Nerve growth factor is an essential growth regulatory polypeptide which promotes the survival and differentiation of immature neuroblasts in the developing nervous system. Unlike most growth hormones which stimulate cell replication, NGF inhibits cell proliferation and induces the maturation of cells bearing specific NGF receptors. Although many of the morphological and biochemical effects which occur following NGF binding have been described in detail, the molecular mechanisms by which NGF elicits these effects are still not well understood. The physiological effects of NGF on selective neuronal populations in both the central and peripheral nervous system result from its initial interaction with a specific, high affinity cell surface receptor. Recent experimental evidence suggests that the high affinity receptor requires co-expression of two low affinity binding proteins, the low affinity NGF receptor, p75, and the trk proto-oncogene product, p140, a receptor tyrosine kinase. Binding of NGF to the high affinity receptor then results in the tyrosine phosphorylation of p140, and subsequent internalization of the receptor complex. The identification of a receptor tyrosine kinase which conveys a differentiative rather than a mitogenic signal provides a unique opportunity to define specific mechanisms controlling growth and differentiation. Furthermore, this receptor motif, in which a tyrosine kinase receptor interacts with another low affinity binding protein to form a high affinity complex is unique, as most receptor tyrosine kinases function independently of other binding proteins. The goal of the studies proposed below is to characterize the interactions of the two receptor subunits and assess their roles in signal transduction. First, we will define the subunit composition of the high affinity receptor complex using kinetic analysis of NGF binding, chemical crosslinking and sucrose gradient analysis. We will verify the results obtained from biochemical studies, using mutational analysis of each receptor subunit to define the domains of each subunit which are required for high affinity site generation. We will then examine in detail two functional consequences of high affinity receptor binding, by studying the initial signalling event, p140 tyrosine phosphorylation, and receptor internalization, which is required for the long term differentiative function of NGF. Specifically we will determine the domains of p75 and p140 which influence p140 tyrosine phosphorylation, and we will define the role of each receptor subunit in mediating internalization and retrograde transport of the ligand-receptor complex to the cell body.
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