(518c) Probing Human Transferrin-Receptor Interactions Using Linear Epitope Mapping

Transferrin (Tf) is a carrier protein involved in the transport of iron (Fe3+). Tf binds to transmembrane transferrin receptors (TfRs) expressed on cell surfaces and transports Fe3+ by receptor mediated transcytosis (RMT). The Tf-TfR complex is an interesting system as it provides a natural biological pathway for delivery of Tf-bound cargo across cells and tissues. It has also been demonstrated that cancer cells over-expressing TfR can be selectively targeted with Tf-conjugated cytotoxic drugs. Understanding the interactions between Tf and TfR is therefore valuable for designing better therapeutic modalities for a broad range of physiological diseases. A few studies have highlighted putative amino acid residues involved in the formation of the Tf-TfR complex. The crystal structures of the N-lobe of Tf and the ectodomain of TfR have also been resolved independently. We propose to use peptide microarrays for epitope mapping of the Tf-TfR complex to probe the interactions between Tf and TfR. Linear epitope mapping has been widely used as a technique for identifying antigenic regions in proteins in a fast, reliable high-throughput fashion. We have employed peptide microarrays of short peptides that are linear, continuous fragments of TfR to capture the interactions between TfR and its ligand. Microarrays printed with peptide fragments that span the entire sequence of TfR were tested against Tf to screen for regions on TfR that are involved in binding to Tf. Our results have confirmed the prior findings on the Tf-TfR interaction and complex formation. Additionally, in order to probe what regions of Tf interact with TfR we have prepared microarrays of peptides that are short fragments of Tf. We are currently conducting cell-based screening assays where these microarrays are incubated with varying densities of live cells over-expressing TfRs on their surface. Not only will this cell-based screening technique provide insight into the regions of Tf involved in interaction with TfR but also serve as a platform for probing ligand-receptor interactions of other cell-surface receptors. We anticipate that our studies along with the existing knowledge about the Tf-TfR system will further advance the understanding about this important transport and regulatory pathway.