Molecular Inflammation Research Center for Aging Intervention (MRCA) and College of Pharmacy, Pusan National University, Busan, Korea.
Br J Pharmacol. 2018 Jan;175(1):154-161. doi: 10.1111/bph.14082. Epub 2017 Dec 8.
GPR35 has long been considered an orphan GPCR, because no endogenous ligand of GPR35 has been discovered. CXCL17 (a chemokine) has been reported to be an endogenous ligand of GPR35, and it has even been suggested that it be called CXCR8. However, at present there is no supporting evidence that CXCL17 does interact with GPR35.
We applied two assay systems to explore the relationship between CXCL17 and GPR35. An AP-TGF-α shedding assay in GPR35 over-expressing HEK293 cells was used as a gain-of-function assay. GPR35 knock-down by siRNA transfection was performed in endogenously GPR35-expressing THP-1 cells.
In the AP-TGF-α shedding assay, lodoxamide, a well-known synthetic GPR35 agonist, was confirmed to be the most potent agonist among other reported agonists. However, neither human nor mouse CXCL17 had an effect on GPR35. Consistent with previous findings, G proteins Gα and Gα were found to couple with GPR35. Furthermore, lodoxamide-induced activation of GPR35 was concentration-dependently inhibited by CID2745687 (a selective GPR35 antagonist). In endogenously GPR35-expressing THP-1 cells, lodoxamide concentration-dependently inhibited migration and this inhibitory effect was blocked by CID2745687 treatment or GPR35 siRNA transfection. However, even though CXCL17 stimulated the migration of THP-1 cells, which is consistent with a previous report, this stimulatory effect of CXCL17 was not blocked by CID2745687 or GPR35 siRNA.
The present findings suggest that GPR35 functions as a migration inhibitory receptor, but CXCL17-stimulated migration of THP-1 cells is not dependent on GPR35.
GPR35 长期以来被认为是孤儿 GPCR，因为尚未发现 GPR35 的内源性配体。趋化因子 CXCL17 已被报道为 GPR35 的内源性配体，甚至有人建议将其称为 CXCR8。然而，目前尚无支持 CXCL17 确实与 GPR35 相互作用的证据。
我们应用了两种检测系统来探讨 CXCL17 与 GPR35 之间的关系。在过表达 GPR35 的 HEK293 细胞中，AP-TGF-α 脱落测定被用作功能获得测定。通过 siRNA 转染在内源性表达 GPR35 的 THP-1 细胞中敲低 GPR35。
在 AP-TGF-α 脱落测定中，证实了洛度胺（一种众所周知的合成 GPR35 激动剂）是报告的其他激动剂中最有效的激动剂。然而，无论是人源还是鼠源 CXCL17 都对 GPR35 没有作用。与先前的发现一致，发现 G 蛋白 Gα 和 Gα 与 GPR35 偶联。此外，洛度胺诱导的 GPR35 激活被 CID2745687（一种选择性 GPR35 拮抗剂）浓度依赖性抑制。在内源性表达 GPR35 的 THP-1 细胞中，洛度胺浓度依赖性地抑制迁移，而这种抑制作用可被 CID2745687 处理或 GPR35 siRNA 转染阻断。然而，尽管 CXCL17 刺激 THP-1 细胞迁移，这与先前的报告一致，但 CXCL17 的这种刺激作用不受 CID2745687 或 GPR35 siRNA 的阻断。
本研究结果表明，GPR35 作为迁移抑制受体发挥作用，但 CXCL17 刺激 THP-1 细胞迁移不依赖于 GPR35。
