Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, The Panum Institute, University of Copenhagen, Copenhagen, Denmark.
Br J Pharmacol. 2014 Mar;171(6):1566-79. doi: 10.1111/bph.12553.
A conserved amino acid within a protein family indicates a significance of the residue. In the centre of transmembrane helix (TM)-5, position V:13/5.47, an aromatic amino acid is conserved among class A 7TM receptors. However, in 37% of chemokine receptors - a subgroup of 7TM receptors - it is a leucine indicating an altered function. Here, we describe the significance of this position and its possible interaction with TM-3 for CCR5 activity.
The effects of [L203F]-CCR5 in TM-5 (position V:13/5.47), [I116A]-CCR5 in TM-3 (III:16/3.40) and [L203F;G286F]-CCR5 (V:13/5.47;VII:09/7.42) were determined in G-protein- and β-arrestin-coupled signalling. Computational modelling monitored changes in amino acid conformation.
[L203F]-CCR5 increased the basal level of G-protein coupling (20-70% of Emax ) and β-arrestin recruitment (50% of Emax ) with a threefold increase in agonist potency. In silico, [I116A]-CCR5 switched χ1-angle in [L203F]-CCR5. Furthermore, [I116A]-CCR5 was constitutively active to a similar degree as [L203F]-CCR5. Tyr(244) in TM-6 (VI:09/6.44) moved towards TM-5 in silico, consistent with its previously shown function for CCR5 activation. On [L203F;G286F]-CCR5 the antagonist aplaviroc was converted to a superagonist.
The results imply that an aromatic amino acid in the centre of TM-5 controls the level of receptor activity. Furthermore, Ile(116) acts as a gate for the movement of Tyr(244) towards TM-5 in the active state, a mechanism proposed previously for the β2 -adrenoceptor. The results provide an understanding of chemokine receptor function and thereby information for the development of biased and non-biased antagonists and inverse agonists.
在蛋白质家族中，保守的氨基酸表明该残基具有重要意义。在跨膜螺旋（TM）-5 的中心位置 V：13/5.47，A 类 7TM 受体中的芳香族氨基酸是保守的。然而，在 37%的趋化因子受体（7TM 受体的一个亚群）中，它是亮氨酸，表明其功能发生了改变。在这里，我们描述了该位置的意义及其与 TM-3 相互作用对 CCR5 活性的可能影响。
研究了 TM-5（位置 V：13/5.47）中的[L203F]-CCR5、TM-3（III：16/3.40）中的[I116A]-CCR5 和 [L203F;G286F]-CCR5（V：13/5.47；VII：09/7.42）的作用，这些作用涉及 G 蛋白偶联和β-抑制蛋白偶联信号转导。计算建模监测了氨基酸构象的变化。
[L203F]-CCR5 增加了 G 蛋白偶联的基础水平（20-70%的 Emax）和β-抑制蛋白募集（50%的 Emax），并使激动剂的效力增加了三倍。在计算机模拟中，[I116A]-CCR5 改变了 [L203F]-CCR5 的 χ1 角。此外，[I116A]-CCR5 与 [L203F]-CCR5 一样具有组成性活性。在计算机模拟中，TM-6（VI：09/6.44）中的 Tyr（244）向 TM-5 移动，这与之前研究表明的其对 CCR5 激活的作用一致。在 [L203F;G286F]-CCR5 上，拮抗剂 aplaviroc 转化为超激动剂。
研究结果表明，TM-5 中心的芳香族氨基酸控制受体活性的水平。此外，Ile（116）作为 Tyr（244）在活性状态下向 TM-5 移动的门控，这是先前提出的β2-肾上腺素受体的机制。研究结果提供了对趋化因子受体功能的理解，从而为开发偏性和非偏性拮抗剂和反向激动剂提供了信息。
