BINDING OF A BIOTINYLATED NEUROTROPHIC ACTH(4-9) ANALOG, ORG-2766, TONEUROFILAMENT-POSITIVE CELLS IN PRIMARY OR CELL-LINE CULTURES

To study the putative binding sites of the neurotrophic peptide Org 27 66, an analogue of ACTH(4-9) [H-Met(O2)-Glu-His-Phe-D-Lys-Phe-OH], bio tinylated forms of the peptide were used. After fixation, cultures of rat spinal cord and dorsal root ganglia were incubated with 4-10 muM o f biotinyl-Org 2766 (b-Org 2766). Binding of both N- and C-terminally biotinylated Org 2766 was seen to phase-bright, round cells with thin processes, but not to flat, orthogonal-shaped cells with tapering proc esses. The b-Org 2766 binding was displaceable by an excess of nonbiot inylated Org 2766. Light and electron microscopy showed that the bioti nylated peptide binds to a cytoplasmatic component as well as to the c ell membrane. Double-labeling experiments with b-Org 2766 and an antib ody (RT-97) to a high molecular weight neurofilament protein in dorsal root ganglion cultures showed, using fluorescence and confocal scanni ng laser microscopy, that all b-Org 2766 binding cells were neurofilam ent positive. Biotinylated Org 2766 did also bind to the neuronally di fferentiated cells in cultures of the human neuroblastoma cell line SK -N-SH, but not to those differentiated into epithelial cells. The pres ent data suggest that the neurotrophic peptide Org 2766 binds specific ally to cell types with neuronal characteristics.