGF. Interestingly, enzymatic activity on an artificial substrate, and on NGF, was inhibited by NGF1-8 and by bradykinin. These studies further supported the view that beta-NGF-endopeptidase acts on both NGF and kininogen. The first 30 NH2-terminal amino acids of beta-NGF-endopeptidase were sequenced. This analysis demonstrated that the enzyme is encoded by the gene designated mGK-22 (Evans et al., 1987). The sequence of this gene corresponds to that of EGF-BP type A (Anundi et al., 1982; Drinkwater et al., 1987), and so studies were performed to determine whether or not beta-NGF-endopeptidase participates in EGF complex formation. Chromatographic and kinetic data gave no evidence that beta-NGF-endopeptidase is an EGF-binding protein. Our studies suggest that contamination of high molecular weight (HMW) EGF preparations with beta-NGF-endopeptidase erroneously led to earlier designation of the product of mGK-22 as an EGF-BP.(ABSTRACT TRUNCATED AT 250 WORDS)     

In situ localization of mRNA for epidermal growth factor in the submandibular gland of the mouse

Epidermal growth factor (EGF) is a polypeptide originally isolated from the mouse submandibular gland, where it is localized immunocytochemically in cells of the granular convoluted tubules (GCT). cDNAs encoding the precursor of mouse submandibular EGF have been cloned (Scott et al. Science 221:236, 1983; Gray et al. Nature 303:722, 1983). A fragment of one of these clones, pmegf10, containing the EGF coding region, was tritium-labeled by nick-translation and used as a probe for in situ hybridization to EGF mRNA. A specific hybridization signal for EGF mRNA was seen only in mature or developing GCT cells. The intensity of the signal was stronger in glands of intact males than in females or in castrated males. In glands of castrates treated with testosterone, or of intact females treated with triiodothyronine (T3), the signal was comparable to that in intact males. In glands of males treated with T3 the intensity of the signal was stronger than in untreated males. A weak to moderate signal was seen in developing GCT cells of 20-day-old males but not females. Hybridization for 3 days gave a stronger signal than that for 1 day. No signal was seen in either sex at 10 days of age, or in control preparations exposed to labeled DNA of pBR322. The presence of EGF mRNA exclusively in GCT cells provides strong evidence that these cells are the only site of synthesis of EGF in the submandibular gland. In situ hybridization with this cDNA probe will provide a sensitive method to determine possible cellular sites of EGF production outside of the submandibular gland.     

A Mr = 190,000 glycoprotein phosphorylated on tyrosine residues in epidermal growth factor stimulated KB cells is the product of the c-erbB-2 gene

In human epidermoid carcinoma KB cells, a glycoprotein of Mr = 190,000 (gp190) has been shown to be phosphorylated on tyrosine residues upon EGF stimulation (Kadowaki et al., 1987, J. Biol. Chem. in press). Using a specific antibody to the c-terminal portion of the human c-erbB-2 gene product, we have found that gp190 is the human c-erbB-2 gene product which is structurally closely related to the epidermal growth factor (EGF) receptor. Since monoclonal antibody specific for the EGF receptor abolished both EGF binding to its receptor and tyrosine phosphorylation of the c-erbB-2 gene product, we have concluded that activation of EGF receptor tyrosine kinase activity upon EGF binding leads to the phosphorylation of the c-erbB-2 gene product on its tyrosine residues.     

Regulation of extracellular signal-regulated kinase activity by p120 RasGAP does not involve its pleckstrin homology or calcium-dependent lipid binding domains but does require these domains to regulate cell proliferation.

The gene encoding for p120 RasGAP, has been disrupted in mice (M. Henkemeyer et al., Nature (Lond.), 377: 695-701, 1995).In this study, using fibroblasts derived from these mouse embryos (Gap-/-; P. van der Geer et al., Mol. Cell Biol., 17: 1840-1847, 1997), we demonstrate that mitogen-activated protein kinase (MAPK) activation is prolonged after epidermal growth factor (EGF), but not lysophosphatidic acid, stimulation as compared with wild-type cells. Furthermore, these cells exhibited a moderate increase in their proliferative rate and saturation density, as well as a limited ability to form colonies in soft agar. Stable cell lines expressing full-length p120GAP not only restored the ability to down-regulate MAPK after EGF stimulation but also lowered their saturation densities. Similarly, expression of p120GAP, missing either its pleckstrin homology (PH) or its calcium-dependent lipid binding (CaLB)/C2 domain, restored MAPK down-regulation and retained the ability to associate with p190 RhoGAP and to be phosphorylated by v-src but exhibited higher saturation densities similar to Gap-/- cells. Our results, therefore, suggest that p120GAP functions not only by down-regulating the Ras/MAPK pathway after growth factor stimulation but is also important in regulating cell proliferation that involves its PH and CaLB domains.     

