The interaction between a growth factor and the extracellular region of its receptor is the first step in triggering the intracellular signalling pathways that induce a change in cell phenotype. Several platelet-derived growth factors (PDGFs) each bind to and activate PDGF receptor (PDGFR) alpha, a transmembrane receptor tyrosine kinase that simulates proliferation and migration in cells of mesenchymal origin. PDGFR alpha itself activates several intracellular signalling pathways. Thus, the possibility exists that different growth factors interact with PDGFR alpha in distinct ways to elicit divergent cellular effects. This possibility was investigated by examining the interactions that occur between the extracellular region of PDGFR alpha and three growth factors: PDGF-AA, PDGF-BB and vascular endothelial growth factor-A165 (VEGF-A165). The extracellular region of PDGFR alpha, and fragments thereof, were recombinantly expressed by mammalian 293-EBNA cells. The affinities of the protein fragments for each of the three growth factors were assayed by solid phase binding analysis. Each growth factor had a different affinity both for the extracellular region of PDGFR alpha and the fragments of that region, indicating that they differentially interact with the receptor.These different growth factor/receptor interactions were further investigated by substituting charged amino acids for uncharged residues in the putative ligand binding domain of PDGFR alpha. The amino acids selected for substitution were analogous to residues that had been shown to form direct contacts with growth factors in growth factor/receptor systems that are evolutionarily related to PDGFR alpha. Mutants of the PDGFR alpha ectodomain were generated by site-directed mutagenesis, and their growth factor binding properties were subsequently assayed. Particular mutants had differential effects on growth factor binding to PDGFR alpha. For example, the K209A mutant had no effect on PDGF-AA binding to the receptor, compared to wild-type, but it had a lower affinity for PDGF-BB and a greater affinity for VEGF-A165, compared to wild-type.These data demonstrated that the examined growth factors form subtly different interactions with PDGFR alpha. Thus, it is likely that each growth factor induces a distinct conformational change in the receptor upon binding. In this way, different growth factors may elicit divergent cellular effects through the same receptor.
|Date of Award||1 Aug 2012|
- The University of Manchester
|Supervisor||Catherine Kielty (Supervisor)|