A Broad G Protein-Coupled Receptor Internalization Assay that Combines SNAP-Tag Labeling, Diffusion-Enhanced Resonance Energy Transfer, and a Highly Emissive Terbium Cryptate.
A Broad G Protein-Coupled Receptor Internalization Assay that Combines SNAP-Tag Labeling, Diffusion-Enhanced Resonance Energy Transfer, and a Highly Emissive Terbium Cryptate.
复制标题
DOI：
10.3389/fendo.2015.00167
复制
发表时间：
2015
影响因子：
5.2
通讯作者：
Bachelerie F
中科院分区：
文献类型：
--
作者：
Levoye A;Zwier JM;Jaracz-Ros A;Klipfel L;Cottet M;Maurel D;Bdioui S;Balabanian K;Prézeau L;Trinquet E;Durroux T;Bachelerie F
尽管G蛋白偶联受体（GPCR）内化长期以来被认为是调节配体反应性的脱敏过程的一个主要方面，但内化也参与受体重敏和信号传导，以及一些非典型受体的配体清除功能。因此，内化有助于 GPCR 依赖性信号传导的多样性，其在活细胞中的动态和定量引起了人们极大的兴趣。我们开发了一种稳健且灵敏的检测方法，用于跟踪和量化配体诱导和组成型诱导的 GPCR 内化以及活细胞中的受体循环。该测定基于用发光铽穴状化合物供体标记的细胞表面 GPCR 和培养基中存在的荧光素受体之间的扩散增强共振能量转移 (DERET)。 GPCR 内化导致能量转移的可量化减少。由于时间分辨测量，该方法产生高信噪比。对于属于不同类别的各种GPCR，我们证明可以将组成型和配体诱导的内化作为时间和配体浓度的函数进行监测，从而允许准确定量测定受体内化的动力学以及化合物的半最大有效或抑制浓度。除了其选择性和灵敏度之外，我们还提供了证据表明基于 DERET 的内化测定特别适合表征偏向配体。此外，确定 Z' 因子值为 0.45 表明基于 DERET 的内化测定对于可调节 GPCR 内化的化合物的高通量筛选 (HTS) 的质量和适用性。
Although G protein-coupled receptor (GPCR) internalization has long been considered as a major aspect of the desensitization process that tunes ligand responsiveness, internalization is also involved in receptor resensitization and signaling, as well as the ligand scavenging function of some atypical receptors. Internalization thus contributes to the diversity of GPCR-dependent signaling, and its dynamics and quantification in living cells has generated considerable interest. We developed a robust and sensitive assay to follow and quantify ligand-induced and constitutive-induced GPCR internalization but also receptor recycling in living cells. This assay is based on diffusion-enhanced resonance energy transfer (DERET) between cell surface GPCRs labeled with a luminescent terbium cryptate donor and a fluorescein acceptor present in the culture medium. GPCR internalization results in a quantifiable reduction of energy transfer. This method yields a high signal-to-noise ratio due to time-resolved measurements. For various GPCRs belonging to different classes, we demonstrated that constitutive and ligand-induced internalization could be monitored as a function of time and ligand concentration, thus allowing accurate quantitative determination of kinetics of receptor internalization but also half-maximal effective or inhibitory concentrations of compounds. In addition to its selectivity and sensitivity, we provided evidence that DERET-based internalization assay is particularly suitable for characterizing biased ligands. Furthermore, the determination of a Z′-factor value of 0.45 indicates the quality and suitability of DERET-based internalization assay for high-throughput screening (HTS) of compounds that may modulate GPCRs internalization.