TOGETHER IS BETTER: PHYSICAL INTERACTION OF NGF RECEPTORS TRKA AND P75

”. IACARUSO MF, MARTI M, VILLALTA JI, ESTRIN D, JARES-ERIJMAN EA, GALLI S, PIETRASANTA LI

Hotel Casa Serrana, Huerta Grande, Cordoba

Congreso; I reunión conjunta neurociencias (IRCN); 2009

Nerve Growth Factor (NGF) mediates survival, differentiation and maintenance of responsive neurons through activation of signalling events from the nerve terminal plasma membrane to the nucleus. NGF engages two structurally distinct transmembrane receptors, p75 and TrkA, which have been proposed to create a “high affinity” NGF binding site through the formation of a ternary NGF-p75-TrkA complex. Evidence from a variety of systems suggests that these receptors may cooperate in NGF signal transduction: the expression patterns of these two receptors overlap extensively, neurons co-expressing p75 and TrkA respond to lower concentrations of NGF than cells expressing TrkA alone, and p75 knockout mice require higher NGF concentrations than normal for survival. However, the mechanism by which these receptors produce synergistic effects has been difficult to discern. It has been proposed that p75 and TrkA form complexes, though an interaction through the extracellular domains has been discarded by crystallography studies. We determined that p75 and TrkA interact by their intracellular domains by Förster resonance energy transfer (FRET) and by computational modelling and protein interaction prediction. We observed energy transfer between the recombinant receptors fused to fluorescent proteins, TrkA-CFP and p75-YFP, in PC12 and COS-7 cells. In addition, we detected FRET among the endogenous receptors in PC12 cells by labelling p75 and TrkA with antibodies conjugated with the suitable fluorophores. We obtained an accurate structure of TrkA intracellular domain by homology modelling using MODELLER, and predicted three potential TrkA homodimers and several TrkA-p75 dimers with the program ClusPro. We examined the stability of selected dimers by molecular dynamics using AMBER force fields and calculated the relative binding free energies for different homo and hetero dimers. We propose that the cooperation in NGF signal transduction is achieved by the formation of a complex between TrkA and p75 intracellular domains.