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-D-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 microM 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.
van Huizen
F
F
Organon International BV, Department of Neuropharmacology, Oss, The Netherlands.
Philipsen
H L
HL
Draaijer
J
J
Hermkens
P H
PH
ten Kortenaar
P B
PB
Tonnaer
J A
JA
D000324:Adrenocorticotropic Hormone / Q000031:analogs & derivatives* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001710:Biotin; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000502:physiology; D005727:Ganglia, Spinal / Q000166:cytology / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009447:Neuroblastoma; D009454:Neurofibrils / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013116:Spinal Cord / Q000166:cytology / Q000378:metabolism; D014407:Tumor Cells, Cultured
D010446:Peptide Fragments; C013765:Org 2766; D001710:Biotin; D000324:Adrenocorticotropic Hormone