Roles of Meltrin beta /ADAM19 in the processing of neuregulin.

Meltrin beta/ADAM19 is a member of ADAMs (a disintegrin and metalloproteases), which are a family of membrane-anchored glycoproteins that play important roles in fertilization, myoblast fusion, neurogenesis, and proteolytic processing of several membrane-anchored proteins. The expression pattern of meltrin beta during mouse development coincided well with that of neuregulin-1 (NRG), a member of the epidermal growth factor family. Then we examined whether meltrin beta participates in the proteolytic processing of membrane-anchored NRGs. When NRG-beta1 was expressed in mouse L929 cells, its extracellular domain was constitutively processed and released into the culture medium. This basal processing activity was remarkably potentiated by overexpression of wild-type meltrin beta, which lead to the significant decrease in the cell surface exposure of extracellular domains of NRG-beta1. Furthermore, expression of protease-deficient mutants of meltrin beta exerted dominant negative effects on the basal processing of NRG-beta1. These results indicate that meltrin beta participates in the processing of NRG-beta1. Since meltrin beta affected the processing of NRG-beta4 but not that of NRG-alpha2, meltrin beta was considered to have a preference for beta-type NRGs as substrate. Furthermore, the effects of the secretory pathway inhibitors suggested that meltrin beta participates in the intracellular processing of NRGs rather than the cleavage on the cell surface.

A metalloprotease-disintegrin, MDC9/meltrin-gamma/ADAM9 and PKCdelta are involved in TPA-induced ectodomain shedding of membrane-anchored heparin-binding EGF-like growth factor.

The ectodomains of many proteins located at the cell surface are shed upon cell stimulation. One such protein is the heparin-binding EGF-like growth factor (HB-EGF) that exists in a membrane-anchored form which is converted to a soluble form upon cell stimulation with TPA, an activator of protein kinase C (PKC). We show that PKCdelta binds in vivo and in vitro to the cytoplasmic domain of MDC9/meltrin-gamma/ADAM9, a member of the metalloprotease-disintegrin family. Furthermore, the presence of constitutively active PKCdelta or MDC9 results in the shedding of the ectodomain of proHB-EGF, whereas MDC9 mutants lacking the metalloprotease domain, as well as kinase-negative PKCdelta, suppress the TPA-induced shedding of the ectodomain. These results suggest that MDC9 and PKCdelta are involved in the stimulus-coupled shedding of the proHB-EGF ectodomain.

Spatially- and temporally-restricted expression of meltrin alpha (ADAM12) and beta (ADAM19) in mouse embryo.

The cloning of the full-length cDNA encoding meltrin beta (ADAM19), one of the metalloprotease-disintegrins expressed in mouse myogenic cells, revealed that the meltrin beta gene encodes a membrane protein closely related to meltrin alpha (ADAM12) which participates in myotube formation in vitro. To delineate the functions of meltrin alpha and beta, we examined the expression patterns of their transcripts during embryogenesis. The meltrin alpha gene is activated in condensed mesenchymal cells that give rise to skeletal muscle, bones and visceral organs. Meltrin beta mRNA, in contrast, is markedly expressed in craniofacial and dorsal root ganglia and ventral horns of the spinal cord, where peripheral neuronal cell lineages differentiate. Heart, skeletal muscle, intestine and lung also express meltrin beta mRNA transiently. Although the meltrin alpha and beta transcripts exhibit distinct expression patterns during embryogenesis, both genes are mainly activated in mesenchymal cells that are derived from both mesoderm and ectoderm.

Immunocytochemical analysis of MDM2 protein expression and its relevance to tumor angiogenesis in primary breast cancer.

MDM2 once-protein is known to be involved in the inactivation of p53 and RE functions. We have investigated the expression of MDM2 protein in breast cancer by immunocytochemical analysis. Out of 103 primary tumors 39 (38%) showed positive nuclear staining of MDM2, although its gene amplification was infrequent. There was no correlation between MDM2 expression and various clinicopathological factors. However, MDM2 expression was significantly associated with expression of angiogenesis factors including vascular endothelial growth factor and platelet-derived endothelial cell growth factor. Anti-endothelial immunostaining demonstrated a correlation between MDM2 expression and the increment of intratumoral microvessel density. It is suggested that altered MDM2 protein expression might be involved in the promotion of angiogenesis in human breast cancer.

A metalloprotease-disintegrin participating in myoblast fusion.

