Wilson, Lynn; (1990) Expression of functional human growth factors in insect cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Peptide growth factors are important in a wide variety of physiological and pathological processes as a consequence of their ability to generate a mitogenic signal via their interaction with and activation of a specific membrane-associated receptor. The subversion of the normal growth factor signalling pathway, albeit by the enhanced expression of the growth factor or receptor, the unregulated activity of the receptor protein-tyrosine kinase or the uncoupling of intracellular pathways can lead to uncontrolled cellular proliferation resulting in cellular transformation and the formation of tumours in vivo. The major part of this thesis was devoted to the development of baculoviral expression systems producing large amounts of biologically active EGF and TGFa for the assessment of the structural and functional basis of ligand-receptor interactions. Insect cells infected with the recombinant baculoviruses expressed growth factors with the correct putative molecular weights which were immunologically indistinguishable from their authentic counterparts. The recombinant growth factors were able to bind to the EGF receptor expressed in insect cells and NR6+ fibroblasts and activate the receptor protein-tyrosine kinase activity, Furthermore, they induced a mitogenic response in NR6+ and Swiss 3T3 fibroblasts. It is anticipated that milligram quantities of these biologically important growth factors will be available for crystallisation. The elucidation of the three-dimensional structures of the growth factors by diffractional analysis of the crystals and structural mutational analysis will establish the nature of the receptor binding face. This information will lead to the design of clinically important EGF and TGFa antagonists. The recombinant ligands will allow the continued biophysical characterisation of the conformation changes occurring in the extracellular domain of the EGF receptor in response to ligand binding. Cocrystallisation will ultimately lead to the determination of the ligand binding site of the receptor. The biosynthetic precursors for EGF and TGFa are integral membrane glycoproteins suggesting that they may function to mediate physiological cell-cell recognition events. The TGFa precursor expressed in insect cells was able to stimulate the EGF receptor protein-tyrosine kinase activity. Characterisation of the precursor in insect cells will determine the relevance of this mechanism for the production of secreted growth factors and investigate further potential roles for their membrane- associated precursors. The expression of c-erbB-2 in insect cells has allowed the evaluation of a putative growth factor receptor which is highly homologous to the EGF receptor. Furthermore, an in vivo association of the EGF receptor and p185c-erbB-2 been demonstrated. This system will provide a valuable tool for the determination of the mechanism responsible for the transmembrane translocation of the mitogenic signal. It is anticipated that baculoviral coexpression of growth factor receptors and their cognate ligands, in conjunction with intracellular substrates will play an important role in the identification of their intracellular signalling pathways. The elucidation of the growth factor signalling pathway is crucial to the understanding of how oncogenic activation of anyone of the components can result in cellular transformation.

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