Membranes of human neuroblastoma SH-SY5Y cells contain specific binding sites for [3H]K-252A.
K-252a and the structurally similar compound staurosporine promote neurotrophic responses in several cell lines (PC12, SH-SY5Y human neuroblastoma) and in cultures of primary neurons. The molecular mechanisms involved in the induction of these neurotrophic activities are unknown. It is demonstrated in this report that [3H]K-252a binds to SH-SY5Y membranes and that the binding is specific and saturable with a Kd of 2.7 nM and a Bmax of 100,000 sites per cell. The association of [3H]K-252a with its binding site is rapid and reversible, and the binding was inhibited by unlabeled K-252a and by staurosporine. Binding of [3H]K-252a was not inhibited by the potent protein kinase C (PKC) inhibitor GF109203X. Down regulation of PKC by treating SH-SY5Y cells with a phorbol ester did not cause a reduction in the specific binding of [3H]K-252a to membranes, suggesting that the binding is not to PKC. Treatment of the SH-SY5Y membranes with trypsin and by boiling destroyed all specific binding of [3H]K-252a. These results suggest that the [3H]K-252a binds to a specific protein site that is associated with membranes of SH-SY5Y cells.
Knight
E
E
Cephalon Inc., West Chester, Pennsylvania 19380, USA.
Connors
T J
TJ
Hudkins
R
R
Maroney
A C
AC
Neff
N
N
D000470:Alkaloids / Q000494:pharmacology; D001665:Binding Sites; D002227:Carbazoles / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D026121:Indole Alkaloids; D007211:Indoles / Q000494:pharmacology; D007700:Kinetics; D008301:Maleimides / Q000494:pharmacology; D009447:Neuroblastoma; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D019311:Staurosporine; D013379:Substrate Specificity; D014316:Tritium; D014407:Tumor Cells, Cultured
D000470:Alkaloids; D002227:Carbazoles; D026121:Indole Alkaloids; D007211:Indoles; D008301:Maleimides; D014316:Tritium; C049985:staurosporine aglycone; D011493:Protein Kinase C; D019311:Staurosporine; C070515:bisindolylmaleimide I