STRUCTURE-FUNCTION DYNAMICS OF NGF RECEPTORS

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There is a fundamental tripartite relationship between a proteins's structure, its function, and its ability to diffuse relative to the molecules with which it must interact. This proposal concentrates on this relationship for the nerve growth factor receptor (NGFR) which plays an important role in neural development and repair. NGFR exists in both high- and low-affinity forms. The low copy number high-affinity form is required for function. Recently, a second component, gp140trk, has been identified, which is essential for high-affinity binding. We have shown that gp75 is homogeneously distributed and diffuses freely on nonresponsive cells, but is aggregated and immobilized on responsive cells. Our hypothesis is that gp140trk complexes with gp75 to form the high affinity NGFR and is responsible for immobilizing and aggregating gp75 on responsive cells. To test this hypothesis we will ask: 1. What are the diffusion and distribution properties of gp140trk? a. How does gp140trk distribution and diffusibility relate to responsiveness? b. Does addition of NGF alter the distribution or diffusion of gp140trk? c. What structures are associated with gp75 and/or gp140trk complexes in responsive cells? d. Are the diffusion properties of high- and low-affinity NGFR gp75 and gp140trk different. Distribution of the components of gp140trk will be studied by fluorescence microscopy. Diffusion measurements will be made using fluorescence recovery after photobleaching (FRAP). 2. Is the high-affinity NGFR a complex of gp75 and gp140trk? (a.) Does the complex pre-exist or does it form in response to NGF? (b.) Is there more than one gp75 or gp140trk in this complex? The relative amounts of NGFR and its components gp75 and gp140trk will be determined on responsive and nonresponsive cells by radioactive binding analysis. Three methods will then be used to address the question of aggregation: (1.) FRAP; (2.) receptor solubilization; and (3.) fluorescence resonance energy transfer (FRET). 3. Is gp75-gp140trk complexing responsible for the large scale aggregation of gp75 observed on responsive cells? We will further develop a new method using video imaging FRET microscopy to determine the spatial distribution of gp75-gp140trk complexes on the cell surface. 4. Which domains of gp75 and gp140trk control the function and physical properties of NGFR? Using NGFR mutants we will ask, a. Is the cytoplasmic domain of gp75 required for complex formation and gp75- gp140trk immobilization? b. Is the intracellular domain of gp140trk required for gp75-gp140trk complex formation and immobilization? c. Is a functional kinase domain required? d. Is the consensus sequence for association with coated pits required?