Génier Samuel, Degrandmaison Jade, Moreau Pierrick, Labrecque Pascale, Hébert Terence E, Parent Jean-Luc
Service de Rhumatologie, Département de Médecine, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Centre de Recherche du Centre Hospitalier Universitaire de Sherbrooke (CR-CHUS), and Institut de Pharmacologie de Sherbrooke, Sherbrooke, QC J1H 5N4, Canada.
Department of Pharmacology and Therapeutics, McGill University, Montréal, QC H3G 1Y6, Canada.
Mol Biol Cell. 2016 Dec 1;27(24):3800-3812. doi: 10.1091/mbc.E16-04-0224. Epub 2016 Oct 5.
Mechanisms that prevent aggregation and promote folding of nascent G protein-coupled receptors (GPCRs) remain poorly understood. We identified chaperonin containing TCP-1 subunit eta (CCT7) as an interacting partner of the β-isoform of thromboxane A receptor (TPβ) by yeast two-hybrid screening. CCT7 coimmunoprecipitated with overexpressed TPβ and β-adrenergic receptor (βAR) in HEK 293 cells, but also with endogenous βAR. CCT7 depletion by small interfering RNA reduced total and cell-surface expression of both receptors and caused redistribution of the receptors to juxtanuclear aggresomes, significantly more so for TPβ than βAR. Interestingly, Hsp90 coimmunoprecipitated with βAR but virtually not with TPβ, indicating that nascent GPCRs can adopt alternative folding pathways. In vitro pull-down assays showed that both receptors can interact directly with CCT7 through their third intracellular loops and C-termini. We demonstrate that Trp in the TPβ C-terminus is critical for the CCT7 interaction and plays an important role in TPβ maturation and cell-surface expression. Of note, introducing a tryptophan in the corresponding position of the TPα isoform confers the CCT7-binding and maturation properties of TPβ. We show that an interaction with a subunit of the CCT/TCP-1 ring complex (TRiC) chaperonin complex is involved in regulating aggregation of nascent GPCRs and in promoting their proper maturation and expression.
防止新生G蛋白偶联受体（GPCRs）聚集并促进其折叠的机制仍知之甚少。我们通过酵母双杂交筛选，鉴定出含TCP-1亚基eta的伴侣蛋白（CCT7）是血栓素A受体β亚型（TPβ）的相互作用伙伴。在HEK 293细胞中，CCT7与过表达的TPβ和β肾上腺素能受体（βAR）共免疫沉淀，也与内源性βAR共免疫沉淀。通过小干扰RNA耗尽CCT7会降低这两种受体的总表达和细胞表面表达，并导致受体重新分布到近核聚集体中，TPβ比βAR更明显。有趣的是，Hsp90与βAR共免疫沉淀，但几乎不与TPβ共免疫沉淀，这表明新生GPCRs可以采用不同的折叠途径。体外下拉实验表明，这两种受体都可以通过其第三个细胞内环和C末端直接与CCT7相互作用。我们证明TPβ C末端的色氨酸对于CCT7相互作用至关重要，并且在TPβ成熟和细胞表面表达中起重要作用。值得注意的是，在TPα亚型的相应位置引入色氨酸可赋予TPβ与CCT7结合和成熟的特性。我们表明，与伴侣蛋白CCT/TCP-1环复合物（TRiC）亚基的相互作用参与调节新生GPCRs的聚集，并促进其正确成熟和表达。
