While other laboratories have made many mutations of NGF, our laboratory continues to characterize mutant proteins with biophysical methods in order to test the integrity of the conformation and quaternary structure after mutation. In some cases, these approaches have supported the interpretation that the receptor interactions are directly with residues in the N-terminus and particularly around residues 9-13 or lower (Woo et al, 1995). For a structure of the NGF N-terminus interacting with the d5 IgG-C2 subdomain of the TrkA receptor (as solved by Weismann, et al 1999), click here. In other cases, our structural studies have suggested that the effects on receptor binding are indirect through conformational effects or destabilization of the dimer, e.g. H75, H84, and F53 (Woo and Neet, 1996; Guo, et al, 1996).
We have prepared about 40 mutants over the past 7 years, characterized activity of more than 25 of them, and extensively characterized binding and/or biophysical properties of about 20 of these (click here for Table "NGF mutants studied in the Neet lab"). We have provided further evidence for an important biological role for the C-terminus of NGF by showing that a monoclonal antibody directed to the C-terminus of NGF is important in differential signaling through the TrkA receptor to distinct signal transduction pathways (Saragovi, et al, 1998). C-terminal extension mutations have a significant affect on biological activity (Bhat, et al, in preparation). The D9/13 mutant that had been previously shown to only bind p75NTR (Woo et al, 1995) promotes PC12 cell survival and protects against serum-deficient apoptosis but does not support differentiation or cell cycle arrest (Hughes, et al,2001), suggesting a key role for p75NTR in cell survival. These studies have positioned us to develop old or new NGF mutants with unusual and useful signaling properties that separate the signaling transduction pathways within the cell. For a model of NGF with all significant mutations and relevant citations, click here. For related references, click here.
We have prepared a recombinant TrkA extracellular domain and shown that it dimerizes in the presence of NGF and that a small conformational change occurs (Woo et al, 1998). Also, we have characterized the kinetic and equilibrium binding properties of NGF to the TrkA extracellular domain with BiaCore biosensor (SPR) technology. This receptor preparation will be extremely useful for characterizing the binding of NGF mutants.
Cell biology techniques have also been developed in order to exploit our mutants in signaling studies, since there is a great interest in distinguishing between different signaling pathways within the cell by events triggered at the cell surface receptor. This work has led to a focus on the role of p53 in signaling for cessation of proliferation and apoptosis in PC21 cells (Gollapudi and Neet, 1997). We have now generated a unique PC12 cell line that contains a dominant negative, temperature sensitive p53 gene. This cell line differentiates in response to NGF but still remains in the cell cycle to replicate DNA (Hughes, et al, 2000), showing that p53 plays a key role in NGF-regulated cell cycle progression. For a picture of cells that are in the process of differentiating as well as replicating their DNA click here.