A Mutagenic and Biological Study of Rat Insulin-Like Growth Factor-Binding Protein-2

We have undertaken a mutagenic and cell biological study of the properties of the rat insulin like growth factor binding protein-2 (IGFBP-2). This 32kDa protein belongs to a family of six high affinity IGFBPs (IGFBP 1-6) which are well characterised in both rodents and humans. In initial studies we mutated two highly conserved proline residues in the N-terminal region of IGFBP-2 to create the mutant proteins P20A-IGFBP-2 and P22A-IGFBP-2 and expressed these proteins along with wild type rat IGFBP-2 in the baculovirus/insect cell system. We found that this expression system was able to produce correctly folded, functional rat IGFBP-2 and using solution phase equilibrium binding techniques we demonstrated that the affinity of these mutated proteins for both IGF-I and IGF-II was similar to that of the wild type protein. We conclude that, although highly conserved in all IGFBP species, these proline residues are not involved in the IGF ligand binding site of rat IGFBP-2. In order to investigate the biological functions of IGFBP-2 in the regulation of IGF action we undertook experiments to establish a cell culture model in which the appropriate components of the IGF axis were present to allow such an investigation to take place. In initial experiments we screened several cell lines for the presence of cell or extra-cellular matrix associated IGFBP-2 (a phenomenon which limited reports in the literature had previously described). For one of these cell lines Clone 9 - a cell line derived from rat liver- we demonstrated clearly by Western blotting, ligand blotting and immunoprecipitation the association of IGFBP-2 with the surface of the cell line although experiments to determine the chemical nature of this interaction proved inconclusive. In addition to the demonstration of cell membrane associated IGFBP-2 we also identified the presence of IGF-I receptor on these cells and we were also able to show that these cells secrete IGF-I (but not IGF-II) into the surrounding medium. The identification of these components of the IGF axis in Clone 9 cells encouraged us in biological experiments to examine the possible modulatory role of cell-membrane associated and secreted IGFBP-2 on the modulation of IGF-I activity at the IGF-I receptor. We initially established that Clone 9 cells do indeed respond to exogenous IGF-I both in short term signalling events (acute tyrosine phosphorylation of a 180kDa protein) and in longer term metabolic/mitogenic assays as determined by MTT-formazan dye conversion. Further, we demonstrate, by the use of the IGF-I analogue des(l -3) IGF-I (which does not interact with IGFBPs but retains wild type potency at the IGF-I receptor) that IGF-I activity is inhibited both acutely and in longer term assays by the presence of IGFBP-2. We postulate that the former inhibition may be due to the association of IGFBP-2 with the plasma membrane of Clone 9 cells while the latter may be more related to the accumulation of IGFBP-2 in the conditioned medium. In order to further test and clarify this hypothesis we finally report on some preliminary experiments aimed at stably transfecting Clone 9 cells with sense or antisense IGFBP-2 constructs. Initial characterisation of mixed populations of such cells show no difference in basal growth rates between control, antisense and sense transfected cells although we observed reduced secretion of IGFBP-2 into conditioned medium by antisense transfected cells. We believe that the use of these cells will aid in the clarification of the role of IGFBP-2 in the regulation of the autocrine and perhaps paracrine role of IGF-I. In the final chapter of this thesis we expand on those areas of the current work which we feel would benefit from further research