The functional importance of Leu15 of human epidermal growth factor in receptor binding and activation |
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Authors: | Nandagopal, Krishnadas Tadaki, Douglas K. Lamerdin, John A. Serpersu, Engin H. Niyogi, Salil K. |
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Affiliation: | 2Department of Biochemistry, Cellular and Molecular Biology, University of Tennessee Knoxville, TN 37796, USA Protein Engineering and Molecular Mutagenesis Program and University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences, Biology Division, Oak Ridge National Laboratory PO Box 2009, Oak Ridge, TN 37831-8080 |
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Abstract: | The biological importance of Leu15 of epidermal growth factor(EGF) is suggested by its conservation through evolution, itscritical location in the domaindomain interface of EGFand its close proximity to Arg41, a residue that is crucialfor receptor binding and activation. Mutagenesis of Leu15 ofhuman EGF (hEGF) was employed to examine the role of this residuein the ligand-receptor interaction. The relative receptor affinitiesof the hEGF variants, as determined by radioreceptor competitionassays, varied depending on the amino acid substitution. TheL15F, L15W and L15V hEGF analogues had receptor affinities 45,26 and 18% respectively of wild type hEGF. The L15A and L15Ranalogues displayed receptor affinities of only 2.4 and 1.6%relative to wild type hEGF. No binding of the L15E analoguewas detected. The relative agonist activities, as measured byreceptor tyrosine kinase stimulation assays, generally followeda similar trend. The L15F, L15W and L15V analogues stimulatedthe receptor kinase to a level (Vmax) similar to that for wildtype hEGF. A striking difference was observed between the L15Aand L15R variants; although having similar binding affinities,the L15A mutant activated the receptor to only {small tilde}5%of the wild type Vmax in contrast to 53% for the L15R mutant1H-NMR analysis of the L15R and L15A mutants showed only minorstructural alterations that were not sufficient to account forthe dramatic losses in binding and agonist activities. The resultsindicate that both the size and hydrophobicity of the -branchedaliphatic side chain of Leu15 of hEGF are important in the formationof a catalytically active ligandreceptor complex. |
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Keywords: | Leu 15 of human EGF/ mutagenesis/ NMR analysis/ partial agonist/ receptor binding and activation |
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