Mutation of Asn293 to Asp in transmembrane helix VI abolishes agonist-induced but not constitutive activity of the β2-adrenergic receptor
Mutation of Asn293 to Asp in transmembrane helix VI abolishes agonist-induced but not constitutive activity of the β2-adrenergic receptor
复制标题
DOI：
10.1124/mol.62.6.1431
复制
发表时间：
2002-12-01
影响因子：
3.6
通讯作者：
Lohse, MJ
中科院分区：
文献类型：
--
作者：
Hannawacker, A;Krasel, C;Lohse, MJ
除了激动剂诱导的激活外，β(2)-肾上腺素能受体已被证明具有显著的结构性活性(即，在没有激动剂的情况下)。各种研究表明，跨膜螺旋VI的运动在激活各种G蛋白偶联受体中起着作用。在这里，我们表明，在这个区域的β(2)-肾上腺素能受体突变取消激动剂激活，但不是结构性的活性。人β(2)-肾上腺素能受体突变体Asn293Asp在COS-7细胞中瞬时表达，在中国仓鼠卵巢细胞中稳定表达。突变的受体不能与G(S)偶联，表现为缺乏高亲和力激动剂的结合以及与其内在活性相关的几种激动剂亲和力的降低。突变受体仅引起腺苷环化酶的极少量激活(野生型活性的2.5%)，也不能被G蛋白偶联受体激酶2激动剂诱导的磷酸化。相反，突变受体的构成活性受到的影响要小得多：瞬时将野生型和突变型受体导入COS-7细胞后，细胞内cAMP水平增加，这取决于受体的表达水平，突变型和野生型受体的最大水平分别是5.4倍和6.8倍(野生型受体活性的67%)。将Asn293Asp突变引入到具有结构性活性的突变体受体中，并不影响该突变体的结构性活性。这些结果强调了跨膜螺旋VI在控制激动剂诱导的受体激活中的重要性，并表明结构性活性不同于激动剂诱导的活性。此外，它们表明Asn293是将构象信息从激动剂结合部位转移到细胞内表面的关键残基。
The beta(2)-adrenergic receptor has been shown to display significant constitutive activity (i.e., in the absence of agonist) in addition to agonist-induced activation. Various studies have suggested that a movement in transmembrane helix VI plays a role in activation of various G-protein-coupled receptors. Here we show that a mutation in this domain of the beta(2)-adrenergic receptor abolishes agonist activation but not constitutive activity. An Asn293Asp mutant of the human beta(2)-adrenergic receptor was expressed either transiently in COS-7 cells or stably in Chinese hamster ovary cells. The mutant receptors were unable to couple to G(s), as seen by the lack of high-affinity agonist binding as well as a reduction of the affinities of several agonists correlating with their intrinsic activities. The mutant receptors caused only minimal activation of adenylyl cyclase (2.5% of wild-type activity) and also failed to show agonist-induced phosphorylation by G-protein-coupled receptor kinase 2. In contrast, the mutant receptors were much less affected in their constitutive activity: transient transfection of wild-type and mutant receptors into COS-7 cells caused an increase in intracellular cAMP-levels that was dependent on the level of receptor expression and was maximally 5.4-fold for the mutant and 6.8-fold for the wild-type receptors (67% of wild-type activity). Introduction of the Asn293Asp mutation into a constitutively active mutant receptor did not affect the constitutive activity of this mutant. These results underscore the importance of transmembrane helix VI in controlling agonist-induced activation of the receptor and suggest that constitutive activity is different from agonist-induced activity. Furthermore, they indicate that Asn293 is a key residue in transferring conformational information from the agonist-binding site to the intracellular surface.