Showing papers by "Luigi Naldini published in 1993"

Molecular characteristics of HGF-SF and its role in cell motility and invasion.

Abstract: Scatter factor (SF), a secretory protein of fibroblasts, dissociates and increases the motility of epithelial cells and may be involved in cell migration processes during embryogenesis and tumor progression. Hepatocyte growth factor (HGF) is a potent mitogen for hepatocytes and other cells, and is thought to play a role in liver regeneration. We have presented structural and functional evidence that human SF and human HGF are identical proteins encoded by a single gene, since (i) no differences could be found by protein sequencing, by cDNA analysis, or by immunological comparison, and (ii) SF acts as a hepatocyte growth factor--i.e., stimulates DNA synthesis of primary hepatocytes and is a morphogen of kidney epithelial cells--whereas HGF exhibits SF activity--i.e., dissociates and induces invasiveness of various epithelial cells. Furthermore, there exists only one gene for human HGF-SF which is located on chromosome 7, bands q11.2-21 (Weidner et al., Proc. Natl. Acad. Sci. USA 88, 7001-7005, 1991). HGF-SF has been found to be the ligand of the c-met receptor tyrosine kinase (Naldini et al., EMBO J. 10, 2867-2878, 1991b). We have recently used transient expression of naturally occurring and in vitro mutagenized cDNAs of HGF-SF in order to delineate the protein domains necessary for biological activity and c-met receptor activation. (i) A single-chain HGF-SF resulting from the destruction of the protease cleavage site between heavy and light chain (Arg494 to Gln) was largely inactive, indicating that proteolytic cleavage is essential for acquisition of the biologically active conformation. (ii) A HGF-SF splice variant encoding a protein with a 5 amino acid deletion in the first kringle domain was as highly active as the wild type molecule. (iii) The separately expressed light chain (with serine protease homology) was inactive in all assays tested. (iv) The separate heavy chain as well as a naturally occurring splice variant consisting of the N-terminus and the first two kringle domains bound the met receptor, stimulated its tyrosine phosphorylation, and induced dissociation of epithelial cells but not mitogenesis. These data indicate that a functional domain in the N-terminal region and/or the first two kringle domains of HGF-SF is sufficient for binding to and activation of the met receptor (Hartmann et al., Proc. Natl. Acad. Sci. USA, in press).

Structure, Biosynthesis and Biochemical Properties of the HGF/SF Receptor

Abstract: Hepatocyte Growth Factor and Scatter Factor are indistinguishable factors (HGF/SF) secreted by mesodermal cells and acting on epithelial cells. HGF/SF stimulates mitogenesis, cell motility and dissociation of epithelial sheets, it promotes matrix invasion and is considered a major mediator of liver regeneration in vivo. HGF/SF is a potent angiogenic factor in vivo and in vitro induces endothelial cells to proliferate and migrate. HGF/SF is secreted as a single-chain biologically inactive precursor (pro-HGF/SF) and maturation into the active aβ heterodimer results from a proteolytic cleavage by urokinase which acts as a pro-HGF/SF convertase. HGF/SF is the ligand for p190 MET , the receptor tyrosine kinase encoded by the MET proto-oncogene. This is sustained by following evidence: (1) it binds to immunopurified p190 MET , (2)the chemical cross-linking of the radiolabelled ligand, (3) HGF/SF-induced tyrosine phosphorylation of p190 MET and (4) the reconstitution of high-affinity binding sites for HGF/SF into insect ceUs infected with a recombinant baculovirus carrying the human MET cDNA, The structure of p190 MET consists of a heterodimer of two (α, β) disulfide-linked protein subunits. The α subunit is solely extracellular, while the β subunit contains an extracellular portion involved in ligand binding, a transmembrane domain, and a cytoplasmic tyrosine kinase domain bearing phosphorylation sites which regulate its activity. The mature p190 MET results from glycosylation and proteolytic cleavage of the precursor of 170 kDa. Alternative post-transcriptional processing originates two truncated Met proteins, endowed with ligand binding activity and lacking the cytoplasmic kinase domain of the β subunit. Only one form is soluble and released from the cells. The interaction of HGF/SF with p190 MET in intact cells stimulates tyrosine autophosphorylation of the receptor β subunit. The autophosphorylation of the receptor upregulates its kinase activity. The tyrosine kinase activity of the receptor is downmodulated by PKC activity and also the rise of intracellular Ca2+. The signal transduction pathways of the target cells are also stimulated by the autophosphorylation of the HGF/SF receptor β subunit. The phosphorylated receptor associates rasGAP, phospholipase C-γ, and src-related tyrosine kinase in vitro. Phosphosphatidylinositol 3-kinase associates autophosphorylated p190 MET in vitro and in vivo, indicating that the generation of the D-3 phosphorylated inositol lipids is involved in effecting the motility and/or the growth response to HGF/SF.

Solubilization and Characterization of the Receptor for Gastrin-Releasing Peptide

Abstract: Publisher Summary Gastrin-releasing peptide (GRP), the mammalian counterpart of bombesin (an amphibian hormone), is a neurohormone that exerts a number of different effects in vivo and in vitro . An autocrine loop involving GRP and its receptor play an important role in the proliferation of small cell carcinoma of the lung . The GRP receptor is a G protein-coupled receptor that exerts its effects through the stimulation of phospholipase C. The involvement of a G protein in the GRp receptor signaling pathway and the molecular cloning of the GRP receptor confirm that the GRP receptor has seven putative transmembrane regions and is a member of the G protein-coupled receptor superfamily. The purification of the GRP receptor necessitates the solubilization of the receptor from the membrane to purify it away from other protein and lipids in the membrane environment. The first step toward purification or analysis of a membrane receptor is the successful solubilization of the receptor in an active form. The choice of detergent for effective solubilization is empirical. A number of nonionic detergents are available for the solubilization of membrane proteins.