Homo- and hetero-oligomeric interactions between G-protein-coupled receptors in living cells monitored by two variants of bioluminescence resonance energy transfer (BRET): hetero-oligomers between receptor subtypes form more efficiently than between less closely related sequences
Homo- and hetero-oligomeric interactions between G-protein-coupled receptors in living cells monitored by two variants of bioluminescence resonance energy transfer (BRET): hetero-oligomers between receptor subtypes form more efficiently than between less closely related sequences
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DOI：
10.1042/bj20020251
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发表时间：
2002-07-15
影响因子：
4.1
通讯作者：
Milligan, G
中科院分区：
文献类型：
--
作者：
Ramsay, D;Kellett, E;Milligan, G
用两种生物发光共振能量转移(BRET)技术检测了HEK-293细胞中G蛋白偶联受体(GPCRs)的同源和异源寡聚。BRET2(BRET的一种变体)与传统的BRET相比，提供了更好的给体和受体部分发射光谱的分离。之前记录的人类增量阿片受体的同源齐聚作用通过BRET2得到了证实。用两种BRET技术观察到kappa-阿片受体的同源寡聚。同时含有β-阿片受体和kappa-阿片受体的同型和异型低聚物都不受受体配体的影响。对阿片受体同型和异型寡聚体的BRET检测要求每个细胞表达50000-100000个拷贝的受体能量受体结构。β-kappa-阿片受体异低聚物形成的效果与同聚体相互作用的效果一样好。两种阿片受体与β(2)-肾上腺素能受体形成低聚复合体的能力也被评估。尽管检测到了这种相互作用，但每个细胞的能量受体至少需要250000个拷贝。在试图测量kappa-阿片受体和促甲状腺激素释放激素受体-1之间的异寡体形成时，对高水平受体表达的要求同样明显。这些研究表明，在含有不到100000个阿片受体拷贝的活细胞中，可以检测到构成形成的阿片受体亚型的同种和异种低聚物。然而，尽管可以检测到某些不太相关的GPCR之间的异寡体相互作用，但它们的亲和力似乎低于包含密切相关序列的同源或异源低聚物。在异源表达系统中记录的某些GPCR家族成员之间的相互作用很可能是极端水平的过度表达的人工制品。
Homo- and hetero-oligomerization of G-protein-coupled receptors (GPCRs) were examined in HEK-293 cells using two variants of bioluminescence resonance energy transfer (BRET). BRET2 (a variant of BRET) offers greatly improved separation of the emission spectra of the donor and acceptor moieties compared with traditional BRET. Previously recorded homo-oligomerization of the human delta-opioid receptor was confirmed using BRET2. Homo-oligomerization of the kappa-opioid receptor was observed using both BRET techniques. Both homo- and hetero-oligomers, containing both delta- and kappa-Opioid receptors, were unaffected by the presence of receptor ligands. BRET detection of opioid receptor homo- and hetero-oligomers required expression of 50000-100000 copies of the receptor energy acceptor construct per cell. The effectiveness of delta-kappa-opioid receptor hetero-oligomer formation was as great as for homomeric interactions. The capacity of the two opioid receptors to form oligomeric complexes with the beta(2)-adrenoceptor was also assessed. Although such interactions were detected, at least 250000 copies per cell of the energy acceptor were required. Requirement for high levels of receptor expression was equally pronounced in attempts to measure hetero-oligomer formation between the kappa-Opioid receptor and the thyrotropin-releasing hormone receptor-1. These studies indicate that constitutively formed homo- and hetero-oligomers of opioid receptor subtypes can be detected in living cells containing less than 100000 copies of the receptors. However, although hetero-oligomeric interactions between certain less closely related GPCRs can be detected, they appear to be of lower affinity than homo- or hetero-oligomers containing closely related sequences. Interactions recorded between certain GPCR family members in heterologous expression systems are likely to be artefacts of extreme levels of overexpression.