GPR18 undergoes a high degree of constitutive trafficking but is unresponsive to N-Arachidonoyl Glycine.
GPR18 undergoes a high degree of constitutive trafficking but is unresponsive to N-Arachidonoyl Glycine.
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作者：
Finlay DB;Joseph WR;Grimsey NL;Glass M
孤儿受体GPR 18是一种内源性激动剂，被称为N-花生四烯酰甘氨酸（NAGly）。NAGly和内源性大麻素anandamide之间的化学相似性表明GPR 18是第三种大麻素受体的假设。GPR 18介导的细胞信号传导通过抑制环腺苷一磷酸（cAMP）和细胞外信号调节激酶（ERK）的磷酸化，除了生理学后果如调节细胞迁移和增殖/凋亡之外，已经描述了对NAGly和花生四烯酸的响应。然而，也有不一致的调查结果。在这里，我们试图描述GPR 18在异源表达HEK细胞中激活的功能后果。在稳定转染的细胞系中，GPR 18主要在细胞内表达，但在瞬时转染的细胞中也可实现适度的细胞表面表达，瞬时转染的细胞也具有较高的总体表达。采用测定来验证NAGly或花生四烯酸酰胺抑制cAMP或通过GPR 18诱导ERK磷酸化或诱导受体运输的能力。使用表达hCB 1受体（hCB 1 R）的细胞进行阳性对照实验，以验证试验设计和性能。虽然在GPR 18表达细胞中的这些功能途径在用一组推定的GPR 18配体处理时没有被修饰，但是发现了该受体的组成型表型。我们的数据显示，GPR 18经历快速组成型受体膜运输-比hCB 1 R（一种高度组成型活性受体）快几倍。为了提高检测激动剂介导的受体信号传导反应的可能性，我们增加了GPR 18蛋白的表达（通过用催乳素前体信号序列标记），并通过在氨基酸位点108（丙氨酸至天冬酰胺）突变hGPR 18基因来产生推定的组成型失活受体。该A108 N突变体确实导致表面受体表达增加（这可能是组成型活性降低的原因），但未检测到配体介导的效应。测定了两种多形性胶质母细胞瘤细胞系（内源性表达GPR 18）的NAGly诱导的pERK磷酸化，结果为阴性。尽管在所有测定中缺乏配体介导的反应，但GPR 18的组成性运输仍然是受体功能的一个有趣方面，并将对理解GPR 18在生理学中的作用产生影响。
The orphan receptor GPR18 has become a research target following the discovery of a putative endogenous agonist, N-arachidonoyl glycine (NAGly). Chemical similarity between NAGly and the endocannabinoid anandamide suggested the hypothesis that GPR18 is a third cannabinoid receptor. GPR18-mediated cellular signalling through inhibition of cyclic adenosine monophosphate (cAMP) and phosphorylation of extracellular signal-regulated kinase (ERK), in addition to physiological consequences such as regulation of cellular migration and proliferation/apoptosis have been described in response to both NAGly and anandamide. However, discordant findings have also been reported. Here we sought to describe the functional consequences of GPR18 activation in heterologously-expressing HEK cells. GPR18 expression was predominantly intracellular in stably transfected cell lines, but moderate cell surface expression could be achieved in transiently transfected cells which also had higher overall expression. Assays were employed to characterise the ability of NAGly or anandamide to inhibit cAMP or induce ERK phosphorylation through GPR18, or induce receptor trafficking. Positive control experiments, which utilised cells expressing hCB1 receptors (hCB1R), were performed to validate assay design and performance. While these functional pathways in GPR18-expressing cells were not modified on treatment with a panel of putative GPR18 ligands, a constitutive phenotype was discovered for this receptor. Our data reveal that GPR18 undergoes rapid constitutive receptor membrane trafficking—several-fold faster than hCB1R, a highly constitutively active receptor. To enhance the likelihood of detecting agonist-mediated receptor signalling responses, we increased GPR18 protein expression (by tagging with a preprolactin signal sequence) and generated a putative constitutively inactive receptor by mutating the hGPR18 gene at amino acid site 108 (alanine to asparagine). This A108N mutant did cause an increase in surface receptor expression (which may argue for reduced constitutive activity), but no ligand-mediated effects were detected. Two glioblastoma multiforme cell lines (which endogenously express GPR18) were assayed for NAGly-induced pERK phosphorylation, with negative results. Despite a lack of ligand-mediated responses in all assays, the constitutive trafficking of GPR18 remains an interesting facet of receptor function and will have consequences for understanding the role of GPR18 in physiology.