MECHANISM(S) OF ACTION OF NERVE GROWTH FACTOR

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The importance of nerve growth factor (NGF) in the development and maintenance of the nervous system is well documented. While much is known about its chemistry and structure and about the biological processes in which NGF participates, little progress has been made toward understanding the mechanism(s) by which NGF acts. The overall objective of this proposal is to investigate two major questions in hormone-receptor biochemistry with respect to NGF: (I) is the internalization and subsequent processing of an NGF-receptor complex necessary for elicitation of the physiological responses to this hormone (i.e., NGF)? and (II) can a hormone, in this case NGF, exert its physiological responses if it is introduced into a target cell by a mechanism which by-passes its native receptor (the NGF receptor)? The experimental approach will take advantage of "chimeric toxin technology", a new and powerful methodology, which will be used in this project for the selection of cell variants with altered NGF-receptor functions. A rat pheochromocytoma-derived clonal cell line, PC-12, which responds to NGF by extensive neurite outgrowth and acquisition of neuron-like differentiated properties will be used as a tissue culture model system for these studies. To answer the first question, a "chimeric" or hybrid conjugate consisting of NGF and purified, highly-toxic ricin A chain will be constructed. This receptor-specific conjugate (NGF-A) will be used to select variant PC-12 lines (clones) which are resistant to killing by toxin. Two resulting variant phenotypes, NGF-receptor negative (R-) and NGF-receptor positive-internalization negative (R+I-), will be distinguished by standard receptor-binding assays. Internalization-negative variants (R+I-) will then be tested to determine if NGF binding alone is sufficient to elicit responses to NGF. To answer the second question, a "chimeric" conjugate (NGF-B) consisting of NGF and the purified B chain of ricin, the non-toxic galactose-binding subunit will be constructed, and used to introduce NGF into (R-) cells via ricin receptors. The NGF-B conjugate will then be tested on the receptor-negative variants (R-). These clones will be used to directly test for the importance of the NGF-receptor in cellular responsiveness to NGF, by assaying for NGF-dependent phenomena such as neurite outgrowth and known pleiotypic responses.