Contacts between extracellular loop two and transmembrane helix six determine basal activity of the thyroid-stimulating hormone receptor
Contacts between extracellular loop two and transmembrane helix six determine basal activity of the thyroid-stimulating hormone receptor
复制标题
DOI：
10.1074/jbc.m606176200
复制
发表时间：
2007-01-05
影响因子：
4.8
通讯作者：
Krause, Gerd
中科院分区：
文献类型：
--
作者：
Kleinau, Gunnar;Claus, Maren;Krause, Gerd
与野生型受体相比，促甲状腺激素受体（TSHR）细胞外环2（ECL 2）中的一些丙氨酸突变被发现增加或减少基础活性。K565A被鉴定为具有降低的基础活性和强烈受损的激素诱导的信号传导活性的突变体。为了深入了解ECL 2突变体如何影响基础活性，我们专注于ECL 2中的组成型激活致病突变体1568V，其表现出升高的基础活性。因为我们的分子模型表明，Ile-568嵌入在由跨膜螺旋束提供的疏水残基的环境中，我们测试了该区域的突变体以鉴定Ile-568的潜在相互作用伴侣。事实上，双突变体1568V/1640L（ECL 2/TMH6）抑制了由单独的1568V表现出的增加的基础活性。我们提出了ECL 2和TMH6之间的空间和功能关系，其中Ile-568和Ile-640之间的侧链相互作用将受体限制在具有低基础活性的构象中。虽然单突变体1640L表现出低于野生型的基础活性，但其不同分支和更大的侧链补充了1568V中减少的侧链体积，恢复了双突变体的野生型基础活性。这种情况被相互双突变体1640V/I568L证实。基础增加的1640V活性和基础降低的突变体1568L活性的组合也恢复了野生型TSHR的基础活性。这些和其他突变表型报告支持TMH6和ECL 2之间的动态界面。通过在TSHR中引入突变来破坏这种关键的信号传导界面可以增加或减少基础活性。
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 1568V 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 1568V/1640L (ECL2/TMH6) suppresses the increased basal activity exhibited by 1568V 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 1640L exhibits basal activity lower than wild type, its differently branched and bulkier side chain complements the reduced side chain bulk in 1568V, restoring wild type basal activity to the double mutant. This scenario is confirmed by the reciprocal double mutant 1640V/I568L. The combination of basally increased activity of 1640V and basally decreased activity of mutant 1568L 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.