Quantitative analysis of the formation and diffusion of A1-adenosine receptor-antagonist complexes in single living cells
Quantitative analysis of the formation and diffusion of A1-adenosine receptor-antagonist complexes in single living cells
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DOI：
10.1073/pnas.0400420101
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发表时间：
2004-03-30
影响因子：
11.1
通讯作者：
Hill, SJ
中科院分区：
文献类型：
--
作者：
Briddon, SJ;Middleton, RJ;Hill, SJ
A（1）-腺苷受体（A（1）-AR）是一种G蛋白偶联受体，介导腺苷在脑、心脏、肾脏和脂肪细胞中的许多生理作用。目前，配体与A（1）-AR的相互作用只能通过使用放射性配体结合在大细胞群上定量。为了提高这些测量的分辨率，我们设计并表征了以前未描述的A（1）-AR的荧光拮抗剂XAC-BY 630，其基于黄嘌呤胺同源物（XAC）。该化合物已被用于使用荧光相关光谱（FCS）在单细胞水平上定量配体-受体结合。XAC-BY 630是A（1）-AR介导的cAMP蓄积抑制[亲和常数（pK（B））的log（10）= 6.7]和磷酸肌醇蓄积刺激（pK（B）= 6.5）的竞争性拮抗剂。通过使用共聚焦显微镜，还可以在活的中国仓鼠卵巢（CHO）-A1细胞中观察到XAC-BY 630与细胞表面A（1）-AR的特异性结合。XAC-BY 630与CHO-A1细胞膜结合的FCS分析揭示了三种组分，扩散时间（tau（D））为62 μ s（tau（D1），游离配体）、17 ms（tau（D2），A（1）-AR-配体）和320 MS（tau（D3））。从荧光A（1）-AR-Topaz融合蛋白观察到的相似扩散时间（15 ms）证实tau（D2）是由配体-受体复合物扩散产生的。tau蛋白（D2）的定量显示，受体-配体复合物的数量随着游离配体浓度的增加而增加，并被选择性A（1）-AR拮抗剂8-环戊基-1，3-二丙基黄嘌呤减少。FCS与XAC-BY 630的组合将成为表征健康和疾病中单个活细胞中配体-A（1）-AR相互作用的有力工具。
The A(1)-adenosine receptor (A(1)-AR) is a G protein-coupled receptor that mediates many of the physiological effects of adenosine in the brain, heart, kidney, and adipocytes. Currently, ligand interactions with the A(1)-AR can be quantified on large cell populations only by using radioligand binding. To increase the resolution of these measurements, we have designed and characterized a previously undescribed fluorescent antagonist for the A(1)-AR, XAC-BY630, based on xanthine amine congener (XAC). This compound has been used to quantify ligand-receptor binding at a single cell level using fluorescence correlation spectroscopy (FCS). XAC-BY630 was a competitive antagonist of A(1)-AR-mediated inhibition of cAMP accumulation [log(10) of the affinity constant (pK(b)) = 6.7)] and stimulation of inositol phosphate accumulation (pK(b) = 6.5). Specific binding of XAC-BY630 to cell surface A(1)-AR could also be visualized in living Chinese hamster ovary (CHO)-A1 cells by using confocal microscopy. FCS analysis of XAC-BY630 binding to the membrane of CHO-A1 cells revealed three components with diffusion times (tau(D)) of 62 mus (tau(D1), free ligand), 17 ms (tau(D2), A(1)-AR-ligand), and 320 MS (tau(D3)). Confirmation that tau(D2) resulted from diffusion of ligand-receptor complexes came from the similar diffusion time observed for the fluorescent A(1)-AR-Topaz fusion protein (15 ms). Quantification of tau(D2) showed that the number of receptor-ligand complexes increased with increasing free ligand concentration and was decreased by the selective A(1)-AR antagonist, 8-cyclopentyl-1,3-dipropylxanthine. The combination of FCS with XAC-BY630 will be a powerful tool for the characterization of ligand-A(1)-AR interactions in single living cells in health and disease.