DataSheet1_Cholesterol Is a Dose-Dependent Positive Allosteric Modulator of CCR3 Ligand Affinity and G Protein Coupling.pdf

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 E. coli. 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 in vitro. 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 α inhibitory subunit 3 (Gαi3). 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）的变构调节剂，其功能已得到充分证实。这种典型的哺乳动物脂质可促进受体-配体相互作用及多聚化状态的形成。从功能层面而言，这为GPCR信号转导的稳态调控引入了复杂的调控机制。
趋化因子受体属于A类GPCR，通过结合内源性肽配体介导免疫细胞的趋化迁移。CCR3是表达于嗜酸性粒细胞与嗜碱性粒细胞的CC基序趋化因子受体，其通过转导CC基序趋化因子第一信使11、24和26所激活的信号，介导上述两类细胞的趋化迁移。这些过程与人体免疫应答密切相关，因此CCR3与癌症转移及多种炎症性疾病密切相关。然而，目前尚缺乏将CCR3的功能状态与胆固醇-受体协同作用的分子机制相关联的实验证据。
为此，我们开发了一种结合密码子适配与麦芽糖结合蛋白融合标签的方法，可从大肠杆菌中表达制备CCR3蛋白。该技术可在每升基础培养基中获得约2.6毫克具有生物活性的GPCR蛋白。我们借助这一蛋白制备能力，在体外探究了胆固醇对CCR3功能的影响。我们发现，在苯乙烯-马来酸脂质颗粒（SMALPs）与蛋白脂质体两种体系中，CCR3对内源性配体CCL11的亲和力均随胆固醇浓度升高呈剂量依赖性增强。这种增强的受体激活可直接转化为信号转导效率的提升，这可通过结合的G蛋白抑制性α亚基3（Gαi3）的GTP酶活性得以验证。本研究为理解胆固醇-GPCR变构作用在信号转导调控中的功能提供了关键进展。