Examination of ligand receptor interactions and resulting function using nerve growth factor homodimeric and heterodimeric muteins.

摘要

The development and maintenance of the nervous system is largely dependent on a conserved family of proteins called neurotrophins. The nerve growth factor (NGF) is the founding member of the neurotrophin family. It is a non-covalently bound dimer that binds to two distinct receptors; TrkA (tyrosine kinase receptor family) and p75 (TNF receptor family). NGF supports survival and differentiation through these receptors and can also induce apoptosis through p75 alone. NGF exhibits high (∼10 pM) and low affinity (∼1 nM) binding sites on cells expressing these receptors. The individual receptors do not generally exhibit high affinity binding. The high affinity site is observed on cells expressing both receptors and thus is believed to be made up of a heteroreceptor complex. The picomolar affinity observed with the high affinity site plays a significant role in vivo, due to the scarce availability of NGF, especially during development. The NGF signals via its receptors by binding two receptors at two identical surfaces present at the dimer interface. Thus NGF induces receptor dimerization, by binding one on either side.;In chapter 1, we have studied the mechanism of action for a novel mutein 7-84-103 (F7A, H84A, R103A), which demonstrated a selectiveness of survival over wtNGF in earlier experiments. The purified mutein gave a similar effect as was observed with the unpurified supernatant extracts. This mutein showed a selectiveness for survival with an EC50 of 0.95 nM for survival compared to an EC50 of 10 nM for differentiation. The selectiveness for survival was less dependent on binding via affinity to TrkA, than differentiation. Signal transduction analysis revealed that the selectiveness for survival was largely dependent on selective activation of Akt over MAPK. Activation of Akt is responsible for induction of survival and that of MAPK for differentiation. Thus the 7-84-103 mutein selectively activates the survival pathway and thus promoting survival over differentiation.;The exact nature of the high affinity site has been a point of controversy for decades. Two models exist to explain the nature of the high affinity. The first model proposes that p75 interacts with TrkA and presents TrkA to NGF as a high affinity receptor. The second model proposes a ligand passing model, wherein the NGF initially binds p75, gets concentrated at the cell surface and then gets transferred to TrkA, via a transient heteroreceptor complex mediated by simultaneous binding to NGF. In Chapter 2, we developed a heterodimer of two NGF muteins (heteromutein): 7-84-103 and Delta9/13. This heterodimer has the ability to bind p75 on one side and TrkA on the other, but not two TrkA receptors. The heteromutein thus allowed us to study the heteroreceptor complex binding and function, in the theoretical absence of NGF induced TrkA dimerization. Binding studies with both surface plasmon resonance and cellular binding indicated that the heteromutein does not bind TrkA efficiently, but has better affinity than either of the parent muteins. The functional bioassays also show a reduced response for neurite outgrowth compared to wtNGF, but better than the parent muteins. The heteromutein, 7-84-103 and &