Transforming growth factor alpha: mutation of aspartic acid 47 and leucine 48 results in different biological activities.

Abstract

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.

Substitution of lysine for arginine at position 42 of human transforming growth factor-alpha eliminates biological activity without changing internal disulfide bonds.

“…In either case, a careful structure-function analysis of TGF-a is needed to understand how to exploit the therapeutic potential of TGF-a. Prior studies from our laboratory (5) and others (13,14) have shown that nonconservative amino acid substitutions with alanines at positions His-12, Phe-15, Tyr-38, Arg-42, Asp-47, and Leu-48 decrease the receptorbinding and growth-promoting activities of TGF-a. By contrast, conservative amino acid substitutions at positions Lys-29 and Tyr-38 have little or no effect on the biological properties of TGF-a.…”

Transforming growth factor alpha: an aromatic side chain at position 38 is essential for biological activity.

“…Mutants of Tyr-38 were further purified as described previously (18). The elution profiles on a Biogel P30 column for the mutant TGF-cx proteins were identical to that shown for wild-type TGF-a in a previous paper (18).…”

“…The maximum activities in both radioreceptor and soft-agar assays were found in the same fraction, called the peak fraction. The activities were expressed in terms of EGF equivalence as reported previously (18). The peak fractions were also tested in thymidine incorporation assays and in radioimmunoassays (with the Biotope polyclonal antibody in denaturing conditions).…”

“…The sequence of the mutated gene has been determined (26) along the entire sequence of TGF-ot for each mutant. The mutated sequences were inserted into the yeast shuttle vector pyTE1 (18) and expressed in Saccharomyces cerevisiae 20B-12 (MATa trpl pep4-3) (16). Selected clones were grown to saturation, and the yeast media were dialyzed thoroughly against 1 M acetic acid in 3,000-molecular-weight cutoff dialysis tubing.…”

Replacement of N‐terminal portions of TGF‐α with corresponding heregulin sequences affects ligand‐induced receptor signaling and intoxication of tumor cells by chimeric growth‐factor toxins

“…13 Also for TGF-␣ residues from both the N-terminal and the Cterminal loops are important for receptor binding. 38,39 The Cterminal fragment of H194T20 is derived from TGF-␣ and might be functional in a manner similar to the wild-type ligand. The N-terminal binding site of H194T20 is composed of both HRG-␤1 and TGF-␣ sequences.…”