It is proposed to define in molecular terms the effect of nerve growth factor (NGF) binding to the specific NGFR and the structural changes of NGFR in response to the interaction. The signalling events associated with NGF/NGFR interaction may involve a direct action of the NGFR to generate a signal, its interaction with other plasma membrane or cytoplasmic proteins, and/or its internalization. This project will be greatly facilitated by using two cloned cultured cell lines: (i) A875 human melanoma cells which possess large numbers of NGFR and can be easily grown in large quantities. (ii) PC12 rat pheochromocytoma cells which respond to NGF with the induction of neurite outgrowth and increased activity of several enzymes. In addition, we can identify an NGFR peptide of similar Mr in both PC 12 and A875 cells by covalently attaching 125i-NGF to the NGFR using hydroxysuccinimidyl-p-azidobenzoate (HSAB). The similarity in crosslinked peptides in A875 and PC12 cells indicates the receptor is structurally similar in the two cell types. We will purify the NGFR from a plasma membrane-enriched preparation from A875 cells that have 18 pmol NGFR/mg protein, corresponding to approximately 0.25 percent of the total membrane protein being NGFR. Thus, a 400-500-fold purification of NGFR is required for homogeneity. The purified receptor will be characterized structurally and used to prepare antibodies recognizing the NGFR. The purified NGFR will be reconstituted into phospholipid vesicles and used for studies to determine the events that occur immediately after binding of NGF. Our working hypothesis is that NGF binding to the NGFR stimulates the interaction of NGFR with other cellular proteins, either membrane, cytoplasmic or cytoskeleton associated, that are involved in mediating the cascade of events regulated by NGF in responsive cells. Antibodies agains the NGFR will be used to indirectly immunoprecipitate these proteins. The proteins associated with the NGFR will be characterized in regard to their function, interaction with the NGFR and the effects of NGF binding on their interaction, subcellular localization, and fate during internalization of NGF/NGFR complexes.
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