Mueller Anja, Mahmoud Nasir G, Strange Philip G
School of Animal and Microbial Sciences, University of Reading, PO Box 228, Reading, RG6 6AJ, UK.
Biochem Pharmacol. 2006 Sep 14;72(6):739-48. doi: 10.1016/j.bcp.2006.06.001. Epub 2006 Jul 17.
We have investigated the signalling properties of the chemokine receptor, CCR5, using several assays for agonism: stimulation of changes in intracellular Ca(2+) or CCR5 internalisation in CHO cells expressing CCR5 or stimulation of [(35)S]GTPgammaS binding in membranes of CHO cells expressing CCR5. Four isoforms of the chemokine CCL3 with different amino termini (CCL3, CCL3(2-70), CCL3(5-70), CCL3L1) were tested in these assays in order to probe structure/activity relationships. Each isoform exhibited agonism. The pattern of agonism (potency, maximal effect) was different in the three assays, although the rank order was the same with CCL3L1 being the most potent and efficacious. The data show that the amino terminus of the chemokine is important for signalling. A proline at position 2 (CCL3L1) provides for high potency and efficacy but the isoform with a serine at position 2 (CCL3(2-70)) is as efficacious in some assays showing that the proline is not the only determinant of high efficacy. We also increased the sensitivity of CCR5 signalling by treating cells with sodium butyrate, thus increasing the receptor/G protein ratio. This allowed the detection of a change in intracellular Ca(2+) after treatment with CCL7 and Met-RANTES showing that these ligands possess measurable but low efficacy. This study therefore shows that sodium butyrate treatment increases the sensitivity of signalling assays and enables the detection of efficacy in ligands previously considered as antagonists. The use of different assay systems, therefore, provides different estimates of efficacy for some ligands at this receptor.
我们使用了多种激动作用检测方法，对趋化因子受体CCR5的信号传导特性进行了研究：检测表达CCR5的CHO细胞内钙离子（Ca(2+)）变化、CCR5内化情况，或检测表达CCR5的CHO细胞膜中[(35)S]GTPγS结合情况。为了探究结构/活性关系，我们在这些检测中对具有不同氨基末端的趋化因子CCL3的四种同工型（CCL3、CCL3(2 - 70)、CCL3(5 - 70)、CCL3L1）进行了测试。每种同工型均表现出激动作用。尽管在三种检测中激动作用的排序相同，CCL3L1最为强效且有效，但激动作用模式（效力、最大效应）有所不同。数据表明趋化因子的氨基末端对信号传导很重要。第2位为脯氨酸的同工型（CCL3L1）具有高效力和高活性，但第2位为丝氨酸的同工型（CCL3(2 - 70)）在某些检测中同样有效，这表明脯氨酸并非高效力的唯一决定因素。我们还通过用丁酸钠处理细胞来提高CCR5信号传导的敏感性，从而增加受体/G蛋白比率。这使得在用CCL7和甲硫氨酸-RANTES处理后能够检测到细胞内钙离子（Ca(2+)）的变化，表明这些配体具有可测量但较低的活性。因此，本研究表明丁酸钠处理可提高信号传导检测的敏感性，并能够检测到先前被视为拮抗剂的配体的活性。所以，使用不同的检测系统会对某些配体在该受体上的活性给出不同的评估。
