Role of disordered domains in dimerization and ligand binding of epidermal growth factor receptor

In cell biology protein kinases have integral role in regulating the biological mechanisms. Tyrosine kinase receptors are a large versatile group, which perform their action when bind to a ligand and have an intrinsic kinase activity. Understanding the physiological and biochemical properties of receptor protein-kinases has been the focus of many molecular biologists over a span of past three decades. Epidermal Growth Factor Receptor (EGFR) belongs to receptor tyrosine kinase family; the ligands of EGFR have been recognized as molecules of essential importance to regulate female reproduction in terms of sexual maturity, follicle maturation and embryonic implantation in the horn of uterus. Overexpression of this receptor results in generation of stronger signals which activate the further signaling cascade, consequently cells grow aggressively and metastasize in surrounding tissues and organs. Ligand binding to EGFR results in rearrangement of major domains which uncover the dimerization arm. This research was conducted to see the effect of site-specific mutagenesis on the dimerization of EGFR. To analyze the specific role of sequences in extracellular domain for dimerization pattern, four mutant EGFRs were constructed in which threonine, glutamine, lysine, aspartate were replaced with glycine, phenylalanine, glutamine and asparagine at 274,276,278 and 284th position respectively. According to the sequence analysis of our collaboration partner these mutations may result in weaker interactions of receptors (impaired dimerization) due to changes in the fuzziness (disordered structure) of certain receptor parts. For this purpose, generation of predicted mutation sites was done by site directed mutagenesis. Molecular cloning was done and mutants were inserted in pcDNA3.Transfection was carried out with lipofectamine in Chinese hamster ovary cells. After labelling of transfected CHO cells with monoclonal antibodies, confocal microscopic examination of transiently transfected cells revealed that CHO cells successfully expressed the mutant EGFRs and emphasized the fact that the mutant EGFRs were present on the cell surface like a wild type receptor.MSc/MAmolekuláris biológiaangolBiokémia-genomik