The implication of the transmembrane signalling Receptor Tyrosine Kinases
(RTKs) in cancer has accelerated the pursuit for drugs to target these
molecules. In the process our understanding of how these membrane bound
molecules are entangled in cell signalling has significantly expanded. There is
now evidence that RTKs can facilitate the formation of a lattice-type network of
signalling molecules to elicit whole cell responses to external ligand stimuli.
Although beginning to be unravelled, knowledge pertaining to the mechanisms
of molecular control that initiate these signalling pathways is still in its infancy.
In this thesis, a random mutagenesis approach allowed the identification of the
crucial interaction surfaces between membrane-bound EphA3 and its
preferential binding partner ephrinA5, that are required to induce the formation
of higher-order Eph signalling complexes. Modelling and experimental
dissection of this co-ordinated receptor aggregation has provided detailed
insights into the molecular mechanisms of Eph receptor activation, which in
some aspects may also apply to other members of the RTK family. In particular,
the importance of certain molecular interfaces in determining preferential and
non-preferential Eph/ephrin interactions, suggests their role in the selection of
biologically important binding partners.
In addition to the assignment of the ephrin-interaction surfaces, the random
mutagenesis strategy also identified a continuous conformational epitope as
binding site for an anti-EphA3 monoclonal antibody. Fortuitously, antibody
binding to this site functionally mimics ephrin stimulation of EphA3 positive cells,
and in particular together with divalent ephrinA5, yields synergistically enhanced
EphA3 activation. Elucidation of the underlying mechanism has provided
opportunities to develop an efficient EphA3 targeting mechanism that is based
on increased affinity and accelerated ephrinA5 uptake as consequence of this
unique activation mechanism. On a genetic level, novel oligonucleotide
analogues known as Peptide Nucleic Acids (PNAs) were analysed for their
ability to sterically inhibit EphA3 DNA transcription and suggest a dosedependent
downregulation of EphA3 expression, in malignant melanoma cells.
Combined, ephrinA5, the anti-EphA3 MAb (IIIA4) and PNA, offer the possibility to investigate the specific machinery involved in Eph receptor expression and
signalling for the specific targeting of EphA3 expressing tumour cells.
| Identifer | oai:union.ndltd.org:ADTP/216513 |
| Date | January 2005 |
| Creators | Vearing, Christopher John, chris.vearing@med.monash.edu.au |
| Publisher | Swinburne University of Technology. |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://www.swin.edu.au/), Copyright Christopher John Vearing |
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