Skeletal muscle development involves the formation of multi-nucleated myotubes. This is thought to proceed by the induction of differentiation (acquisition of fusion competence) of myoblast cells, their aggregation, and union of their plasma membranes. Various membrane proteins including N- and M-cadherins, N- and V-CAMs and integrins participate in myotube formation, but the molecular mechanisms of muscle cell fusion are poorly understood. Here we report the identification of three new, myoblast-expressed gene products, meltrin-alpha, beta and gamma, with homology to both viper haemorrhagic factors and fertilin (PH-30), a membrane protein involved in egg-sperm fusion. Meltrin-alpha, a member of the metalloproteinase/disintegrin protein family, appears to be required for myotube formation. Involvement of a fertilin-related protein in myogenesis suggests that there are common mechanisms in gamete and myoblast fusion.

Effects of the protein phosphatase inhibitors, tautomycin and calyculin-A, on protein phosphorylation and cytoskeleton of human platelets.

Effects of the protein phosphatase inhibitors, tautomycin and calyculin A on protein phosphorylation and cytoskeleton of human platelets. It has been discovered recently that many cytotoxic compounds isolated from a variety of sources are potent phosphatase inhibitors. Two of these, tautomycin (TM) and calyculin-A (CL-A) were applied to human platelets to investigate the role of protein phosphorylation on cytoskeletal structure and function. Exposure to 10 microM TM or 0.1 microM CL-A induced marked morphological changes. The granules were centralized and surrounded by actin filaments, but there was no evidence of granule release. Myosin became more centralized, was occluded from the granulomere, but was not confined to the microfilament ring. These changes occurred without an increase in cytosolic Ca2+ concentrations, as determined by measurements with fura-2. TM and CL-A induced an overall increase in protein phosphorylation. Phosphorylation of the 20,000 dalton light chain of myosin increased markedly and multiple phosphorylation sites were indicated. Cytoskeletons were prepared from control, thrombin- and TM-treated platelets, the latter prepared in the absence of external calcium. The major difference in protein composition was the increased content of myosin associated with the cytoskeleton from TM-treated platelets where the dominant phosphoprotein was the 20,000 dalton light chain. These results suggest that myosin phosphorylation drives the initial shape changes, and via a contractile process results in the formation of the microfilament ring and centralization of granules.

Melastin, a novel product of Streptomyces that selectively inhibits leukemia cell growth.

In the course of our screening for new immunomodulators, a novel compound, melastin, was purified from the culture broth of Streptomyces. Melastin was purified through absorption to Diaion HP-20, ethanol precipitation, and anion exchange column as a brown powder. The molecular weight was estimated as 5000 +/- 3000 by gel filtration HPLC. Melastin suppressed lipopolysaccharide (LPS)-induced blastogenesis of B cells more profoundly than concanavalin A (con A)- or phytohemagglutinin (PHA)-induced blastogenesis of T cells. Moreover, it selectively inhibited the growth of several leukemia cells as compared with interleukin-dependent nontransformed leukocytes. No selectivity was observed between nontransformed fibroblasts and their oncogene-transformed variants. Melastin did not selectively inhibit macromolecule synthesis of leukemia cells.

Morphological changes and reorganization of actinfilaments in human myeloid leukemia cells induced by a novel protein phosphatase inhibitor, tautomycin.

A protein phosphatase inhibitor, tautomycin induces blebs on the surface of human myeloid leukemia K562 cells within 10 min. In this paper we examined the cytoskeleton of tautomycin-treated cells. In the presence of tautomycin, cells with blebs turned into segmented forms with smooth surfaces after 15 min and into smooth round shapes without microprotuberance after 60 min. Observation with fluorescence microscopy showed that F-actin detached from the plasma membrane at the site of the blebs. Further treatment with tautomycin induced the accumulation of F-actin at the segmentation centers. Under electron microscopy, an electron dense ring-structure was detected at the segmentation center. Tautomycin did not induce major changes of the microtubule network although F-actin accumulated near the microtubule organizing center. The amount of F-actin increased in tautomycin-treated cells. These results indicate that the morphological changes are induced by reorganization of actinfilaments.

Effects of tautomycin, a protein phosphatase inhibitor, on recycling of mammalian cell surface molecules.

The effects of tautomycin, a protein phosphatase inhibitor, on recycling of cell surface molecules were studied with transferrin receptor (TFR) of human myeloid leukemia K562 cells and with CD4 of murine thymocytes. Tautomycin increased expression of TFR of K562 cells whereas phorbol dibutylate (PDBu) decreased it. Tautomycin inhibited PDBu-induced down-regulation of CD4 although it did not induce up-regulation. Okadaic acid also inhibited down-regulation of CD4 which was induced by PDBu. The results suggest that certain inhibitors of protein phosphatases preferentially inhibit endocytosis of cell surface molecules.