Sturm M A, Conover C A, Pham H, Rosenfeld R G
Department of Pediatrics, Stanford University Medical Center, California 94305.
Endocrinology. 1989 Jan;124(1):388-96. doi: 10.1210/endo-124-1-388.
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 micrograms/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 cross-linking [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 alpha-subunit and an incompletely reduced alpha-alpha-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 IGF-II, 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.
B104是一种已确立的具有特定神经元特性的大鼠神经母细胞瘤细胞系，被选作研究胰岛素样生长因子（IGF）在中枢神经系统中的结合及作用的模型。与[125I]IGF-I的结合量平均为10.1±2.9%相比，[125I]IGF-II与B104细胞膜的特异性结合平均为12.2±4.0%/100微克/毫升蛋白质。在以[125I]IGF-II作为放射性配体的竞争性结合研究中，显示出对IGF-II具有高亲和力（在2.7纳克/毫升时50%被置换），对IGF-I或胰岛素则无亲和力。在还原条件下将[125I]IGF-I与细胞膜交联后，观察到两条明显的条带，表观分子量（Mr）分别为135,000和280,000。这两条条带均受到IGF和胰岛素的抑制，但不受针对2型受体的多克隆抗体R-II-PABI的抑制。这些条带可能代表1型IGF受体的α亚基和未完全还原的α-α二聚体。在还原条件下将[125I]IGF-II与细胞膜交联时，主要标记条带的表观Mr为260,000和280,000。这些条带受到IGF-II和R-II-PABI的抑制，但不受胰岛素的抑制，可能代表单体2型受体。此外，我们在表观Mr为35,000处观察到一条小条带，它受到IGF的抑制但不受胰岛素的抑制。通过一种改良的交联技术，我们证实了在B104培养物的无血清条件培养基中存在一种小的IGF结合蛋白，以两条表观Mr为33,000 - 39,000的条带形式迁移。这些蛋白对IGF-I和IGF-II具有高亲和力，但对胰岛素无亲和力。总之，本研究证明在B104大鼠神经母细胞瘤细胞中存在1）丰富的经典1型和2型IGF受体，以及2）一种分泌型和膜相关的小IGF结合蛋白。
