Specific binding sites for rat prepro-TRH-(160–169) on C6 glioma and BN1010 clonal neural cells

A connecting decapeptide corresponding to rat prepro-TRH-(160–169) (Ps4) displays several biological activities that are related or unrelated to TRH. We have previously characterized pituitary binding sites for this connecting peptide and elucidated structural determinants for high peptide binding affinity. In the current study, a series of cell lines was screened for the presence of specific binding sites with a highly potent derivative of Ps4, the monoiodinated radioligand [ 125I-Tyr 0]Ps4. Neuroblastoma×glioma hybrid NG108-15, glioma C6 and neuroblastoma BN1010 cell lines were found to have high-affinity [ 125I-Tyr 0]Ps4 binding sites containing 600, 9700 and 130 000 sites/cell, respectively. The specific binding of [ 125I-Tyr 0]Ps4 was rapid, time-dependent, reversible and proportional to the amount of C6 and BN1010 membrane preparation. Furthermore, Scatchard or Hill analysis revealed that [ 125I-Tyr 0]Ps4 was bound by a single population of non-interacting sites with dissociation constants in the subnanomolar range. Competition studies made with Ps4 analogues indicated that [ 125I-Tyr 0]Ps4 binding sites on C6 and BN1010 cells were similar to those previously described on rat pituitary membranes. It is concluded that C6 and BN1010 cells are suited for studies on the intracellular events following binding of the Ps4 and for the molecular characterization of the Ps4 binding sites. © 1997 Federation of European Biochemical Societies.