Structure-function analysis of human transforming growth factor-alpha by site-directed mutagenesis.
Site-directed mutants of transforming growth factor-alpha (TGF-alpha) were expressed in an Escherichia coli outer membrane protein A (ompA) expression/secretion vector under the transcriptional control of the lambda PL promoter. TGF-alpha mutant proteins were isolated from cell pellets using alkaline extraction with 0.1 M-Tris (pH 10.5). The levels of protein expression of 23 TGF-alpha mutants were comparable with those of wild-type TGF-alpha, as determined by immunoblotting and radioimmunoassay. An analysis of biological activity using as assays radioreceptor binding competition and colony formation in soft agar showed that the following mutations destroy the activity of TGF-alpha: Gly-19 to Val, Val-33 to Pro and Gly-40 to Val. Mutations of Arg-42 to Lys, Leu-48 to Ala, Tyr-38 to Trp or Phe-17 to Tyr significantly decrease, but do not destroy, biological activity when compared with the wild-type. Mutations in 14 other residues did not significantly alter receptor binding or colony-forming activity. These studies suggest that two domains localized at the surface of TGF-alpha are important in receptor binding and colony-forming activity. Domain I involves amino acid residues which include Tyr-38 and Leu-48; domain II includes residues Phe-15, Phe-17 and Arg-42.
Feild
J A
JA
Department of Cell Sciences, SmithKline Beecham Pharmaceuticals, Research and Development, King of Prussia, PA 19406.
Reid
R H
RH
Rieman
D J
DJ
Kline
T P
TP
Sathe
G
G
Greig
R G
RG
Anzano
M A
MA
D000595:Amino Acid Sequence; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009154:Mutation; D011487:Protein Conformation; D012689:Sequence Homology, Nucleic Acid; D013329:Structure-Activity Relationship; D016211:Transforming Growth Factor alpha / Q000235:genetics* / Q000502:physiology
D046911:Macromolecular Substances; D016211:Transforming Growth Factor alpha