Doctoral Dissertations

Date of Award

5-1995

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

Salik K. Niyogi

Committee Members

John S. Cook, K. Bruce Jacobson, Peggy Terzaghi-Howe

Abstract

Epidermal growth factor (EGF) is a small peptide mitogen that upon binding to its specific cell-surface receptor initiates a signal transduction cascade. An early nuclear magnetic resonance (NMR) study of EGF identified a clustering of aromatic residues on the surface of the protein (Mayo et al, 1986). It was suggested that this aromatic cluster provided a hydrophobic surface through which the formation of the receptor-ligand complex was mediated. Therefore, it was of interest to aromatic residues roles of the conserved aromatic residues of EGF in receptor binding Five conserved in EGF at positions 13, 22, 29, 37, and 44. A previous study from this laboratory demonstrated that the tyrosine at position 37 was not required for receptor binding and was probably a structural residue. Site-directed mutagenesis was employed to analyze the structure/function of the other four tyrosines in human EGF (hEGF)

The tyrosine at position 13 of BGF has been implicated as playing a role in receptor binding due to its close proximity to the critical arginine 41 residue as well as its high degree of conservation in EGF and EGF-like proteins bind to the BGF receptor. Site-directed mutagenesis of Tyr13 in EGF was employed to examine the role of this residue in ligand-receptor interaction and receptor tyrosine kinase stimulation. The effects of removal of the hydroxyl moiety of the tyrosine analyzed by substitution of the tyrosine with phenylalanine did not affect receptor binding indicating that it is not involved in any crucial hydrogen bonds with either the receptor or with other regions of the EGF molecule. The substitution of the aromatic tyrosine side-chain with nonpolar leucine side-chain caused the receptor affinity to decrease only slightly, indicating that aromaticity of the also not critical. Substitutions with other hydrophobic residues, isoleucine, valine, and alanine, resulted in significant decreases in receptor affinity a function of decreasing hydrophobicity. Substitution of Tyr 13 with the polar residues histidine and arginine markedly decreased receptor binding affinity, and complete removal of the side-chain by substitution with glycine dramatically lowered the binding affinity to 0.3% as compared to wild type. The relative agonist activity as measured by the tyrosine kinase stimulation assay showed a trend similar to that seen with the radioreceptor competition assay. Analysis of three EGF mutants, Tyr13→Leu, Tyr13→Arg, and Tyr13→Gly, by circular dichroism (CD) showed that the major structural features of hEGF were not significantly altered. These results demonstrate that the decreased binding affinities of hEGF matanis due disruption of the functional contribution(s) of the tyrosine 13 residues rather than alteration(s) in the overall structural integrity. The results suggest that the hydrophobicity of the Tyr13 side-chain may be the critical characteristic of this residue despite the highly conserved aromatic nature of the amino acid at this site of the EGF protein

The aromatic moiety of the residue at either position 22 or 29 in the major anti-parallelB-sheet of EGF is fairly well conserved in all EGF species. Two mutations, Tyr22→Asp andTyr29→Gly, were generated in an earlier study and showed decreased binding affinity to 17% and8%, respectively, of wild-type. This early study suggested a possible structural role for these residues in the maintenance of a proper tertiary conformation (Engler et al., 1988, Campion etal., 1990). The following single-site substitutions made at both the 22 and 29 positions: Phe,Leu, Ala, and Lys. The relative binding affinities of these mutants examined by radioreceptor competition assay. The results show that all the mutants retain significant activity compared with wild-type hBGF. Tyrosine kinase stimulation by these mutants also demonstrated no significant loss in relative agonist activity. In addition, substitutions with Tipand Pro at the 22 position exhibited little change in the relative binding affinity or agonist activityThe data suggest that tyrosines 22 and 29 are not involved in direct receptor binding due the that a large variety of substitutions tolerated at bothOne other substitution at the29 site, Tyr29→Pro, showed dramatic losses in binding and kinase stimulatory activity. Three dimensional computer modeling and HPLC elution profiles suggest that the intolerance of the Tyr22→Asp, Tyr29→Gly, or Tyr29→Pro mutations could be due to the disruption of the local structure caused by charge repulsion by a cluster of acidic residues in the case of the Tyr22→Asp analogue and changes in the B-sheet structure by the Tyr29→Gly and Tyr29→Pro mutants.

Residue 44 is conserved as a tyrosine, histidine or threonine in the EGF family. Mutagenesis of this residue indicates that neither aromaticity nor hydrophobicity is required for receptor binding. A significant loss in binding affinity was seen with only one mutant, Y44K, which may be due to structural disturbances caused by charge repulsion with the neighboring Arg45.

Computer modeling studies have permitted visualization of the hEGF molecule in three dimensions and is important for bridging the gap from experimental data to rational drug design. Mutagenesis studies have identified several residues that are important for receptor binding and modeling studies have shown that all of these residues exist on one surface of the EGF molecule. This information has aided us in the formulation of experiments examining the effects of double- site substitutions. The model, with inputs from the positions of electrostatic residues, shows a putative divalent metal binding pocket in the N-terminal domain of the protein. Preliminary studies of metal binding by electron paramagnetic resonance (EPR) and NMR demonstrate the capacity of hEGF to bind Mn2+; with high affinity and the existence of two or more divalent metal binding sites that act in a positively cooperative manner.

Download
Files over 3MB may be slow to open. For best results, right-click and select "save as..."

Share

COinS