Structure-function analysis of synthetic and recombinant derivatives of transforming growth factor alpha.
Transforming growth factor alpha (TGF-alpha) is a 50-amino-acid peptide that stimulates cell proliferation via binding to cell surface receptors. To identify the structural features of TGF-alpha that govern receptor-ligand interactions, we prepared synthetic peptide fragments and recombinant mutant proteins of TGF-alpha. These TGF-alpha derivatives were tested in receptor binding and mitogenesis assays. Synthetic peptides representing the N terminus, the C terminus, or the individual disulfide constrained rings of TGF-alpha did not exhibit receptor-binding or mitogenic activity. Replacement of the cysteines with alanines at positions 8 and 21, 16 and 32, and 34 and 43 or at positions 8 and 21 and 34 and 43 yielded inactive mutant proteins. However, mutant proteins containing substitutions or deletions in the N-terminal region retained significant biologic activity. Conservative amino acid changes at residue 29 or 38 or both and a nonconservative amino acid change at residue 12 had little effect on binding or mitogenesis. However, nonconservative amino acid changes at residues 15, 38, and 47 produced dramatic decreases in receptor binding (23- to 71-fold) and mitogenic activity (38- to 125-fold). These studies indicate that at least three distinct regions of TGF-alpha contribute to biologic activity.
Defeo-Jones
D
D
Department of Cancer Research, Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486.
Tai
J Y
JY
Wegrzyn
R J
RJ
Vuocolo
G A
GA
Baker
A E
AE
Payne
L S
LS
Garsky
V M
VM
Oliff
A
A
Riemen
M W
MW
D000595:Amino Acid Sequence; D000818:Animals; D002455:Cell Division / Q000187:drug effects*; D002460:Cell Line; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D015291:Transforming Growth Factors / Q000138:chemical synthesis / Q000235:genetics / Q000494:pharmacology*
D011994:Recombinant Proteins; D015291:Transforming Growth Factors; D004262:DNA Restriction Enzymes