Insulin-like growth factor receptors and binding protein in rat neuroblastoma cells

B104, an established rat neuroblastoma cell line exhibiting specific neuronal qualities, was chosen as a model to study insulin-like growth factor (IGF) binding and action in the central nervous system. Specific binding of [¹²⁵I]IGF-II to B104 membranes averaged 12.2 ± 4.0% (mean ± SD)/100 μg/ml protein compared with [¹²⁵I]IGF-I binding of 10.1 ± 2.9%. In competitive binding studies employing [¹²⁵I]IGF-II as the radioligand, high affinity for IGF-II was demonstrated (50% displacement at 2.7 ng/ml), with none for IGF-I or insulin. Upon crosslinking [¹²⁵I]IGF-I to membranes under reducing conditions, two prominent bands were observed, migrating with apparent mol wt (M_r) of 135, 000 and 280, 000. Both bands were inhibited by IGFs and insulin, but not by R-II-PABI, a polyclonal antibody to the type 2 receptor. These bands presumably represent the αsubunit and an incompletely reduced a-a-dimer of the type 1 IGF receptor. When cross-linking [¹²⁵I]IGF-II to membranes under reducing conditions, the primary labeled bands migrated with apparent M_r of 260, 000 and 280, 000. These bands were inhibited by IGF-II and R-II-PABI, but not by insulin, and probably represent the monomeric type 2 receptor. In addition, we observed a minor band at apparent M_r 35, 000, which was inhibited by IGF but not by insulin. By a modified cross-linking technique, we confirmed the existence of a small IGF-binding protein in the serum-free conditioned medium of B104 cultures, migrating as two bands with apparent M_r of 33, 000†39, 000. These proteins demonstrated high affinity for IGF-I and IGFII, but none for insulin. In summary, this study demonstrates the presence in B104 rat neuroblastoma cells of 1) abundant classical type 1 and type 2 IGF receptors, and 2) a secreted and membrane-associated small IGF-binding protein.