Nerve growth factor-induced changes in the intracellular localization of the protein kinase C substrate B-50 in pheochromocytoma PC12 cells.
High levels of the neuron-specific protein kinase C substrate, B-50 (= GAP43), are present in neurites and growth cones during neuronal development and regeneration. This suggests a hitherto nonelucidated role of this protein in neurite outgrowth. Comparable high levels of B-50 arise in the pheochromocytoma PC12 cell line during neurite formation. To get insight in the putative growth-associated function of B-50, we compared its ultrastructural localization in naive PC12 cells with its distribution in nerve growth factor (NGF)- or dibutyryl cyclic AMP (dbcAMP)-treated PC12 cells. B-50 immunogold labeling of cryosections of untreated PC12 cells is mainly associated with lysosomal structures, including multivesicular bodies, secondary lysosomes, and Golgi apparatus. The plasma membrane is virtually devoid of label. However, after 48-h NGF treatment of the cells, B-50 immunoreactivity is most pronounced on the plasma membrane. Highest B-50 immunoreactivity is observed on plasma membranes surrounding sprouting microvilli, lamellipodia, and filopodia. Outgrowing neurites are scattered with B-50 labeling, which is partially associated with chromaffin granules. In NGF-differentiated PC12 cells, B-50 immunoreactivity is, as in untreated cells, also associated with organelles of the lysosomal family and Golgi stacks. B-50 distribution in dbcAMP-differentiated cells closely resembles that in NGF-treated cells. The altered distribution of B-50 immunoreactivity induced by differentiating agents indicates a shift of the B-50 protein towards the plasma membrane. This translocation accompanies the acquisition of neuronal features of PC12 cells and points to a neurite growth-associated role for B-50, performed at the plasma membrane at the site of protrusion.
Van Hooff
C O
CO
Division of Molecular Neurobiology, Rudolf Magnus Institute for Pharmacology, University of Utrecht, The Netherlands.
Holthuis
J C
JC
Oestreicher
A B
AB
Boonstra
J
J
De Graan
P N
PN
Gispen
W H
WH
D000818:Animals; D001369:Axons / Q000032:analysis; D003994:Bucladesine / Q000494:pharmacology; D002454:Cell Differentiation; D002460:Cell Line; D002462:Cell Membrane / Q000032:analysis; D002838:Chromaffin System / Q000032:analysis / Q000166:cytology / Q000648:ultrastructure; D019922:GAP-43 Protein; D006056:Golgi Apparatus / Q000032:analysis; D007150:Immunohistochemistry; D008247:Lysosomes / Q000032:analysis; D008565:Membrane Proteins / Q000032:analysis*; D008854:Microscopy, Electron; D008871:Microvilli / Q000032:analysis; D009414:Nerve Growth Factors / Q000494:pharmacology*; D009419:Nerve Tissue Proteins / Q000032:analysis*; D009474:Neurons / Q000032:analysis* / Q000648:ultrastructure; D010673:Pheochromocytoma / Q000032:analysis* / Q000648:ultrastructure; D011554:Pseudopodia / Q000032:analysis
D019922:GAP-43 Protein; D008565:Membrane Proteins; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D003994:Bucladesine