Bioluminescent CXCL12 fusion protein for cellular studies of CXCR4 and CXCR7.
Bioluminescent CXCL12 fusion protein for cellular studies of CXCR4 and CXCR7.
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DOI：
10.2144/000113126
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发表时间：
2009-07
期刊：
影响因子：
2.7
通讯作者：
Luker G
中科院分区：
文献类型：
--
作者：
Luker K;Gupta M;Luker G
趋化因子CXCL12及其两种已知受体CXCR4和CXCR7可能在包括肿瘤生长和转移、动脉粥样硬化以及艾滋病病毒感染等疾病中发挥作用。因此，这些分子可能是药物开发的有前景的靶点。由于发光检测具有高灵敏度和高信噪比，细胞信号传导研究以及用于药物研发的高通量筛选越来越多地基于发光检测，但目前尚无针对趋化因子 - 趋化因子受体结合的生物发光检测方法。为了开发一种用于趋化因子结合和细胞摄取的生物发光探针，我们将CXCL12与高斯荧光素酶融合，高斯荧光素酶是一种不依赖ATP的酶，是已知最小的荧光素酶。将CXCL12与高斯荧光素酶融合（CXCL12 - GL）不会改变生物发光发射光谱，并且在不同的pH、温度和NaCl条件下对酶的功能影响极小。CXCL12 - GL还能在与未融合的CXCL12相当的程度上激活依赖于CXCR4的信号传导。通过多孔板检测，我们确定CXCR7使细胞相关的CXCL12增加的程度明显大于CXCR4。我们还表明，CXCL12 - GL可用于量化特异性靶向CXCR7的化合物对趋化因子受体结合的抑制作用。这些数据验证了CXCL12 - GL作为一种生物发光探针，可用于研究CXCR4和CXCR7的分子功能以及筛选调节配体 - 受体结合的化合物。
Chemokine CXCL12 and its two known receptors, CXCR4 and CXCR7, may play a role in diseases including tumor growth and metastasis, atherosclerosis, and HIV infection. Therefore, these molecules may be promising targets for drug development. While studies of cell signaling and high-throughput screening for drug discovery increasingly are based on luminescent assays because of their high sensitivity and signal-to-background ratio, there currently is no bioluminescent assay for chemokine-chemokine receptor binding. To develop a bioluminescent probe for chemokine binding and cellular uptake, we fused CXCL12 to Gaussia luciferase, an ATP-independent enzyme that is the smallest known luciferase. Fusing CXCL12 to Gaussia luciferase (CXCL12-GL) did not alter the bioluminescence emission spectrum and only minimally affected enzyme function under varying conditions of pH, temperature, and NaCl. CXCL12-GL also activated CXCR4-dependent signaling to a comparable extent as unfused CXCL12. Using multiwell plate assays, we established that CXCR7 increases cell-associated CXCL12 to a significantly greater extent than CXCR4. We also showed that CXCL12-GL can be used to quantify inhibition of chemokine receptor binding by compounds specifically targeting CXCR7. These data validate CXCL12-GL as a bioluminescent probe to investigate molecular functions of CXCR4 and CXCR7 and screen for compounds that modulate ligand-receptor binding.
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影响因子：
3.7
作者：
Badr CE;Hewett JW;Breakefield XO;Tannous BA
通讯作者：
Tannous BA
影响因子：
6.7
作者：
Braunersreuther, Vincent;Mach, Francois;Steffens, Sabine
通讯作者：
Steffens, Sabine
DOI：
10.1073/pnas.2235846100
发表时间：
2003-11-11
影响因子：
11.1
作者：
Rubin, JB;Kung, AL;Segal, RA
通讯作者：
Segal, RA
影响因子：
3.6
作者：
Fredriksson, R;Schiöth, HB
通讯作者：
Schiöth, HB
影响因子：
7.4
作者：
Luker, Kathryn E.;Gupta, Mudit;Luker, Gary D.
通讯作者：
Luker, Gary D.