CC趋化因子受体2和CCL2高度参与癌症的生长和转移以及免疫逃逸。实体瘤中钠离子浓度升高也与转移和免疫调节相关。钠离子可以通过钠离子结合位点调节 A 类 G 蛋白偶联受体，该结合位点以高度保守的天冬氨酸残基 (D2.50) 为特征，该残基也存在于 CCR2 中。因此，我们通过放射性配体结合研究和诱变进一步探索了 CCR2 中的这个结合位点。研究了钠离子和阿米洛利衍生物对三种明显结合的放射性配体的调节。观察到钠离子是相对较弱的拮抗剂结合调节剂，但显着增加 125 I-CCL2 从 CCR2 的解离。 6-取代的六亚甲基阿米洛利 (HMA) 调节所有测试的放射性配体。 HMA 与 CCR2 的假定钠离子结合位点的诱导拟合对接证实了其结合位点。最后，对钠离子结合位点（癌症相关）突变的研究表明，与野生型相比，表达显着降低。只有两个突变体，G123A3.35和G127K3.39，能够被[ 3 H]INCB3344和[ 3 H]CCR2-RA-[R]结合。因此，诱变表明，钠离子结合位点残基不同于其他 A 类 GPCR，且与趋化因子受体进化相关，对于受体完整性至关重要。此外，测试的突变似乎对 HMA 观察到的调节没有影响，或者对测试的放射性配体的氯化钠调节影响较小。总而言之，这些结果需要进一步探索（癌症）生物学中的 CCR2 钠离子结合位点，并有可能作为第三个可药物结合位点。版权所有 © 2024。由 Elsevier Inc. 出版。

CC chemokine receptor 2 and CCL2 are highly involved in cancer growth and metastasis, and immune escape. Raised sodium ion concentrations in solid tumours have also been correlated to metastasis and immune modulation. Sodium ions can modulate class A G protein-coupled receptors through the sodium ion binding site characterized by a highly conserved aspartic acid residue (D2.50), also present in CCR2. Hence, we further explored this binding site in CCR2 by radioligand binding studies and mutagenesis. Modulation of three distinctly binding radioligands by sodium ions and amiloride derivates was investigated. Sodium ions were observed to be relatively weak modulators of antagonist binding, but substantially increased 125I-CCL2 dissociation from CCR2. 6-Substituted Hexamethylene Amiloride (HMA) modulated all tested radioligands. Induced-fit docking of HMA in the presumed sodium ion binding site of CCR2 confirmed its binding site. Finally, investigation of (cancer-associated) mutations in the sodium ion binding site showed a markedly decreased expression compared to wild type. Only two mutants, G123A3.35 and G127K3.39, were able to be bound by [3H]INCB3344 and [3H]CCR2-RA-[R]. Thus, mutagenesis showed that the sodium ion binding site residues, which are distinct from other class A GPCRs and related to chemokine receptor evolution, are crucial for receptor integrity. Moreover, the tested mutations appeared to have no effect on modulation observed by HMA or a minor effect on sodium chloride modulation on the tested radioligands. All in all, these results invite further exploration of the CCR2 sodium ion binding site in (cancer) biology, and potentially as a third druggable binding site.Copyright © 2024. Published by Elsevier Inc.