Multiple mechanisms of self-association of chemokine receptors CXCR4 and CCR5 demonstrated by deep mutagenesis.

The Journal of Biological Chemistry Pub Date : 2023-10-01 Epub Date: 2023-09-09 DOI:10.1016/j.jbc.2023.105229
Kevin S Gill, Kritika Mehta, Jeremiah D Heredia, Vishnu V Krishnamurthy, Kai Zhang, Erik Procko
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Abstract

Chemokine receptors are members of the rhodopsin-like class A GPCRs whose signaling through G proteins drives the directional movement of cells in response to a chemokine gradient. Chemokine receptors CXCR4 and CCR5 have been extensively studied due to their roles in leukocyte development and inflammation and their status as coreceptors for HIV-1 infection, among other roles. Both receptors form dimers or oligomers of unclear function. While CXCR4 has been crystallized in a dimeric arrangement, available atomic resolution structures of CCR5 are monomeric. To investigate their dimerization interfaces, we used a bimolecular fluorescence complementation (BiFC)-based screen and deep mutational scanning to find mutations that change how the receptors self-associate, either via specific oligomer assembly or alternative mechanisms of clustering in close proximity. Many disruptive mutations promoted self-associations nonspecifically, suggesting they aggregated in the membrane. A mutationally intolerant region was found on CXCR4 that matched the crystallographic dimer interface, supporting this dimeric arrangement in living cells. A mutationally intolerant region was also observed on the surface of CCR5 by transmembrane helices 3 and 4. Mutations predicted from the scan to reduce BiFC were validated and were localized in the transmembrane domains as well as the C-terminal cytoplasmic tails where they reduced lipid microdomain localization. A mutation in the dimer interface of CXCR4 had increased binding to the ligand CXCL12 and yet diminished calcium signaling. There was no change in syncytia formation with cells expressing HIV-1 Env. The data highlight that multiple mechanisms are involved in self-association of chemokine receptor chains.

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通过深度诱变证明了趋化因子受体CXCR4和CCR5的多种自缔合机制。
趋化因子受体是视紫红质样A类GPCR的成员,其通过G蛋白的信号传导驱动细胞响应趋化因子梯度的定向运动。趋化因子受体CXCR4和CCR5由于其在白细胞发育和炎症中的作用以及其作为HIV-1感染的辅助受体的地位等作用而被广泛研究。两种受体都形成功能不明确的二聚体或低聚物。虽然CXCR4已经以二聚体排列结晶,但CCR5的可用原子分辨率结构是单体的。为了研究它们的二聚化界面,我们使用了基于双分子荧光互补(BiFC)的筛选和深度突变扫描,以发现通过特定的低聚物组装或近距离聚集的替代机制改变受体自我结合方式的突变。许多破坏性突变促进了非特异性的自缔合,表明它们聚集在膜中。在CXCR4上发现了一个与晶体二聚体界面匹配的突变不耐受区,支持活细胞中的这种二聚体排列。跨膜螺旋3和4在CCR5的表面上也观察到突变不耐受区。通过扫描预测的减少BiFC的突变得到了验证,并定位在跨膜结构域以及C末端细胞质尾部,在那里它们减少了脂质微结构域的定位。CXCR4二聚体界面的突变增加了与配体CXCL12的结合,但减少了钙信号传导。与表达HIV-1Env的细胞的合胞体形成没有变化。这些数据强调,趋化因子受体链的自缔合涉及多种机制。
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