Signalling by CXC-chemokine receptors 1 and 2 expressed in CHO cells:: a comparison of calcium mobilization, inhibition of adenylyl cyclase and stimulation of GTPγS binding induced by IL-8 and GROα
Signalling by CXC-chemokine receptors 1 and 2 expressed in CHO cells:: a comparison of calcium mobilization, inhibition of adenylyl cyclase and stimulation of GTPγS binding induced by IL-8 and GROα
复制标题
DOI：
10.1038/sj.bjp.0702329
复制
发表时间：
1999-02-01
影响因子：
7.3
通讯作者：
Giles, H
中科院分区：
文献类型：
--
作者：
Hall, DA;Beresford, IJM;Giles, H
1白细胞介素-8（IL-8）和生长相关癌基因α的作用（GRO α）对[S-35]-鸟苷5 '-O-（3-硫代三磷酸）（[S-35]GTP γ S）结合，在表达HA标记的CXC趋化因子受体1和2的重组CHO细胞中测定毛喉素刺激的环AMP积累和胞浆钙浓度（CXCR 1和CXCR 2）。2放射性配体结合试验证实，重组受体的结合特征与天然蛋白相似。IL-8取代[I-125]-IL-8与CXCR 1和CXCR 2的结合，pK（1）值分别为8.89 +/- 0.05和9.27 +/- 0.03。GRO α是一种选择性CXCR 2配体，在CXCR 2上的pK（1）值为9.66 +/- 0.39，但在CXCR 1上的pK（i）> 8。钙动员实验也与先前关于天然受体的报告一致。3两种受体的激活导致刺激[S-35]GTP γ S结合和抑制腺苷酸环化酶。4 CXCR 1的功能数据比较显示，在所有三种测定中获得了相似的效力顺序（IL-8 > > GRO α）。然而，在CXCR 2，虽然钙动员和腺苷酸环化酶抑制的效力顺序相似（IL-8大于或等于GRO α），但刺激[S-35]GTP γ S结合的顺序相反（GRO α> IL-8）。5两种受体的所有功能反应均受到百日咳毒素（PTX）抑制，表明与Gi/Go蛋白偶联。然而，IL-8在CXCR 1诱导的钙动员并没有被PTX完全抑制，这表明与Gq家族的G蛋白的相互作用。我们对百日咳毒素的研究结果还表明，在[S-35]GTP γ S结合试验中，CXCR 1显示出一定的组成性活性。我们已经表征了HA标记的CXCR 1和2的结合和几种功能反应，并表明它们的药理学与天然受体的药理学完全一致。我们也有初步的证据表明，CXCR 1在我们的细胞系中表现出组成型活性，CXCR 2可能在不同的PTX敏感性G蛋白之间运输。
1 The effect of interleukin-8 (IL-8) and growth-related oncogene alpha (GRO alpha) on [S-35]-guanosine 5'-O-(3-thiotriphosphate) ([S-35]GTP gamma S) binding, forskolin-stimulated cyclic AMP accumulation and cytosolic calcium concentration were determined in recombinant CHO cells expressing HA-tagged CXC-chemokine receptors 1 and 2 (CXCR1 and CXCR2).2 Radioligand binding assays confirmed that the binding profiles of the recombinant receptors were similar to those of the native proteins. IL-8 displaced [I-125]-IL-8 binding to CXCR1 and CXCR2 with pK(1) values of 8.89 +/- 0.05 and 9.27 +/- 0.03, respectively. GRO alpha, a selective CXCR2 ligand, had a pK(1) value of 9.66 +/- 0.39 at CXCR2 but a pK(i) > 8 at CXCR1. Calcium mobilization experiments were also consistent with previous reports on native receptors.3 Activation of both receptors resulted in stimulation of [S-35]GTP gamma S binding and inhibition of adenylyl cyclase.4 A comparison of the functional data at CXCR1 showed that a similar potency order (IL-8 > > GRO alpha) was obtained in all three assays. However, at CXCR2 whilst the potency orders for calcium mobilization and inhibition of adenylyl cyclase were similar (IL-8 greater than or equal to GRO alpha), the order was reversed for stimulation of [S-35]GTP gamma S binding (GRO alpha > IL-8).5 All of the functional responses at both receptors were inhibited by pertussis toxin (PTX), suggesting coupling to a Gi/Go protein. However, the calcium mobilization induced by IL-8 at CXCR1 was not fully inhibited by PTX, suggesting an interaction with a G-protein of the Gq family. Our results with pertussis toxin also suggested that, in the [S-35]GTP gamma S binding assay, CXCR1 displays some constitutive activity.6 Thus. we have characterized the binding and several functional responses at HA-tagged CXCRs 1 and 2 and have shown that their pharmacology agrees well with that of the native receptors. We also have preliminary evidence that CXCR1 displays constitutive activity in our cell line and that CXCR2 may traffic between different PTX sensitive G-proteins.