Binding of a biotinylated neurotrophic ACTH(4–9) analogue, Org 2766, to neurofilament-positive cells in primary or cell line cultures

To study the putative binding sites of the neurotrophic peptide Org 2766, an analogue of ACTH(4–9) [H-Met(O2)-Glu-His-Phe- -Lys-Phe-OH], biotinylated forms of the peptide were used. After fixation, cultures of rat spinal cord and dorsal root ganglia were incubated with 4–10 μM of 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 processes. The b-Org 2766 binding was displaceable by an excess of nonbiotinylated Org 2766. Light and electron microscopy showed that the biotinylated peptide binds to a cytoplasmatic component as well as to the cell membrane. Double-labeling experiments with b-Org 2766 and an antibody (RT-97) to a high molecular weight neurofilament protein in dorsal root ganglion cultures showed, using fluorescence and confocal scanning laser microscopy, that all b-Org 2766 binding cells were neurofilament positive. Biotinylated Org 2766 did also bind to the neuronally differentiated cells in cultures of the human neuroblastoma cell line SK-N-SH, but not to those differentiated into epithelial cells. The present data suggest that the neurotrophic peptide Org 2766 binds specifically to cell types with neuronal characteristics.