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

Marlene A. Sturm, Cheryl A. Conover, Hung Pham, Ron G. Rosenfeld

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Abstract

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 (Mr) 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 Mr 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 Mr 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 Mr 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.

Original languageEnglish (US)
Pages (from-to)388-396
Number of pages9
JournalEndocrinology
Volume124
Issue number1
DOIs
StatePublished - Jan 1989

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ASJC Scopus subject areas

  • Endocrinology

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