Molecular mechanism of TrkA activation and pain - Project Summary/Abstract The binding of nerve growth factor (NGF) to the receptor tyrosine kinase (RTK), TrkA is critical for neuronal survival, growth, maintenance, and pain perception. Mutations in the TrkA:NGF signaling axis result in neuronal defects and pain imperception found in populations with Hereditary Sensory and Autonomic Neuropathies (HSAN). However, those with a subtype of this condition, HSAN-V, have a point mutation in NGF (NGFpainless) that preserves neurological function but show a loss of pain acuity. The molecular mechanism by which NGF regulates TrkA, and how the mutant NGFpainless alters or biases downstream signaling to guide physiological outcomes, remains elusive. I hypothesize that NGFpainless-mediated disentanglement of neurotrophic and pain signaling is regulated by the structural dynamics and/or the dimer lifetime of the TrkA:NGF-signaling competent complex, which biases downstream effectors and signaling away from pain. I will approach this hypothesis in three aims. In Aim 1, I will employ a structural approach (cryo-EM) to study intact TrkA:NGF/NGFpainless 2:2 complex to identify potential differences in structural or conformational states that may bias downstream signaling. I have already obtained high-resolution structural information for TrkA:NGF and TrkA:NGFpainless. I will also use 3D variability analysis (3DVA) to visualize the heterogeneity underlying the dynamic landscape of these complexes. In Aim 2, I will study the organization and dynamics of TrkA:NGF/NGFpainless in the context of its native membrane environment using single-molecule microscopy. Using Native-nanoBleach, a technique developed in the Bhattacharyya lab that uncovers the oligomeric distribution of protein complexes at equilibrium, I have determined that there is a decrease in the population of dimers/multimers in TrkA:NGFpainless when compared to TrkA:NGF. I will further use single-particle tracking (SPT) to study how varying NGF conditions affect dimer lifetime of the TrkA signaling complex on native, live cell membrane. In Aim 3, I will investigate any changes in phosphorylation and activation status of known downstream effectors of TrkA by quantitative western blots, targeting PLCγ1, ERK, and AKT. My preliminary data shows that there is a significant decrease in PLCγ1(pY783) in SHSY5Y neuroblastoma cells stably expressing TrkA when treated with NGFpainless versus NGF for 15 min. I will also measure these changes over a time course where I will incubate the cells with NGF variants for upto 60 min. In an alternate approach for this aim, I will use unbiased, mass spectrometry-based phosphoproteomics to identify (and quantify) changes in the global phosphorylation network regulated by the TrkA:NGF pathway when treated with NGFpainless. Together, these studies will allow me to understand the molecular basis for how NGFpainless, bearing only a single- point mutation to NGF, biases TrkA-downstream signaling away from pain perception, ultimately providing new ideas and directions to exploit this pathway either for chronic pain management or improved neurogenesis.
