Beguinot F, Smith R J, Kahn C R, Maron R, Moses A C, White M F
Joslin Diabetes Center, Charles A. Dana Research Institute, Harvard-Thorndike Laboratory, Boston, Massachusetts.
Biochemistry. 1988 May 3;27(9):3222-8. doi: 10.1021/bi00409a015.
The interaction between insulin and insulin-like growth factor I (IGF I) receptors was examined by determining the ability of each receptor type to phosphorylate tyrosine residues on the other receptor in intact L6 skeletal muscle cells. This was made possible through a sequential immunoprecipitation method with two different antibodies that effectively separated the phosphorylated insulin and IGF I receptors. After incubation of intact L6 cells with various concentrations of insulin or IGF I in the presence of [32P]orthophosphate, insulin receptors were precipitated with one of two human polyclonal anti-insulin receptor antibodies (B2 or B9). Phosphorylated IGF I receptors remained in solution and were subsequently precipitated by anti-phosphotyrosine antibodies. The identities of the insulin and IGF I receptor beta-subunits in the two immunoprecipitates were confirmed by binding affinity, by phosphopeptide mapping after trypsin digestion, and by the distinct patterns of expression of the two receptors during differentiation. Stimulated phosphorylation of the beta-subunit of the insulin receptor correlated with occupancy of the beta-subunit of the insulin receptor by either insulin or IGF I as determined by affinity cross-linking. Similarly, stimulation of phosphorylation of the beta-subunit of the IGF I receptor by IGF I correlated with IGF I receptor occupancy. In contrast, insulin stimulated phosphorylation of the beta-subunit of the IGF I receptor at hormone concentrations that were associated with significant occupancy of the insulin receptor but negligible IGF I receptor occupancy. These findings indicate that the IGF I receptor can be a substrate for the hormone-activated insulin receptor tyrosine kinase activity in intact L6 skeletal muscle cells.
通过测定完整的L6骨骼肌细胞中每种受体类型使另一种受体上的酪氨酸残基磷酸化的能力，研究了胰岛素与胰岛素样生长因子I（IGF I）受体之间的相互作用。这通过一种顺序免疫沉淀方法得以实现，该方法使用两种不同的抗体，可有效分离磷酸化的胰岛素受体和IGF I受体。在用[32P]正磷酸盐存在下，将完整的L6细胞与不同浓度的胰岛素或IGF I孵育后，用两种人多克隆抗胰岛素受体抗体之一（B2或B9）沉淀胰岛素受体。磷酸化的IGF I受体保留在溶液中，随后用抗磷酸酪氨酸抗体沉淀。通过结合亲和力、胰蛋白酶消化后的磷酸肽图谱以及两种受体在分化过程中不同的表达模式，证实了两种免疫沉淀物中胰岛素受体和IGF I受体β亚基的身份。胰岛素受体β亚基的磷酸化刺激与胰岛素或IGF I对胰岛素受体β亚基的占据相关，这通过亲和交联确定。同样，IGF I对IGF I受体β亚基磷酸化的刺激与IGF I受体的占据相关。相比之下，在与胰岛素受体显著占据但IGF I受体占据可忽略不计相关的激素浓度下，胰岛素刺激了IGF I受体β亚基的磷酸化。这些发现表明，在完整的L6骨骼肌细胞中，IGF I受体可以成为激素激活的胰岛素受体酪氨酸激酶活性的底物。
