Fluorescence Resonance Energy Transfer Reveals Key Binding Domains of Neurotrophin Receptor-Interacting Melanoma-Associated Antigen Homolog in Bone Morphogenetic Protein-Mediated Apoptosis

Apoptosis, one form of programmed cell death, is used by tissues to develop normally and maintain homeostasis. Lack of apoptosis underlies many diseases such as cancer, while an excess can cause neurodegenerative disorders. Understanding the molecular events that initiate either condition is necessary for the development of treatments. Bone morphogenetic proteins (BMP) play profound roles in development, such as regulation of neural progenitor apoptosis and glial differentiation. Binding of ligand to the BMP receptors triggers the canonical and non-canonical pathways involving Smad and TAK1 activation respectively. In the non-canonical pathway, p38 mitogen activated kinase (p38MAPK) is upregulated in P19 cells, a model line of neural progenitors, triggering a signal cascade leading to apoptosis. X-linked inhibitor of apoptosis protein (XIAP) functions as a positive mediator linking TAK1 to the BMP receptors through TAB1. Neurotrophin receptor-interacting MAGE (NRAGE) homolog binds with XIAP in the XIAP-TAB1-TAK1 complex and is necessary in the non-canonical pathway for apoptosis. We have measured fluorescence resonance energy transfer (FRET) between enhanced green fluorescent protein attached to NRAGE deletion mutants and DsRed-monomer attached to XIAP. Results show that the interaction is direct and is facilitated by a unique tryptophan-glutamine-X-proline-X-X (WQXPXX) repeat in NRAGE. We have continued using FRET with enhanced cyan and yellow fluorescent proteins (FPs) in designs with the FP fused to the amino or carboxy termini of NRAGE deletion mutants and XIAP to compare the FRET efficiencies and verify that the FP placement does not affect the NRAGE-XIAP interaction. Additionally, we compare the FRET of overexpressed NRAGE-XIAP with that of the endogenous interaction using antibodies conjugated to Alexa Fluors.