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Imaging GPCR internalization using near-infrared Nebraska red-based reagents
Organic & Biomolecular Chemistry ( IF 2.9 ) Pub Date : 2020/03/06 , DOI: 10.1039/d0ob00043d
Lauren Lesiak 1, 2, 3, 4 , Xinqi Zhou 1, 2, 3, 4 , Yuan Fang 1, 1, 2, 3, 4 , Jia Zhao 1, 2, 3, 4 , Jon R. Beck 1, 2, 3, 4 , Cliff I. Stains 1, 1, 2, 3, 4
Affiliation  

Internalization of G protein-coupled receptor (GPCRs) represents a nearly universal pathway for receptor downregulation. Imaging this process provides a means for the identification of pharmaceutical agents as well as potential ligands for orphan receptors. However, there is a need for the further development of near-infrared (NIR) probes capable of monitoring internalization in order to enable multiplexing with existing green fluorescent GPCR activity assays. Our laboratory has recently described a series of near-infrared (NIR) fluorophores in which a phosphinate functionality is inserted at the bridging position of the xanthene scaffold. These fluorophores, termed Nebraska Red (NR) dyes, provide attractive reagents for imaging protein localization. Herein, we disclose the development of NR-based HaloTag ligands for imaging membrane proteins on living cells. These new probes are utilized to image membrane pools of the human orexin type 2 receptor, an established target for the treatment of insomnia. We demonstrate the ability of fetal bovine serum (FBS) to noncovalently associate with a spirolactonized NR probe, enabling no-wash imaging with a 45-fold enhancement of fluorescence. Furthermore, we characterize the utility of NR-based HaloTag ligands for real-time monitoring of receptor internalization upon agonist stimulation. These new reagents enable potential multiplexing with existing GPCR activity assays in order to identify new modulators of GPCR activity as well as ligands for orphan receptors.

中文翻译:

使用近红外内布拉斯加州红试剂对GPCR内在化进行成像

G蛋白偶联受体(GPCR)的内部化代表了受体下调的几乎普遍途径。对这一过程进行成像,为鉴定药剂以及孤儿受体的潜在配体提供了一种手段。但是,需要进一步开发能够监测内部化的近红外(NIR)探针,以便能够与现有的绿色荧光GPCR活性测定法进行多路复用。我们的实验室最近描述了一系列近红外(NIR)荧光团,其中在the吨骨架的桥接位置插入了次膦酸酯官能团。这些被称为内布拉斯加州红(NR)染料的荧光团为成像蛋白质的定位提供了诱人的试剂。在这里 我们公开了用于成像活细胞上的膜蛋白的基于NR的HaloTag配体的开发。这些新的探针可用于为人类orexin 2型受体的膜池成像,这是治疗失眠的既定目标。我们证明了胎牛血清(FBS)与螺内酰胺化NR探针非共价结合的能力,使免洗成像的荧光强度提高了45倍。此外,我们表征了基于NR的HaloTag配体在激动剂刺激后实时监测受体内在化的效用。这些新试剂能够与现有的GPCR活性测定法进行潜在的多路复用,以鉴定GPCR活性的新调节剂以及孤儿受体的配体。确定的失眠治疗目标。我们证明了胎牛血清(FBS)与螺内酰胺化NR探针非共价结合的能力,使免洗成像的荧光强度提高了45倍。此外,我们表征了基于NR的HaloTag配体在激动剂刺激后实时监测受体内在化的效用。这些新试剂能够与现有的GPCR活性测定法进行潜在的多路复用,以鉴定GPCR活性的新调节剂以及孤儿受体的配体。确定的失眠治疗目标。我们证明了胎牛血清(FBS)与螺内酰胺化NR探针非共价结合的能力,使免洗成像的荧光强度提高了45倍。此外,我们表征了基于NR的HaloTag配体在激动剂刺激后实时监测受体内在化的效用。这些新试剂能够与现有的GPCR活性测定法进行潜在的多路复用,以鉴定GPCR活性的新调节剂以及孤儿受体的配体。我们表征了基于NR的HaloTag配体在激动剂刺激后实时监测受体内在化的效用。这些新试剂能够与现有的GPCR活性测定法进行潜在的多路复用,以鉴定GPCR活性的新调节剂以及孤儿受体的配体。我们表征了基于NR的HaloTag配体在激动剂刺激后实时监测受体内在化的效用。这些新试剂能够与现有的GPCR活性测定法进行潜在的多路复用,以鉴定GPCR活性的新调节剂以及孤儿受体的配体。
更新日期:2020-04-01
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