Transforming growth factor alpha: mutation of aspartic acid 47 and leucine 48 results in different biological activities.
To study the relationship between the primary structure of transforming growth factor alpha (TGF-alpha) and some of its functional properties (competition with epidermal growth factor (EGF) for binding to the EGF receptor and induction of anchorage-independent growth), we introduced single amino acid mutations into the sequence for the fully processed, 50-amino-acid human TGF-alpha. The wild-type and mutant proteins were expressed in a vector by using a yeast alpha mating pheromone promoter. Mutations of two amino acids that are conserved in the family of the EGF-like peptides and are located in the carboxy-terminal part of TGF-alpha resulted in different biological effects. When aspartic acid 47 was mutated to alanine or asparagine, biological activity was retained; in contrast, substitutions of this residue with serine or glutamic acid generated mutants with reduced binding and colony-forming capacities. When leucine 48 was mutated to alanine, a complete loss of binding and colony-forming abilities resulted; mutation of leucine 48 to isoleucine or methionine resulted in very low activities. Our data suggest that these two adjacent conserved amino acids in positions 47 and 48 play different roles in defining the structure and/or biological activity of TGF-alpha and that the carboxy terminus of TGF-alpha is involved in interactions with cellular TGF-alpha receptors. The side chain of leucine 48 appears to be crucial either indirectly in determining the biologically active conformation of TGF-alpha or directly in the molecular recognition of TGF-alpha by its receptor.
Lazar
E
E
Laboratory of Chemoprevention, National Cancer Institute, Bethesda, Maryland 20892.
Watanabe
S
S
Dalton
S
S
Sporn
M B
MB
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics*; D002460:Cell Line; D005796:Genes; D006133:Growth Substances / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007930:Leucine / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D010957:Plasmids; D011863:Radioimmunoassay; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D014170:Transformation, Genetic; D015291:Transforming Growth Factors
D006133:Growth Substances; D010455:Peptides; D001224:Aspartic Acid; D015291:Transforming Growth Factors; D007930:Leucine