Department of Pharmacology, University of Washington School of Medicine, Seattle, WA, USA.
SLAS Discov. 2021 Apr;26(4):570-578. doi: 10.1177/2472555220979793. Epub 2021 Jan 5.
We have developed a novel reporter assay that leverages SNAP-epitope tag/near-infrared (NIR) imaging technology to monitor G protein-coupled receptor (GPCR) degradation in human cell lines. N-terminal SNAP-tagged GPCRs were subcloned and expressed in human embryonic kidney (HEK) 293 cells and then subjected to 24 h of cycloheximide (CHX)-chase degradation assays to quantify receptor degradation half-lives () using LICOR NIR imaging-polyacrylamide gel electrophoresis (PAGE) analysis. Thus far, we have used this method to quantify for all nine adrenergic (ADRA1A, ADRA1B, ADRA1D, ADRA2A, ADRA2B, ADRA2C, ADRB1, ADRB2, ADRB3), five somatostatin (SSTR1, SSTR2, SSTR3, SSTR4, SSTR5), four chemokine (CXCR1, CXCR2, CXCR3, CXCR5), and three 5-HT2 (5HT2A, 5HT2B, 5HT2C) receptor subtypes. SNAP-GPCR-CHX degradation values ranged from 0.52 h (ADRA1D) to 5.5 h (SSTR3). On the contrary, both the SNAP-tag alone and SNAP-tagged and endogenous β-actin were resistant to degradation with CHX treatment. Treatment with the proteasome inhibitor bortezomib produced significant but variable increases in SNAP-GPCR protein expression levels, indicating that SNAP-GPCR degradation primarily occurs through the proteasome. Remarkably, endogenous β2-adrenergic receptor/ADRB2 dynamic mass redistribution functional responses to norepinephrine were significantly decreased following CHX treatment, with a time course equivalent to that observed with the SNAP-ADRB2 degradation assay. We subsequently adapted this assay into a 96-well glass-bottom plate format to facilitate high-throughput GPCR degradation screening. values quantified for the α-adrenergic receptor subtypes (ADRA1A, ADRA1B, ADR1D) using the 96-well-plate format correlated with values quantified using NIR-PAGE imaging analysis. In summary, this novel assay permits precise quantitative analysis of GPCR degradation in human cells and can be readily adapted to quantify degradation for any membrane protein of interest.
我们开发了一种新颖的报告测定法，该方法利用 SNAP- 表位标签/近红外 (NIR) 成像技术来监测人细胞系中的 G 蛋白偶联受体 (GPCR) 降解。N 端 SNAP 标记的 GPCR 被亚克隆并在人胚肾 (HEK) 293 细胞中表达，然后进行 24 小时的环己酰亚胺 (CHX) 追踪降解测定，以使用 LICOR NIR 成像-聚丙烯酰胺凝胶电泳 (PAGE) 分析定量受体降解半衰期 (t1/2)。到目前为止，我们已经使用该方法对所有九个肾上腺素能 (ADRA1A、ADRA1B、ADRA1D、ADRA2A、ADRA2B、ADRA2C、ADRB1、ADRB2、ADRB3)、五个生长抑素 (SSTR1、SSTR2、SSTR3、SSTR4、SSTR5)、四个趋化因子 (CXCR1、CXCR2、CXCR3、CXCR5) 和三个 5-HT2 (5HT2A、5HT2B、5HT2C) 受体亚型进行了定量。SNAP-GPCR-CHX 降解 t1/2 值范围从 0.52 小时 (ADRA1D) 到 5.5 小时 (SSTR3)。相反，单独的 SNAP 标签和 SNAP 标记的内源性 β-肌动蛋白都能抵抗 CHX 处理的降解。用蛋白酶体抑制剂硼替佐米处理会显著但可变地增加 SNAP-GPCR 蛋白表达水平，表明 SNAP-GPCR 降解主要通过蛋白酶体发生。值得注意的是，内源性β2-肾上腺素能受体/ADRB2 对去甲肾上腺素的动态质量重分布功能反应在 CHX 处理后显著降低，与 SNAP-ADRB2 降解测定中观察到的时间过程相当。随后，我们将该测定法改编为 96 孔玻璃底平板格式，以方便高通量 GPCR 降解筛选。使用 96 孔板格式定量的 α-肾上腺素能受体亚型 (ADRA1A、ADRA1B、ADR1D) 的 t1/2 值与使用 NIR-PAGE 成像分析定量的值相关。总之，该新测定法可精确定量分析人细胞中的 GPCR 降解，并可轻易适应于定量任何感兴趣的膜蛋白的降解。
