The α1b-adrenoceptor exists as a higher-order oligomer:: Effective oligomerization is required for receptor maturation, surface delivery, and function
The α1b-adrenoceptor exists as a higher-order oligomer:: Effective oligomerization is required for receptor maturation, surface delivery, and function
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DOI：
10.1124/mol.106.033035
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发表时间：
2007-04-01
影响因子：
3.6
通讯作者：
Milligan, Graeme
中科院分区：
文献类型：
--
作者：
Lopez-Gimenez, Juan F.;Canals, Meritxell;Milligan, Graeme
鉴定G蛋白偶联受体寡聚体而不是二聚体的方法一直缺乏。使用多联体的荧光蛋白，我们建立了监测连续三色荧光共振能量转移（3-FRET）的条件，并使用这些检测寡聚复合物的α（1b）-肾上腺素受体在单个活细胞。跨膜结构域I和IV中推定的关键疏水残基的突变导致连续3-FRET的大幅减少，并且与缺乏蛋白质成熟、防止质膜递送和消除信号传导功能相关。虽然这些突变阻止了细胞表面的交付，双分子荧光互补研究表明，他们没有消融蛋白质-蛋白质相互作用，并确认内质网/高尔基体保留的跨膜结构域I加跨膜结构域IV突变的受体。跨膜结构域I加跨膜结构域IV突变的受体在阻断野生型受体的细胞表面递送中是“显性阴性”的。仅在跨膜结构域I中的突变不导致3-FRET的减少，而限制突变至跨膜结构域IV确实导致3-FRET减少。然而，跨膜结构域I或跨膜结构域IV中的突变足以消除细胞表面递送。末端N-糖基化不足以确定细胞表面递送，因为跨膜结构域I和跨膜结构域IV突变体与野生型受体一样有效地成熟。这些数据表明，α（1b）-肾上腺素受体能够形成寡聚体，而不仅仅是简单的二聚体复合物，并且通过将突变引入跨膜结构域IV来破坏有效的寡聚化对细胞表面递送和功能具有深远的影响。
Approaches to identify G protein-coupled receptor oligomers rather than dimers have been lacking. Using concatamers of fluorescent proteins, we established conditions to monitor sequential three-color fluorescence resonance energy transfer (3-FRET) and used these to detect oligomeric complexes of the alpha(1b)-adrenoceptor in single living cells. Mutation of putative key hydrophobic residues in transmembrane domains I and IV resulted in substantial reduction of sequential 3-FRET and was associated with lack of protein maturation, prevention of plasma membrane delivery, and elimination of signaling function. Although these mutations prevented cell surface delivery, bimolecular fluorescence complementation studies indicated that they did not ablate protein-protein interactions and confirmed endoplasmic reticulum/Golgi retention of the transmembrane domain I plus transmembrane domain IV mutated receptor. The transmembrane domain I plus transmembrane domain IV mutated receptor was a "dominant-negative" in blocking cell surface delivery of the wild-type receptor. Mutations only in transmembrane domain I did not result in a reduction in 3-FRET, whereas restricting mutation to transmembrane domain IV did result in reduced 3-FRET. Mutations in either transmembrane domain I or transmembrane domain IV, however, were sufficient to eliminate cell surface delivery. Terminal N-glycosylation is insufficient to determine cell surface delivery because both transmembrane domain I and transmembrane domain IV mutants matured as effectively as the wild-type receptor. These data indicate that the alpha(1b)-adrenoceptor is able to form oligomeric rather than only simple dimeric complexes and that disruption of effective oligomerization by introducing mutations into transmembrane domain IV has profound consequences for cell surface delivery and function.