Kleinau Gunnar, Claus Maren, Jaeschke Holger, Mueller Sandra, Neumann Susanne, Paschke Ralf, Krause Gerd
Leibniz-Institut für molekulare Pharmakologie (FMP), Robert-Rössle-Str. 10, D-13125 Berlin, Germany.
J Biol Chem. 2007 Jan 5;282(1):518-25. doi: 10.1074/jbc.M606176200. Epub 2006 Nov 1.
A number of alanine mutations in extracellular loop two (ECL2) of the thyroid-stimulating hormone receptor (TSHR) were found to increase or decrease basal activity when compared with the wild type receptor. K565A was identified as a mutant with decreased basal activity, and strongly impaired hormone induced signaling activity. To gain insights into how ECL2 mutants affect basal activity, we focused on constitutively activating pathogenic mutant I568V in ECL2, which exhibits elevated basal activity. Because our molecular model suggests that Ile-568 is embedded in an environment of hydrophobic residues provided by transmembrane helix bundle, we tested mutants in this region to identify potential interaction partner(s) for Ile-568. Indeed, the double mutant I568V/I640L (ECL2/TMH6) suppresses the increased basal activity exhibited by I568V alone. We suggest a spatial and functional relationship between ECL2 and TMH6 in which side chain interaction between Ile-568 and Ile-640 constrains the receptor in a conformation with low basal activity. Although the single mutant I640L exhibits basal activity lower than wild type, its differently branched and bulkier side chain complements the reduced side chain bulk in I568V, restoring wild type basal activity to the double mutant. This scenario is confirmed by the reciprocal double mutant I640V/I568L. The combination of basally increased activity of I640V and basally decreased activity of mutant I568L also restores basal activity of wild type TSHR. These and other mutant phenotypes reported here support a dynamic interface between TMH6 and ECL2. Disruption of this critical interface for signaling by introduction of mutations in TSHR can either increase or decrease basal activity.
与野生型促甲状腺激素受体（TSHR）相比，在促甲状腺激素受体的细胞外环2（ECL2）中发现的一些丙氨酸突变会增加或降低基础活性。K565A被鉴定为基础活性降低的突变体，并且严重损害激素诱导的信号传导活性。为了深入了解ECL2突变体如何影响基础活性，我们聚焦于ECL2中组成型激活的致病突变体I568V，其基础活性升高。因为我们的分子模型表明，Ile-568嵌入在跨膜螺旋束提供的疏水残基环境中，所以我们测试了该区域的突变体，以确定Ile-568的潜在相互作用伙伴。事实上，双突变体I568V/I640L（ECL2/TMH6）抑制了单独的I568V所表现出的基础活性增加。我们提出ECL2和TMH6之间存在空间和功能关系，其中Ile-568和Ile-640之间的侧链相互作用将受体限制在低基础活性的构象中。虽然单突变体I640L的基础活性低于野生型，但其不同分支且更大的侧链补充了I568V中减少的侧链体积，使双突变体恢复了野生型基础活性。相互的双突变体I640V/I568L证实了这一情况。I640V基础活性增加与突变体I568L基础活性降低的组合也恢复了野生型TSHR的基础活性。本文报道的这些及其他突变体表型支持了TMH6和ECL2之间的动态界面。通过在TSHR中引入突变破坏这个关键的信号传导界面可以增加或降低基础活性。
