Moss A M, Livingston J N
Department of Medicine, University of Rochester School of Medicine and Dentistry, NY 14642.
Biochem J. 1993 Sep 15;294 ( Pt 3)(Pt 3):685-92. doi: 10.1042/bj2940685.
Previous work suggests the existence of different isoforms of the insulin-like-growth-factor-1 (IGF-1) receptor in various tissues. In the present study we provide support for the concept that heterogeneous IGF-1 receptors exist in the brain and that part of the heterogeneity is derived from IGF-1 receptor hybrids formed from different beta-subunits. IGF-1 receptors were extracted from adult-rat forebrain synaptosomes and partially purified by wheat-germ agglutinin (WGA) chromatography. Hormone-binding studies in this preparation demonstrate the presence of receptors for IGF-1 and insulin. An antibody, a-RIR, specific for the rat insulin receptor was used to remove insulin receptors from the WGA extract. Studies with the immunodepleted material demonstrated two proteins of 92 and 99 kDa that are phosphorylated on tyrosine during incubation with low concentrations of IGF-1. Both proteins bound with high affinity and specificity to IGF-1 immobilized on agarose, and each underwent phosphorylation when the agarose beads were incubated with [gamma-32P]ATP and MnCl2. Two-dimensional phosphopeptide maps after exhaustive trypsin treatment of the two proteins showed significant differences in their structure as well as differences from the phosphopeptide map for the beta-subunit of the insulin receptor. The relationship of the two proteins to the IGF-1 receptor was further probed by an antibody (a-HF) raised against a specific sequence in the beta-subunit of the human IGF-1 receptor, and a polyclonal antibody raised against the liver insulin receptor (L1) which cross-reacts with the IGF-1 receptor. Both antibodies immunoprecipitated the two phosphorylated proteins. However, reduction of the receptors to form receptor dimers or monomers showed that a-HF precipitated only the 99 kDa protein, whereas L1 precipitated primarily the 92 kDa protein. In conclusion, the brain IGF-1 receptor apparently has two structurally different beta-subunits, one of 92 kDa and a second of 99 kDa. Interestingly, at least a portion of the IGF-1 receptor population has both isoforms in the same receptor.
先前的研究表明，胰岛素样生长因子-1（IGF-1）受体在各种组织中存在不同的亚型。在本研究中，我们支持这样一种观点，即大脑中存在异质性的IGF-1受体，并且这种异质性的一部分源自由不同β亚基形成的IGF-1受体杂合体。从成年大鼠前脑突触体中提取IGF-1受体，并通过麦胚凝集素（WGA）色谱法进行部分纯化。该制剂中的激素结合研究表明存在IGF-1和胰岛素的受体。一种对大鼠胰岛素受体具有特异性的抗体α-RIR，用于从WGA提取物中去除胰岛素受体。对免疫去除后的物质进行的研究表明，有两种分别为92 kDa和99 kDa的蛋白质，在与低浓度IGF-1孵育期间会发生酪氨酸磷酸化。这两种蛋白质都以高亲和力和特异性与固定在琼脂糖上的IGF-1结合，并且当琼脂糖珠与[γ-32P]ATP和MnCl2孵育时，每种蛋白质都会发生磷酸化。对这两种蛋白质进行彻底的胰蛋白酶处理后得到的二维磷酸肽图谱显示，它们的结构存在显著差异，并且与胰岛素受体β亚基的磷酸肽图谱也有所不同。通过针对人IGF-1受体β亚基中的特定序列产生的抗体（α-HF）以及针对肝脏胰岛素受体（L1）产生的与IGF-1受体发生交叉反应的多克隆抗体，进一步探究了这两种蛋白质与IGF-1受体的关系。两种抗体都能免疫沉淀这两种磷酸化蛋白质。然而，将受体还原以形成受体二聚体或单体表明，α-HF仅沉淀99 kDa的蛋白质，而L1主要沉淀92 kDa的蛋白质。总之，大脑中的IGF-1受体显然有两种结构不同的β亚基，一种为92 kDa，另一种为99 kDa。有趣的是，至少一部分IGF-1受体群体在同一受体中同时具有这两种亚型。
