Mobility, clustering, and transport of nerve growth factor in embryonal sensory cells and in a sympathetic neuronal cell line.

We have prepared a fluorescent conjugate of nerve growth factor (NGF) containing 8--10 rhodamine molecules attached to free carboxyl groups of the protein. This analogue retained full binding capacity toward NGF receptors, full antigenic properties, and the potency to stimulate the differentiation of embryonal chicken sensory ganglia cells in vitro. We have used this analogue to study the mobility and distribution of NGF receptors on embryonal chicken sensory cells from dorsal root ganglia and on a pheochromocytoma cell line (PC-12) that responds to NGF by differentiating along a neuronal pathway. The rhodamine conjugate of nerve growth factor (R-NGF) binds initially to diffusely distributed mobile receptors (D approximately 8 X 10(-10) cm2/sec) on immature sensory and PC-12 cells. At 37 degrees C, the NGF receptor complexes cluster and form immobile visible patches. These patches undergo endocytosis in a process that consumes metabolic energy. Methylamine blocks the formation of visible patches of NGF and the receptors remain dispersed and mobile at 37 degrees C. On differentiated chicken sensory cells, R-NGF binds to diffusely distributed mobile receptors and to aggregated immobile binding sites. These clusters are localized at the tip of the axon, along the axon, and in the main body. The NGF molecules that are internalized at the tip of the axon are transported retrogradely from the peripherey to the cell body.