Quantitative Super-Resolution Imaging for the Analysis of GPCR Oligomerization.
Quantitative Super-Resolution Imaging for the Analysis of GPCR Oligomerization.
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DOI：
10.3390/biom11101503
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发表时间：
2021-10-12
期刊：
影响因子：
5.5
通讯作者：
Simoncelli S
中科院分区：
文献类型：
--
作者：
Joseph MD;Tomas Bort E;Grose RP;McCormick PJ;Simoncelli S
G蛋白偶联受体(GPCRs)是已知的形成同源和异源低聚物的受体，它们被认为是调节其功能的关键。然而，研究这些复合体的存在和功能含义并不是一帆风顺的，因为得出有争议的结果取决于所采用的分析方法。在这里，我们使用一种名为qPAINT的定量单分子超分辨率成像技术来量化一个示例GPCR中的复杂形成。基于DNA-PAINT的qPAINT利用荧光标记的DNA成像器链与连接到蛋白质靶向抗体的互补DNA对接链之间的结合动力学来在纳米尺度上定量蛋白质拷贝数。我们通过一条新的管道进行qPAINT分析，以研究在控制、刺激和拮抗条件下，胰腺癌细胞株ASPC-1中高表达的嘌呤能受体Y2(P2Y2)的寡聚。结果表明，在P2Y2受体密度保持不变的情况下，拮抗条件下低聚物的比例减少，复合体中的受体数量减少。然而，受体的寡聚体状态并没有受到激动剂治疗的影响，这与以前的报道一致。了解P2Y2在激动型和拮抗型条件下的寡聚作用将有助于解开P2Y2的作用机制和治疗靶向。
G-protein coupled receptors (GPCRs) are known to form homo- and hetero- oligomers which are considered critical to modulate their function. However, studying the existence and functional implication of these complexes is not straightforward as controversial results are obtained depending on the method of analysis employed. Here, we use a quantitative single molecule super-resolution imaging technique named qPAINT to quantify complex formation within an example GPCR. qPAINT, based upon DNA-PAINT, takes advantage of the binding kinetics between fluorescently labelled DNA imager strands to complementary DNA docking strands coupled to protein targeting antibodies to quantify the protein copy number in nanoscale dimensions. We demonstrate qPAINT analysis via a novel pipeline to study the oligomerization of the purinergic receptor Y2 (P2Y2), a rhodopsin-like GPCR, highly expressed in the pancreatic cancer cell line AsPC-1, under control, agonistic and antagonistic conditions. Results reveal that whilst the density of P2Y2 receptors remained unchanged, antagonistic conditions displayed reduced percentage of oligomers, and smaller numbers of receptors in complexes. Yet, the oligomeric state of the receptors was not affected by agonist treatment, in line with previous reports. Understanding P2Y2 oligomerization under agonistic and antagonistic conditions will contribute to unravelling P2Y2 mechanistic action and therapeutic targeting.
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影响因子：
16.6
作者：
Jagannath A;Varga N;Dallmann R;Rando G;Gosselin P;Ebrahimjee F;Taylor L;Mosneagu D;Stefaniak J;Walsh S;Palumaa T;Di Pretoro S;Sanghani H;Wakaf Z;Churchill GC;Galione A;Peirson SN;Boison D;Brown SA;Foster RG;Vasudevan SR
通讯作者：
Vasudevan SR
影响因子：
48
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Asher WB;Geggier P;Holsey MD;Gilmore GT;Pati AK;Meszaros J;Terry DS;Mathiasen S;Kaliszewski MJ;McCauley MD;Govindaraju A;Zhou Z;Harikumar KG;Jaqaman K;Miller LJ;Smith AW;Blanchard SC;Javitch JA
通讯作者：
Javitch JA
影响因子：
4.8
作者：
Kotevic, I;Kirschner, KM;Baltensperger, K
通讯作者：
Baltensperger, K
Ryanodine受体的可变聚类特性的真实分子尺度可视化。
影响因子：
8.8
作者：
Jayasinghe I;Clowsley AH;Lin R;Lutz T;Harrison C;Green E;Baddeley D;Di Michele L;Soeller C
通讯作者：
Soeller C
影响因子：
3.6
作者：
Choi, Roy C. Y.;Chu, Glanice K. Y.;Tsim, Karl W. K.
通讯作者：
Tsim, Karl W. K.