Within the intestine, the human G protein–coupled receptor (GPCR) GPR35 is involved in oncogenic signaling, bacterial infections, and inflammatory bowel disease. GPR35 is known to be expressed as two distinct isoforms that differ only in the length of their extracellular N-termini by 31 amino acids, but detailed insights into their functional differences are lacking. Through gene expression analysis in immune and gastrointestinal cells, we show that these isoforms emerge from distinct promoter usage and alternative splicing. Additionally, we employed optical assays in living cells to thoroughly profile both GPR35 isoforms for constitutive and ligand-induced activation and signaling of 10 different heterotrimeric G proteins, ligand-induced arrestin recruitment, and receptor internalization. Our results reveal that the extended N-terminus of the long isoform limits G protein activation yet elevates receptor–β-arrestin interaction. To better understand the structural basis for this bias, we examined structural models of GPR35 and conducted experiments with mutants of both isoforms. We found that a proposed disulfide bridge between the N-terminus and extracellular loop 3, present in both isoforms, is crucial for constitutive G₁₃ activation, while an additional cysteine contributed by the extended N-terminus of the long GPR35 isoform limits the extent of agonist-induced receptor–β-arrestin2 interaction. The pharmacological profiles and mechanistic insights of our study provide clues for the future design of isoform-specific GPR35 ligands that selectively modulate GPR35–transducer interactions and allow for mechanism-based therapies against, for example, inflammatory bowel disease or bacterial infections of the gastrointestinal system.

在肠道内，人类 G 蛋白偶联受体 (GPCR) GPR35 参与致癌信号传导、细菌感染和炎症性肠病。已知 GPR35 表达为两种不同的亚型，它们的胞外 N 末端长度仅相差 31 个氨基酸，但缺乏对其功能差异的详细了解。通过免疫和胃肠细胞中的基因表达分析，我们表明这些亚型来自不同的启动子使用和可变剪接。此外，我们在活细胞中使用光学检测来彻底分析 GPR35 亚型的组成和配体诱导的 10 种不同异源三聚体 G 蛋白的激活和信号传导、配体诱导的抑制素募集和受体内化。我们的结果表明，长同种型的扩展 N 末端限制了 G 蛋白的活化，但提高了受体-β-抑制蛋白的相互作用。为了更好地理解这种偏差的结构基础，我们检查了 GPR35 的结构模型，并对两种同种型的突变体进行了实验。我们发现存在于两种异构体中的 N 末端和细胞外环 3 之间的二硫键对组成型 G 至关重要₁₃激活，而由长 ​​GPR35 同种型的扩展 N 端贡献的额外半胱氨酸限制了激动剂诱导的受体-β-抑制蛋白 2 相互作用的程度。我们研究的药理学特征和机制见解为未来设计同种型特异性 GPR35 配体提供了线索，这些配体选择性地调节 GPR35-传感器相互作用，并允许基于机制的治疗，例如炎症性肠病或胃肠道系统的细菌感染.