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.