A specific model for the functional interaction of 75 kD (p75) and tyrosine kinase (trk) neurotrophin receptors will be tested. The model proposes that the functional form of p75 is dimeric, that dimerization is regulated by phosphorylation, that neurotrophins act upon p75 and trk receptors independently, that activation of p75 by neurotrophins follows the Monod model for concerted conformational transitions, and that the active conformation of p75 preferentially binds trk dimers, facilitating trk receptor dimerization and activation. Also, the possibility that p75 facilitates retrograde axonal signaling by regulating retrograde axonal transport of active neurotrophin/trk complexes will be examined. Binding studies and biochemical studies of p75 receptors expressed in various non-neuronal and neuronal cell lines will be performed to examine the relationship of receptor dimerization, phosphorylation, and positively cooperative neurotrophin binding, and relationship of these properties to the ability of p75 to enhance trk receptor activation will be examined. In vitro mutagenesis of p75, followed by expression in non- neuronal and neuronal cell lines, will be employed to define important structural elements involved in p75 function - particularly focusing on putative regulatory phosphorylation sites in the cytoplasmic domain, and a cysteinyl residue within the transmembrane domain which may be required for generation of functional receptor dimers. The effects of p75 on intracellular trafficking of endocytic vesicles bearing neurotrophins and trk receptors will be examined, in non-neuronal cells, and in axons of neurons in culture. Immunohistochemical techniques at the electron microscopic level will be performed to characterize the distribution of p75 and trk receptors among intracellular membrane pools. Confocal fluorescence microscopy, with pH-sensitive dyes, will be performed to determine the timing of acidification of endosomes within axons of chick embryo sympathetic and sensory axons in culture. This information, coupled with the results of examining the pH-dependence of neurotrophin binding to receptors, will reveal whether functionally active neurotrophin/receptor complexes may be transported intact from the nerve terminus to the neuronal cell body in order to convey retrograde trophic signals. These studies will characterize processes which are fundamental to the mechanisms by which neurotrophic factors shape the developing brain and peripheral nervous system, and by which regeneration of the injured nervous system is encouraged.
