Soos M A, Siddle K
Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, U.K.
Biochem J. 1989 Oct 15;263(2):553-63. doi: 10.1042/bj2630553.
The receptors for insulin and insulin-like growth factor-I (IGF-I) are closely related in primary sequence and overall structure. We have examined the immunological relationships between these receptors by testing the reactivity of anti-(insulin receptor) monoclonal antibodies with IGF-I receptors in various tissues and cell lines. Antibodies for six distinct epitopes reacted with a subfraction of IGF-I receptors, as shown by inhibition of 125I-IGF-I binding, precipitation of 125I-IGF-I-receptor complexes or immunodepletion of receptor from tissue extracts before binding assays. Both immunoreactive and non-immunoreactive subfractions displayed the expected properties of 'classical' IGF-I receptors, in terms of relative affinities for IGF-I and insulin. The proportion of total IGF-I receptors which was immunoreactive varied in different cell types, being approx. 40% in Hep G2 cells, 35-40% in placental membranes and 75-85% in IM-9 cells. The immunoreactive fraction was somewhat higher in solubilized receptors than in the corresponding intact cells or membranes. A previously described monoclonal antibody, alpha-IR-3, specific for IGF-I receptors, inhibited IGF-I binding by more than 80% in all preparations. When solubilized placental receptors were pretreated with dithiothreitol (DTT) under conditions reported to reduce intramolecular (class I) disulphide bonds, the immunoreactivity of IGF-I receptors was abolished although total IGF-I binding was little affected. Under the same conditions insulin receptors remained fully immunoreactive. When solubilized receptor preparations were fractionated by gel filtration, both IGF-I and insulin receptors ran as symmetrical peaks of identical mobility. After DTT treatment, the IGF-I receptor was partially converted to a lower molecular mass form which was not immunoreactive. The insulin receptor peak showed a much less pronounced skewing and remained fully immunoreactive in all fractions. It is concluded that the anti- (insulin receptor) antibodies do not react directly with IGF-I receptor polypeptide, and that the apparent immunoreactivity of a subfraction of IGF-I receptors reflects their physical association with insulin receptors, both in cell extracts and in intact cells. The most likely basis for this association appears to be a 'hybrid' receptor containing one half (alpha beta) of insulin receptor polypeptide and the other (alpha' beta') of IGF-I receptor polypeptide within the native (alpha beta beta' alpha') heterotetrameric structure.
胰岛素受体和胰岛素样生长因子-I（IGF-I）受体在一级序列和整体结构上密切相关。我们通过检测抗（胰岛素受体）单克隆抗体与各种组织和细胞系中IGF-I受体的反应性，研究了这些受体之间的免疫关系。六种不同表位的抗体与IGF-I受体的一个亚组分发生反应，这通过抑制125I-IGF-I结合、沉淀125I-IGF-I-受体复合物或在结合测定前从组织提取物中免疫去除受体来证明。就对IGF-I和胰岛素的相对亲和力而言，免疫反应性和非免疫反应性亚组分均表现出“经典”IGF-I受体的预期特性。总IGF-I受体中免疫反应性的比例在不同细胞类型中有所不同，在Hep G2细胞中约为40%，在胎盘膜中为35 - 40%，在IM-9细胞中为75 - 85%。溶解受体中的免疫反应性部分比相应的完整细胞或膜中的略高。一种先前描述的对IGF-I受体特异的单克隆抗体α-IR-3，在所有制剂中抑制IGF-I结合超过80%。当在据报道可还原分子内（I类）二硫键的条件下用二硫苏糖醇（DTT）预处理溶解的胎盘受体时，IGF-I受体的免疫反应性消失，尽管总IGF-I结合几乎未受影响。在相同条件下，胰岛素受体仍保持完全免疫反应性。当通过凝胶过滤对溶解的受体制剂进行分级分离时，IGF-I和胰岛素受体均以相同迁移率的对称峰形式出现。DTT处理后，IGF-I受体部分转化为较低分子量形式，该形式无免疫反应性。胰岛素受体峰的偏斜不太明显，并且在所有级分中均保持完全免疫反应性。结论是抗（胰岛素受体）抗体不直接与IGF-I受体多肽反应，并且IGF-I受体亚组分的明显免疫反应性反映了它们在细胞提取物和完整细胞中与胰岛素受体的物理关联。这种关联最可能的基础似乎是一种“杂合”受体，在天然（αββ'α'）异四聚体结构中包含一半（αβ）胰岛素受体多肽和另一半（α'β'）IGF-I受体多肽。
