Doctoral Dissertations

Date of Award

12-1997

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

Salil K. Niyogi

Committee Members

Audrey Stevens, John S. Cook, John W. Koontz, Stephen J. Kennel

Abstract

The solution structure of human epidermal growth factor (hEGF), as determined by nuclear magnetic resonance (NMR) spectroscopy, indicates that L15 and H16 of hEGF are solvent-exposed and are in spatial proximity to other residues implicated in receptor recognition and activation. Therefore, they may be involved in receptor-ligand interactions. Although site-specific mutagenesis does not indicate an essential role for the imidazole functionality of H16 in mediating biochemical and biological activity, optimal receptor recognition may rely on hydrogen-bond donor/acceptor interactions involving a polar, isosteric side-chain at this site. Furthermore, the increased relative affinity (272%) and agonist activity (263%) of the H16Q mutant suggest that H16 could be a target for the engineering of superagonists with therapeutic potential. The geometry and hydrophobicity of the γ-branched L15 side-chain of hEGF promote formation of a catalytically active receptor-ligand complex. The L15A mutant partially impairs allosteric activation of the EGFR in a (Glu4,Tyr1)n substrate phosphorylation assay and is also defective in stimulating receptor dimerization / oligomerization in vitro. Gross structural perturbations are not evident in the 1H-NMR spectrum of L15A compared to wild- type, thereby indicating a functional role for L15 of hEGF in receptor-ligand interactions. Stimulation of murine keratinocytes with either the partial agonist (L15A) or wild- type hEGF reveals a lack of linear correlation between the activation of p42 MAP kinase and levels of receptor autophosphorylation. The percentages of cells which proliferate in response to 1 nM L15A and their rate of entry into S phase are both decreased relative to the response elicited by 1 nM wild-type. Higher concentrations of L15A reverse this effect, showing that LISA and wild-type differ in the number of receptors that they must activate to induce the threshold response. Biological thresholds may be attained by cooperative activation of pre-existing receptor dimers / oligomers by van der Waal's weak forces of attraction. The receptor reserve in intact cells thus points to an elegant strategy in cell survival, which ensures the mitogenic response to receptor activation by both L15A and wild-type hEGF. This study shows that L15 of hEGF is important for the reception and relay of mitogenic signals by the epidermal growth factor receptor (EGFR).

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