The major current goal of project is to study the biochemistry and cell biology of Macrophage Stimulating Protein (MSP) and its receptor protein tyrosine kinase (RON). Investigations to date suggest that this ligand/ receptor pair may affect a number of cells that participate in the response to tissue injury, including skin cells, macrophages and capillary endothelium. Our major findings this year are: 1. Pro-MSP, the single chain, biologically inactive precursor of MSP, is synthesized constitutively by the liver and secreted into the circulation. Active MSP was found in human wound fluids. These fluids contain a pro-MSP convertase, which cleaves pro-MSP to active MSP. The inhibitor profile of this enzyme suggests that it is a hitherto undescribed enzyme. Purification and characterization of this enzyme is in progress. 2. We studied the interaction of MSP with its receptor (RON) on epithelial cells. Based on computer modeling and a review of the literature, we proposed that ligand induction of receptor dimerization is comparable to that of human growth hormone, with a stoichiometry of one ligand:2 receptors. We identified two RON binding sites on MSP, a high affinity site on the MSP beta chain, and a low affinity site on the alpha chain. Thus, one ligand has the potential to bind two receptors. We also localized the high affinity beta chain binding region, as shown by loss of binding after mutation of a single arginine residue. 3. We have identified kinases that participate in RON signaling. In addition to previously identified involvement of PI3-K, JNK and MAPK, we found that FAK, c-Src and AKT are rapidly and transiently activated by MSP. Two independent pathways operate to prevent apoptosis in epithelial cells, one via MAPK and the other via PI3-K. 4. Time lapse videos show that MSP stimulates macrophage pinocytosis, with the generation of large pinosomes. We quantified this by testing macrophage uptake of different sized fluoresceinated microspheres. Both unstimulated and MSP-stimulated macrophages ingested 20 nm microspheres. A difference in uptake between unstimulated and stimulated macrophages was apparent with 110 nm microspheres; and pinocytosis of 190 nm microspheres was limited to MSP-stimulated macrophages. - pinocytosis, growth, motility, protease, receptor, kinase, - Human Tissues, Fluids, Cells, etc.
