Kroeger K M, Hanyaloglu A C, Seeber R M, Miles L E, Eidne K A
Western Australian Institute for Medical Research, University of Western Australia and Keogh Institute of Medical Research, Sir Charles Gairdner Hospital, Nedlands, Perth, Western Australia 6009, Australia.
J Biol Chem. 2001 Apr 20;276(16):12736-43. doi: 10.1074/jbc.M011311200. Epub 2001 Jan 18.
The ability of G-protein-coupled receptors (GPCRs) to interact to form new functional structures, either forming oligomers with themselves or forming associations with other intracellular proteins, has important implications for the regulation of cellular events; however, little is known about how this occurs. Here, we have employed a newly emerging technology, bioluminescence resonance energy transfer (BRET), used to study protein-protein interactions in living cells, to demonstrate that the thyrotropin-releasing hormone receptor (TRHR) forms constitutive homo-oligomers. This formation of TRHR homo-oligomers in the absence of ligand was shown by demonstration of an energy transfer between TRHR molecules fused to either donor, Renilla luciferase (Rluc) or acceptor, enhanced yellow fluorescent protein (EYFP) molecules. This interaction was shown to be specific, since energy transfer was not detected between co-expressed tagged TRHRs and either complementary tagged gonadotropin-releasing hormone (GnRH) or beta(2)-adrenergic receptors. Furthermore, generation of a BRET signal between the TRHRs could only be inhibited by co-expression of the wild-type TRHR and not by other GPCRs. Agonist stimulation led to a time- and dose-dependent increase in the amount of energy transfer. Inhibition of receptor internalization by co-expression of dynamin mutant K44A did not affect the interaction between TRHRs, suggesting that clustering of receptors within clathrin-coated pits is not sufficient for energy transfer to occur. BRET also provided evidence for the agonist-induced oligomerization of another GPCR, the GnRH receptor (GnRHR), and the presence of an agonist-induced interaction of the adaptor protein, beta-arrestin, with TRHR and the absence of an interaction of beta-arrestin with GnRHR. This study supports the usefulness of BRET as a powerful tool for studying GPCR aggregations and receptor/protein interactions in general and presents evidence that the functioning unit of TRHRs exists as homomeric complexes.
G蛋白偶联受体（GPCRs）相互作用形成新功能结构的能力，即自身形成寡聚体或与其他细胞内蛋白形成缔合，对细胞事件的调控具有重要意义；然而，关于其发生机制却知之甚少。在此，我们采用了一种新兴技术——生物发光共振能量转移（BRET），用于研究活细胞中的蛋白质-蛋白质相互作用，以证明促甲状腺激素释放激素受体（TRHR）形成组成型同源寡聚体。通过证明与供体海肾荧光素酶（Rluc）或受体增强型黄色荧光蛋白（EYFP）分子融合的TRHR分子之间的能量转移，证实了在无配体情况下TRHR同源寡聚体的形成。这种相互作用具有特异性，因为在共表达的标记TRHR与互补标记的促性腺激素释放激素（GnRH）或β2 -肾上腺素能受体之间未检测到能量转移。此外，TRHR之间BRET信号的产生仅能被野生型TRHR的共表达所抑制，而不能被其他GPCRs抑制。激动剂刺激导致能量转移量呈时间和剂量依赖性增加。通过共表达发动蛋白突变体K44A抑制受体内化并不影响TRHR之间的相互作用，这表明网格蛋白包被小窝内受体的聚集不足以发生能量转移。BRET还为另一种GPCR——GnRH受体（GnRHR）的激动剂诱导寡聚化以及衔接蛋白β-抑制蛋白与TRHR的激动剂诱导相互作用提供了证据，同时也证明了β-抑制蛋白与GnRHR之间不存在相互作用。这项研究支持了BRET作为研究GPCR聚集和一般受体/蛋白质相互作用的强大工具的实用性，并提供证据表明TRHR的功能单位以同源复合物形式存在。
