Full characterization of GPCR monomer-dimer dynamic equilibrium by single molecule imaging.
Full characterization of GPCR monomer-dimer dynamic equilibrium by single molecule imaging.
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DOI：
10.1083/jcb.201009128
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发表时间：
2011-02-07
期刊：
影响因子：
--
通讯作者：
Kusumi A
中科院分区：
文献类型：
--
作者：
Kasai RS;Suzuki KG;Prossnitz ER;Koyama-Honda I;Nakada C;Fujiwara TK;Kusumi A
单分子跟踪技术与数学建模相结合，充分确定动态单体-二聚体平衡的分子或质膜上，这将提供一个框架，了解信号转导途径启动和调节的动态二聚体的膜定位受体。受体二聚化对于许多信号传导途径是重要的。然而，单体-二聚体平衡从未被完全表征为具有2D平衡常数以及缔合/解离速率常数（称为超定量）的任何受体。在这里，我们确定了动态平衡的N-甲酰肽受体（FPR），化学引诱物G蛋白偶联受体（GPCR），在活细胞在37°C下，通过开发一个单一的荧光分子成像方法。在配位前后，二聚体-单体的二维平衡保持不变，平衡常数为3.6拷贝/μm2，解离速率常数和二维结合速率常数分别为11.0 s−1和3.1拷贝/μ m2 s −1。在受体拷贝数/μm2的生理表达水平下（约6，000拷贝/细胞），单体每150 ms持续转化为二聚体，二聚体在91 ms内解离为单体，在任何时刻，分别有2，500和3，500个受体分子参与瞬时二聚体和单体。不仅FPR二聚体迅速分解，而且FPR单体也非常迅速地转化为二聚体。
A single-molecule tracking technique coupled with mathematical modeling was developed for fully determining the dynamic monomer–dimer equilibrium of molecules in or on the plasma membrane, which will provide a framework for understanding signal transduction pathways initiated and regulated by dynamic dimers of membrane-localized receptors. Receptor dimerization is important for many signaling pathways. However, the monomer–dimer equilibrium has never been fully characterized for any receptor with a 2D equilibrium constant as well as association/dissociation rate constants (termed super-quantification). Here, we determined the dynamic equilibrium for the N-formyl peptide receptor (FPR), a chemoattractant G protein–coupled receptor (GPCR), in live cells at 37°C by developing a single fluorescent-molecule imaging method. Both before and after liganding, the dimer–monomer 2D equilibrium is unchanged, giving an equilibrium constant of 3.6 copies/µm2, with a dissociation and 2D association rate constant of 11.0 s−1 and 3.1 copies/µm2s−1, respectively. At physiological expression levels of ∼2.1 receptor copies/µm2 (∼6,000 copies/cell), monomers continually convert into dimers every 150 ms, dimers dissociate into monomers in 91 ms, and at any moment, 2,500 and 3,500 receptor molecules participate in transient dimers and monomers, respectively. Not only do FPR dimers fall apart rapidly, but FPR monomers also convert into dimers very quickly.
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