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Mutational analysis of leucine 47 in human epidermal growth factor
Author(s) -
Matsunami Risë K.,
Yette Margaret L.,
Stevens Audrey,
Niyogi Salil K.
Publication year - 1991
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.240460307
Subject(s) - epidermal growth factor , mutant , leucine , tyrosine , amino acid , tyrosine kinase , biochemistry , receptor , stimulation , biology , chemistry , microbiology and biotechnology , endocrinology , gene
Seven site‐specific mutants (including changes to other hydrophobic, charged, and heterocyclic amino acids) of leucine 47 of human epidermal growth factor (EGF) were generated by protein engineering and characterized for their activity in three assays: radioreceptor competition binding in membrane fractions, the stimulation of the EGF receptor's tyrosine kinase activity, and the stimulation of thymidine uptake in tissue culture cells. K ½ (concentration required for half maximum response) values for each of the mutants are reported in the three assays. The results show that the native leucine residue is quite important for EGF activity. Substitutions are tolerated to different degrees, depending upon hydrophobicity and size of the side chain. Substitution with ionic residues led to the most drastic reduction in activity. One‐dimensional nuclear magnetic resonance spectroscopy, at physiological pH, of several of the mutants did not detect any major structural perturbations which would account for the loss of activity. The results suggest that the side chain of leucine 47, because of its charge neutrality, size, and hydrophobicity, is highly important, although not absolutely essential for the interaction of EGF with its receptor. A striking finding was the lower (compared with wild type) V max values of the mutants in the tyrosine kinase reaction, but these low V max mutants, in cell culture experiments, were able to stimulate at high concentrations a growth response equivalent to wild type EGF.