van Aalst Evan, Wylie Benjamin J
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, United States.
Front Mol Biosci. 2021 Aug 20;8:724603. doi: 10.3389/fmolb.2021.724603. eCollection 2021.
Cholesterol as an allosteric modulator of G protein-coupled receptor (GPCR) function is well documented. This quintessential mammalian lipid facilitates receptor-ligand interactions and multimerization states. Functionally, this introduces a complicated mechanism for the homeostatic modulation of GPCR signaling. Chemokine receptors are Class A GPCRs responsible for immune cell trafficking through the binding of endogenous peptide ligands. CCR3 is a CC motif chemokine receptor expressed by eosinophils and basophils. It traffics these cells by transducing the signal stimulated by the CC motif chemokine primary messengers 11, 24, and 26. These behaviors are close to the human immunoresponse. Thus, CCR3 is implicated in cancer metastasis and inflammatory conditions. However, there is a paucity of experimental evidence linking the functional states of CCR3 to the molecular mechanisms of cholesterol-receptor cooperativity. In this vein, we present a means to combine codon harmonization and a maltose-binding protein fusion tag to produce CCR3 from . This technique yields ∼2.6 mg of functional GPCR per liter of minimal media. We leveraged this protein production capability to investigate the effects of cholesterol on CCR3 function . We found that affinity for the endogenous ligand CCL11 increases in a dose-dependent manner with cholesterol concentration in both styrene:maleic acid lipid particles (SMALPs) and proteoliposomes. This heightened receptor activation directly translates to increased signal transduction as measured by the GTPase activity of the bound G-protein inhibitory subunit 3 (G 3). This work represents a critical step forward in understanding the role of cholesterol-GPCR allostery in regulation of signal transduction.
胆固醇作为G蛋白偶联受体（GPCR）功能的变构调节剂已有充分记载。这种典型的哺乳动物脂质促进受体 - 配体相互作用和多聚化状态。在功能上，这为GPCR信号的稳态调节引入了一种复杂机制。趋化因子受体是A类GPCR，通过内源性肽配体的结合负责免疫细胞运输。CCR3是一种由嗜酸性粒细胞和嗜碱性粒细胞表达的CC基序趋化因子受体。它通过转导由CC基序趋化因子主要信使11、24和26刺激的信号来运输这些细胞。这些行为与人类免疫反应密切相关。因此，CCR3与癌症转移和炎症状态有关。然而，将CCR3的功能状态与胆固醇 - 受体协同作用的分子机制联系起来的实验证据很少。在这方面，我们提出了一种将密码子优化和麦芽糖结合蛋白融合标签相结合的方法，以从[具体来源]产生CCR3。该技术每升基本培养基可产生约2.6毫克功能性GPCR。我们利用这种蛋白质生产能力来研究胆固醇对CCR3功能的影响。我们发现，在苯乙烯：马来酸脂质颗粒（SMALP）和蛋白脂质体中，对内源性配体CCL11的亲和力都随着胆固醇浓度呈剂量依赖性增加。这种增强的受体激活直接转化为信号转导增加，这通过结合的G蛋白抑制亚基3（Gαi-3）的GTP酶活性来衡量。这项工作代表了在理解胆固醇 - GPCR变构在信号转导调节中的作用方面向前迈出的关键一步。
