Functional Characterization of Domains Contained in Hepatocyte Growth Factor-like Protein

Susan E. Waltz, Susan A. McDowell, Rebecca S. Muraoka, Ellen L. Air, Leah M. Flick, Ying-Qing Chen, Ming-Hai Wang, Sandra J. Friezner Degen
1997 Journal of Biological Chemistry  
To delineate the functional protein domains necessary for the biological activity of hepatocyte growth factor-like protein (HGFL), we created various site-directed and deletion mutated cDNAs coding for this protein. Wild-type and mutated versions of HGFL were produced after transfection of the corresponding cDNAs into tissue culture cells. The biological importance of the domains within HGFL was then examined by addition of recombinant wild-type or mutant forms of HGFL to assays aimed at
more » ... ting regions involved in the stimulation of DNA synthesis, the induction of shape changes in macrophages, and the ability to stimulate cell scattering. Mutant proteins lacking the serine protease-like domain (light chain) were not biologically active in any of the assays tested and could not compete with wild-type HGFL in cell scattering experiments. These data, in addition to direct enzyme-linked immunosorbent assay analyses, suggest that the light chain may play an important role in the interaction of HGFL with its receptor, Ron. Elimination of the proposed protease cleavage site between the heavy and light chains (by mutation of Arg-483 to Glu) produced a protein with activity comparable to wild-type HGFL. Further studies with this mutated protein uncovered an additional proteolytic cleavage site that produces biologically active protein. Deletion of the various kringle domains or the amino-terminal hairpin loop had various effects in the multiple assays. These data suggest that the heavy chain may play a pivotal role in determining the functional aspects of HGFL. . 1 The abbreviations used are: HGFL, hepatocyte growth factor-like protein; HGF, hepatocyte growth factor; CHO, Chinese hamster ovary; STI, soybean trypsin inhibitor; PBS, phosphate-buffered saline; WT, wild-type; BrdUrd, 5-bromo-2Ј-deoxyuridine; MDCK, Madin-Darby canine kidney; DMEM, Dulbecco's modified Eagle's medium; ELISA, enzyme-linked immunosorbent assay.
doi:10.1074/jbc.272.48.30526 pmid:9374547 fatcat:xlr3vspsdvd3lbtxirmvcrrkee