Possible dissociation of the heparin-binding and mitogenic activities of heparin-binding (acidic fibroblast) growth factor-1 from its receptor-binding activities by site-directed mutagenesis of a single lysine residue.
The fibroblast or heparin-binding growth factors (HBGFs) are thought to be modulators of cell growth and migration, angiogenesis, wound repair, neurite extension, and mesoderm induction. A better understanding of the structural basis for the different activities of these proteins should facilitate the development of agonists and antagonists of specific HBGF activities and identification of the signal transduction pathways involved in the mechanisms of action of these growth factors. Chemical modification studies of Harper and Lobb (Harper, J. W., and R. R. Lobb. 1988. Biochemistry. 27:671-678) implicated lysine 132 in HBGF-1 (acidic fibroblast growth factor) as being important to the heparin-binding, receptor-binding, and mitogenic activities of the protein. We changed lysine 132 to a glutamic acid residue by site-directed mutagenesis of the human cDNA and expressed the mutant protein in Escherichia coli to obtain sufficient quantities for functional studies. Replacement of this lysine with glutamic acid reduces the apparent affinity of HBGF-1 for immobilized heparin (elutes at 0.45 M NaCl vs. 1.1 M NaCl for wild-type). Mitogenic assays established two points: (a) human recombinant HBGF-1 is highly dependent on the presence of heparin for optimal mitogenic activity, and (b) the change of lysine 132 to glutamic acid drastically reduces the specific mitogenic activity of HBGF-1. The poor mitogenic activity of the mutant protein does not appear to be due to a reduced affinity for the HBGF receptor. Similarly, the mutant HBGF-1 can stimulate tyrosine kinase activity and induce protooncogene expression. Differences in the biological properties of the wild-type and mutant proteins were observed in transfection studies. Mutant HBGF-1 expression in transfected NIH 3T3 cells did not induce the same transformed phenotype characteristic of cells expressing wild-type HBGF-1. Together these data indicate that different functional properties of HBGF-1 may be dissociated at the structural level.
Laboratory of Molecular Biology, Jerome H. Holland Laboratory for the Biomedical Sciences, American Red Cross, Rockville, Maryland 20855.
W H
WH
Burgess
A M
AM
Shaheen
M
M
Ravera
M
M
Jaye
P J
PJ
Donohue
J A
JA
Winkles
D000818:Animals; D002478:Cells, Cultured; D004789:Enzyme Activation; D016220:Fibroblast Growth Factor 1 / Q000737:chemistry* / Q000502:physiology; D005786:Gene Expression Regulation; D005971:Glutamates; D018698:Glutamic Acid; D006493:Heparin / Q000378:metabolism*; D008239:Lysine; D008938:Mitosis / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D011956:Receptors, Cell Surface / Q000378:metabolism*; D017468:Receptors, Fibroblast Growth Factor; D014162:Transfection
D005971:Glutamates; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D017468:Receptors, Fibroblast Growth Factor; D016220:Fibroblast Growth Factor 1; D018698:Glutamic Acid; D006493:Heparin; D011505:Protein-Tyrosine Kinases; D008239:Lysine