Verhulstian analysis of the growth of transplantable mammary tumours in sialoadenectomized mice

In this report we have analysed data published in 1989 by Inui et al. (Incidence of precancerous foci of mammary glands and growth rate of transplantable mammary cancers in sialoadenectomized mice. J. Natl Cancer Inst. 81, 1660) involving the effects of perturbation of the epidermal growth factor (EGF) status of mammary tumour-bearing mice on subsequent volumetric responses. Removal of an endogenous EGF stimulus by surgical ablation of the submaxillary glands, the major EGF-producing organ in mice, produced significantly slower growth of rodent mammary neoplasms, decreased success rate of transplantation, and an increase in the latent period before growth occurred. Administration of i.p. EGF (5 micrograms/mouse/day) to sialadectomized tumour-bearing mice would however, increase tumour growth rate. Data were analysed using the Verhulst equation which indicated that the observed effects on tumour volumetrics by either sialoadenectomy or EGF administration could be interpreted as being produced through paracrine pathways. The use of the Verhulstian analysis indicates that it is possible to analyse neoplastic responses and infer whether paracrine or autocrine pathways are involved.     

Tertiary structure of mouse epidermal growth factor determined by two-dimensional 1H NMR

The tertiary structure of mouse epidermal growth factor (EGF) in solution (28 degrees C, pH 2.0) was studied by two-dimensional NMR spectroscopy. Proton-proton distance constraints derived from NOESY spectra were used to construct a mechanical molecular model of mouse EGF, which was subsequently checked by means of a preliminary distance geometry calculation. The chain-folds in the two structural domains of mouse EGF were very similar to those previously reported (Montelione et al. (1987) Proc. Natl. Acad. Sci. U.S. 84, 5226-5230). However, the relative orientations of the two domains were different. Because we could assign much more inter-domain NOEs, the relative orientations of the two domains were well determined in our model. The hollow between the two domains may function as a binding site for the EGF receptor.     

Reversal of indomethacin-induced inhibition of implantation in the mouse by epidermal growth factor.

In the mouse, estriol (E3) can induce implantation as a phase I of estrogen action. E3-induced implantation was attenuated by indomethacin(INDO), an inhibitor of prostaglandin (PG) synthesis. The inhibitory effect of INDO was reversed by administration of epidermal growth factor (EGF), and this EGF effect was dose-dependent. These results suggest that one of the functions of estrogen could be to activate the EGF ligand-receptor signalling in the uterus in generating PGs required for initiation of implantation. This is consistent with the results of EGF stimulation of synthesis of PGs in uterine stromal cells in culture.     

1H NMR assignment and secondary structural elements of human transforming growth factor alpha

The 1H NMR spectrum of human transforming growth factor alpha (hTGF-alpha) has been completely assigned, and secondary structural elements have been identified as a preliminary step in determining the structure of this protein by distance geometry methods. Many of these structural elements closely correspond to those previously found in a truncated human EGF [Cooke et al. (1987) Nature (London) 327, 339-341] and murine EGF [Montelione et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5226-5230]. These include the presence of an antiparallel beta-sheet between residues G19 and C34 with a type I beta-turn at V25-D28, a type II beta-turn at H35-Y38, and another short beta-sheet between residues Y38-V39 and H45-A46.     

Mapping of the human gene for epidermal growth factor receptor (EGFR) on the p13 leads to q22 region of chromosome 7

We previously reported that the structural gene for epidermal growth factor receptor (EGFR) can be mapped to the p22 leads to qter region of human chromosome 7 (Shimizu et al., 1979, 1980). In the present study, we produced two series of human-mouse cell hybrids by fusing mouse A9 cells that are deficient in EGFR with the human diploid fibroblast lines GM1356, 46,XX,t(1;7)(p34;p13), and GM2068, 46,XX,t(6;7)(q27;q22), both of which possess EGF receptors. Expression of EGF binding ability in the former series of cell hybrids was correlated with the retention of the human translocation chromosome containing the 7p13 leads to qter region, and in the latter series of cell hybrid it was correlated with the retention of the human translocation chromosome containing the 7pter leads to q22 region. Therefore, the EGFR gene can be localized in the p13 leads to q22 region of chromosome 7.     

Global uterine genomics in vivo: microarray evaluation of the estrogen receptor alpha-growth factor cross-talk mechanism

Cross-talk between growth factor receptors and the estrogen receptor (ER) has been proposed as a signaling mechanism in estrogen target tissues, with ER(alpha) as a direct target of growth factor receptor-activated signals, leading to regulation of estrogen target genes and estrogen-like biological responses to growth factors. We evaluated whether global genomic changes in the mouse uterus in response to epidermal growth factor or IGF-I mimic those of estradiol (E2), reflecting the cross-talk mechanism. Overlapping responses to growth factors and E2 were expected in the wild type (WT) whereas no response was expected in mice lacking ER(alpha) (ER(alpha) knockout). Surprisingly, although most of the E2 response in the WT also occurred after growth factor treatment, some genes were induced only by E2. Second, although E2 did not induce gene changes in the ER(alpha) knockout, the growth factor response was almost indistinguishable from that of the WT. Differences in response of some genes to IGF-I or epidermal growth factor indicated selective regulation mechanisms, such as phosphatidylinositol 3-kinase or MAPK-dependent responses. The robust ER(alpha)-independent genomic response to growth factor observed here is surprising considering that the biological growth response is ER(alpha) dependent. We propose two mechanisms as alternatives to the cross-talk mechanism for uterine gene regulation. First, E2 increases uterine growth factors, which activate downstream signaling cascades, resulting in gene regulation. Second, growth factors and estrogen regulate similar genes. Our results suggest that the estrogen response in the uterus involves E2-specific ER(alpha)-mediated responses as well as responses resulting from convergence of growth factor and ER-initiated activities.