Insulin receptor/IGF-1 receptor hybrids are widely distributed in mammalian tissues: quantification of individual receptor species by selective immunoprecipitation and immunoblotting
Insulin receptor/IGF-1 receptor hybrids are widely distributed in mammalian tissues: quantification of individual receptor species by selective immunoprecipitation and immunoblotting
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DOI：
10.1042/bj3270209
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发表时间：
1997-10-01
影响因子：
4.1
通讯作者：
Siddle, K
中科院分区：
文献类型：
--
作者：
Bailyes, EM;Nave, BT;Siddle, K
胰岛素受体（IR）和1型胰岛素样生长因子（IGF-I）受体（IGFR）均在哺乳动物组织中广泛表达，并且已知除了经典描述的受体之外，还能够异聚体组装为胰岛素/IGF杂合受体。通过放射性配体/受体复合物的选择性免疫吸附和免疫印迹，我们确定了胰岛素受体和IGF受体在不同组织中作为杂交体出现的分数。从组织匀浆中分离微粒体膜，并用Triton X-100溶解。将溶解的受体与I-125-IGF-I孵育，并定量IR特异性和IGFR特异性单克隆抗体结合的放射性配体/受体复合物。表现为杂交体（抗IR结合/抗IGFR结合）的IGF-I结合位点的分数约为100%。40%在肝脏和脾脏中，70%在胎盘中，85- 90%在骨骼肌和心脏中，在兔和人体组织中获得了类似的结果。不同组织中I-125-胰岛素/I-125-IGF-I结合率与杂交体比例之间无相关性。与杂交体结合的I-125-胰岛素的分数太低，无法准确定量，因为杂交体对胰岛素的亲和力相对较低。因此，通过免疫印迹法测定杂交体中存在的胰岛素受体的分数。用IR特异性或IGFR特异性单克隆抗体沉淀溶解的人胎盘微粒体膜中的受体，SDS/PAGE后，制备印迹，并用IR特异性和IGFR特异性抗血清进行探测。结果表明，杂种后代中IR和IGFR的表达率分别为15%和80%.与这些图一致，通过用相应抗体进行印迹，估计IR的总体水平比IGFR高4倍，所述抗体的浓度显示为与等量的受体产生相等的信号。总体而言，得出的结论是，IR和IGFR两者的显著部分在大多数哺乳动物组织中作为杂合体出现，包括那些被公认为胰岛素和IGF作用靶点的组织。在不同组织中的杂交体的分数不是IR和IGFR的相对水平的简单函数，可能是因为在不同细胞类型中受体表达的异质性。然而，在胎盘中的IR，IGFR和杂交的比例是一致的随机组装的过程中反映IR和IGFR半受体的摩尔比。
The insulin receptor (IR) and type 1 insulin-like growth factor (IGF-I) receptor (IGFR) are both widely expressed in mammalian tissues, and are known to be capable of heteromeric assembly as insulin/IGF hybrid receptors, in addition to the classically described receptors. By selective immunoadsorption of radioligand/receptor complexes and by immunoblotting we have determined the fraction of insulin receptors and IGF receptors occurring as hybrids in different tissues. Microsomal membranes were isolated from tissue homogenates and solubilized with Triton X-100. Solubilized receptors were incubated with I-125-IGF-I, and radioligand/receptor complexes bound by IR-specific and IGFR-specific monoclonal antibodies were quantified. The fraction of IGF-I binding sites behaving as hybrids (anti-IR-bound/anti-IGFR-bound) was approx. 40% in liver and spleen, 70 % in placenta, and 85-90 % in skeletal muscle and heart, similar results being obtained in rabbit and human tissues. There was no correlation between the proportion of hybrids and the ratio of I-125-insulin/I-125-IGF-I binding in different tissues. The fraction of I-125-insulin bound to hybrids was too low for accurate quantification, because of the relatively low affinity of hybrids for insulin. The fraction of insulin receptors present in hybrids was therefore determined by immunoblotting. Receptors in solubilized human placental microsomal membranes were precipitated with IR-specific or IGFR-specific monoclonal antibodies, and after SDS/PAGE, blots were prepared and probed with IR-specific and IGFR-specific antisera. It was found that 15 % of IR and 80 % of IGFR were present in hybrids. Consistent with these figures, the overall level of IR was estimated, by blotting with the respective antibodies at concentrations shown to give equal signals with equal amounts of receptor, to be 4-fold greater than IGFR. Overall it was concluded that a significant fraction of both IR and IGFR occurs as hybrids in most mammalian tissues, including those that are recognized targets of insulin and IGF action. The fraction of hybrids in different tissues was not a simple function of the relative levels of IR and IGFR, possibly because of heterogeneity of receptor expression in different cell types. However, in placenta the proportions of IR, IGFR and hybrids were consistent with a process of random assembly reflecting the molar ratio of IR and IGFR half-receptors.