The initial interaction of a polypeptide hormone, growth factor, or cytokine with its plasma membrane receptor elicits specific molecular recognition and signal transduction. This proposal deals with the interaction of the neurotrophin family, particularly nerve growth factor (NGF), with the Trk receptor family, particularly TrkA, and the influence of all subdomains of the extracellular domain on ligand binding. The overall objective of this proposal is to define the elementary interactions that determine the ability of a Trk receptor to bind a neurotrophin and initiate signaling. The hypothesis being tested is that multiple subdomains of the extracellular domain of the TrkA receptor simultaneously interact with numerous distinct regions of NGF.
The specific aims are: to determine the quantitative contribution of each extracellular subdomain of TrkA to the kinetics and equilibrium of NGF binding; to determine the enthalpy and entropy of interaction between NGF and the critical binding subdomain(s); to deduce the extent of the conformational change upon ligand binding from microcalorimetric data; to determine if subdomains of the TrkA extracellular domain are capable of dimerization; and to determine the role of the arrangement and spacing of the subdomains on binding and signaling within cells. These studies will be done with the purified receptor extracellular domain (RED) of TrkA, and various mutants thereof, and utilize biophysical instrumentation including a surface plasmon resonance biosensor and a microcalorimeter to assay alterations in the kinetics and thermodynamics of binding of the neurotrophins to mutational variants of Trk-REDs, as well as cell signal transduction assays. The results should provide new general insights into ligand-receptor interactions, the basis for receptor specificity, and the initiation of signaling in tyrosine kinase receptors. Understanding the molecular mechanism of neurotrophin-Trk receptor interactions will provide the basis with which allow the design of therapeutic reagents that could eventually lead to novel treatments of Alzheimer's Disease, Parkinson's Disease, related neurological disorders, and childhood neuroblastomas. Delineating the specificity, molecular basis, and dynamics of neurotrophin binding to the Trk receptor extracellular domain will help exploit the meaningful and rational use of soluble receptors, neurotrophins, or their analogs in these disease states.
