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 K d of 2.7 nM and a B max 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.