Doctoral Dissertations

Date of Award

5-1990

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

Salil K. Niyogi

Committee Members

Fred Hartman, Sankar Mitra, Margaret Terzaghi-Howe

Abstract

The polypeptide hormone epidermal growth factor (EGF) stimulates cell division by binding to its specific cell surface receptor and stimulating the receptor's tyrosine kinase activity. A chimeric gene, coding for human EGF fused at its amino terminal end to the signal peptide of bacterial alkaline phosphatase, was synthesized and cloned into an expression vector under the transcriptional control of the tac promoter and introduced into Escherichia coli. Induction with isopropylthiogalactoside led to the synthesis of the precursor form of the EGF polypeptide containing the signal peptide. Secretion of the correctly processed mature EGF into the periplasm was confirmed by the physicochemical properties and biological activity of the purified protein. The role(s) of several highly conserved amino acid residues in the third disulfide loop (loop C) of EGF was investigated via site-directed mutagenesis. Single amino acid substitutions of glycine-36 and glycine-39 resulted in mutant proteins with decreased receptor affinities. Preliminary evidence implies that these glycyl residues are conserved primarily for structural reasons, possibly because of their location in turn regions of loop C. Several mutant proteins with single amino acid substitutions of tyrosine-37 were evaluated for their receptor affinity and ability to stimulate the receptor's tyrosine kinase activity. With respect to these activities, amino acid substitutions were well tolerated at this position, in general favoring aromatic residues above other types, in the following order; Phe > Tyr (wild-type) > His > Ser > Ala > Asp > Arg > Gly. The results indicate that tyrosine-37 is not directly involved in receptor interaction. In contrast, arginine-41 appears to be conserved for functional rather than structural reasons. A series of single amino acid substitutions at this site all resulted in a drastic lowering of receptor affinity, including a conservative lysine substitution which lowered the receptor affinity 2.3 orders of magnitude to 0.43% that of wild-type. Structural analysis utilizing one-dimensional proton nuclear magnetic resonance spectroscopy of several EGF mutants at positions 37 and 41 was used to ascertain whether the decrease of functional activity was due to altered protein conformations induced by the mutations. The spectra obtained were particularly useful for mutations involving arginine-41, and showed that the dramatic decrease in receptor affinity was not due to drastic changes in overall structure of the mutant EGFs. The conclusions drawn from the structure-function studies of these mutant proteins are correlated with the conservation of these loop C residues in other proteins known to bind to the EGF receptor, and to conservation in EGF-like domains of various proteins with no known EGF-like function.

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