Resent Past Studies

While other laboratorieshave made many mutations of NGF, our laboratory continues to characterizemutant proteins with biophysical methods in order to test theintegrity of the conformation and quaternary structure after mutation.In some cases, these approaches have supported the interpretationthat the receptor interactions are directly with residues in theN-terminus and particularly around residues 9-13 or lower (Wooet al, 1995).

For a structure of the NGF N-terminus interactingwith the d5 IgG-C2 subdomain of the TrkA recptor (as solved byWeismann, et al 1999), click here. In other cases, our structural studies have suggestedthat the effects on receptor binding are indirect through conformationaleffects or destabilization of the dimer, e.g. H75, H84, and F53(Woo and Neet, 1996; Guo,et al, 1996).

We have prepared 33 mutants over the past 7years, characterized activity of about 25 of them, and extensivelycharacterized binding and/or biophysical properties of 16 of these(click here for Table "NGF mutants studied in the Neet lab").We have provided further evidence for an important biologicalrole for theC-terminus of NGF by showing that a monoclonal antibodydirected to theC-terminus of NGF is important in differentialsignaling through the TrkA receptor to distinct signal transductionpathways (Saragovi, et al, 1998).C-terminal extension mutations have a significant affect on biologicalactivity (Bhat, et al, in preparation). The D9/13 mutant thathad been previously sown to only bind p75NTR (Wooet al, 1995) promotes PC12 cell survival and protects againstserum-deficient apoptosis but does not support differentiationor 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 mutantswith unusual and useful signaling properties that separate thesignaling transduction pathways within the cell. For a model ofNGF with all significant mutations and relevant citations, click here. For related references, click here.

We have prepareda recombinant TrkA extracellular domain and shown that it dimerizesin the presence of NGF and that a small conformational changeoccurs (Woo et al, 1998). Also, wehave characterized the kinetic and equilibrium binding propertiesof NGF to the TrkA extracellular domain with BiaCore biosensor(SPR) technology. This receptor preparation will be extremelyuseful for future crystallization studies and for characterizingthe binding of NGF mutants.

Cell biology techniqueshave also been developed in order to exploit our mutants in signalingstudies, since there is a current great interestin distinguishingbetween different signaling pathways within the cell byeventstriggered at the cell surface receptor. This work has led to afocus on the role of p53 in signaling for cessation of proliferationand apoptosis in PC21 cells (Gollapudiand Neet, 1997). We have now generated a unique PC12 cellline that contains a dominant negative, temperature sensitivep53 gene. This cell line differentiates in response to NGF butstill remains in the cell cycle to replicate DNA (Hughes,et al, 2000), showing that p53 plays a key role in NGF-regulatedcell cycle progression. For a picture of cells that are in theprocess of differentiating as well as replicating their DNA click here.