Characterization of IRA/IRB hybrid insulin receptors using bioluminescence resonance energy transfer
Characterization of IRA/IRB hybrid insulin receptors using bioluminescence resonance energy transfer
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DOI：
10.1016/j.bcp.2008.07.027
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发表时间：
2008-10-01
影响因子：
5.8
通讯作者：
Issad, Tarik
中科院分区：
文献类型：
--
作者：
Blanquart, Christophe;Achi, Josepha;Issad, Tarik
胰岛素受体（IR）由两条结合配体的a链和两条具有细胞内酪氨酸激酶活性的β链组成。IR在细胞中以含有或不含外显子11的两种亚型表达（分别为IRB和IRA）。几项mRNA研究表明，这两种亚型在不同组织和几种癌细胞中共表达。IRA/IRB杂交受体由来自IRA的α - β链和来自IRB的α - β链组成，可能出现在共表达两种亚型的细胞中，但由于缺乏特异性工具，它们的研究受到阻碍。在之前的工作中，我们使用BRET研究了IR和IGF1R的同二聚体和异二聚体。在这里，我们使用BRET来表征IRA/IRB；混合动力车。BRET饱和实验表明，IRA/IRB杂交体在细胞内是随机形成的。此外，通过用荧光素酶标记的激酶死亡版本和野生型未标记的版本共同转染HEK-293细胞，我们发现IRA/IRB杂交体可以在配体刺激下招募yfp标记的细胞内伴侣。最后，利用BRET，我们研究了配体诱导的IRA/IRB杂化体的构象变化。剂量反应实验表明，与IRA同型二聚体相比，杂交受体结合IGF-2的亲和力相同，而与IGF-1的亲和力较低。总之，我们的数据表明，IRA/IRB杂交受体可以在共表达两种IR亚型的细胞中形成，它们能够在配体刺激下招募细胞内伴侣，并且它们在IGF-2方面具有与IRA更相似的药理特性，而不是IRB同型二聚体。(c) 2008爱思唯尔公司版权所有。
The insulin receptor (IR) is composed of two a-chains that bind ligands and two beta-chains that possess an intracellular tyrosine kinase activity. The IR is expressed in cells as two isoforms containing or not exon 11 (IRB and IRA, respectively). Several mRNA studies have demonstrated that the two isoforms are co-expressed in different tissues and in several cancer cells. IRA/IRB hybrid receptors, constituting of an alpha beta-chain from IRA and an alpha beta-chain from IRB, are likely to occur in cells co-expressing both isoforms, but their study has been hampered by the lack of specific tools. In previous work, we used BRET to study IR and IGF1R homodimers and heterodimers. Here, we have used BRET to characterize IRA/IRB; hybrids. BRET saturation experiments showed that IRA/IRB hybrids are randomly formed in cells. Moreover, by co-transfecting HEK-293 cells with a luciferase-tagged kinase-dead version of one isoform and a wild-type untagged version of the other isoform, we showed that IRA/IRB hybrids can recruit, upon ligand stimulation, a YFP-tagged intracellular partner. Finally, using BRET, we have studied ligand-induced conformational changes within IRA/IRB hybrids. Dose-response experiments showed that hybrid receptors bind IGF-2 with the same affinity than IRA homodimers, whereas they bind IGF-1 with a lower affinity. Altogether, our data indicate that IRA/IRB hybrid receptors can form in cells co-expressing both IR isoforms, that they are capable of recruiting intracellular partners upon ligand stimulation, and that they have pharmacological properties more similar to those of IRA than those of IRB homodimers with regards to IGF-2. (c) 2008 Elsevier Inc. All rights reserved.