Characterization of the insulin-like growth factors (IGF) axis in a cultured mouse Leydig cell line (TM-3)

Abstract

Characterization of the insulin-like growth factor (IGF) axis in a cultured mouse Leydig cell line (TM-3) was performed. Radioimmunoassayable IGF-I and IGF-II concentrations in TM-3 conditioned media (CM) were below the assay detection levels. Affinity cross-linking of IGF-I and IGF-II to crude membranes prepared from TM-3 cells revealed both type 1 and type 2 receptors and a 31 kDa IGF binding protein (IGFBP). Immunoprecipitation of solubilized crude membranes indicated that the 31 kDa protein was membrane associated IGFBP-4. Western ligand blots of CM from TM-3 cells demonstrated the presence of 24 and 28 kDa bands as major IGFBPs, and 25, 40, and 44 kDa as minor ones. The 28 kDa band could be invisible after Endoglycosidase-F treatment, suggesting that the 28 kDa IGFBP was a glycosylated form of IGFBP. The 24 kDa and 28 kDa bands were immunoprecipitated with an antibody to IGFBP-4. Treatment of TM-3 cells with IGF-I increased the levels of the 24 kDa IGFBP-4, as well as 25, 28, 40, and 44 kDa IGFBPs. IGF-I treatment also resulted in the appearance of a 29 kDa IGFBP. Neither the 25 nor 29 kDa bands were deglycosylated with Endoglycosidase-F nor immunoprecipitated by antibodies to rIGFBP-1 and -2. On the other hand, IGF-II treatment resulted in a significant decrease in the 24 kDa IGFBP-4.(ABSTRACT TRUNCATED AT 250 WORDS)