G protein-coupled receptors (GPCRs) are an important protein family of signalling receptors that govern a wide variety of physiological functions. The capacity to transmit extracellular signals and the extent of cellular response are largely determined by the amount of functional receptors at the cell surface that is subject to complex and fine-tuned regulation. Here, we demonstrate that the cell surface expression level of an inhibitory GPCR, the human δ-opioid receptor (hδOR) involved in pain and mood regulation, is modulated by site-specific N-acetylgalactosamine (GalNAc) -type O-glycosylation. Importantly, we identified one out of the 20 polypeptide GalNAc-transferase isoforms, GalNAc-T2, as the specific regulator of O-glycosylation of Ser6, Ser25 and Ser29 in the N-terminal ectodomain of the receptor. This was demonstrated by in vitro glycosylation assays using peptides corresponding to the hδOR N-terminus, Vicia villosa lectin affinity purification of receptors expressed in HEK293 SimpleCells capable of synthesizing only truncated O-glycans, GalNAc-T edited cell line model systems, and site-directed mutagenesis of the putative O-glycosylation sites. Interestingly, a single-nucleotide polymorphism, at residue 27 (F27C), was found to alter O-glycosylation of the receptor in efficiency as well as in glycosite usage. Furthermore, flow cytometry and cell surface biotinylation assays using O-glycan deficient CHO-ldlD cells revealed that the absence of O-glycans results in decreased receptor levels at the plasma membrane due to enhanced turnover. In addition, mutation of the identified O-glycosylation sites led to a decrease in the number of ligand-binding competent receptors and impaired agonist-mediated inhibition of cyclic AMP accumulation in HEK293 cells. Thus, site-specific O-glycosylation by a selected GalNAc-T isoform can increase the stability of a GPCR, in a process that modulates the constitutive turnover and steady-state levels of functional receptors at the cell surface.

G蛋白偶联受体（GPCR）是信号受体的重要蛋白家族，可控制多种生理功能。传递细胞外信号的能力和细胞反应的程度在很大程度上取决于细胞表面功能受体的数量，这些功能受体需要进行复杂且精细的调节。在这里，我们证明了抑制性GPCR的细胞表面表达水平，即参与疼痛和情绪调节的人δ阿片受体（hδOR），是由位点特异性N-乙酰半乳糖胺（GalNAc）-型O-糖基化调控的。重要的是，我们确定了20种多肽GalNAc-转移酶同工型中的一种，即GalNAc-T2，是O的特异性调节剂。受体N-末端胞外域中Ser6，Ser25和Ser29的糖基化。这是通过证实在体外使用对应于hδORN-末端糖基化的肽测定法，毛苕子凝集素在HEK293表达的受体的亲和纯化SimpleCells能够合成的仅截短ø -glycans，将GalNAc-T编辑细胞系模型系统，以及位点定向O-糖基化位点的诱变。有趣的是，发现残基27（F27C）处的单核苷酸多态性可改变受体的O-糖基化效率和糖位。此外，流式细胞术和细胞表面生物素化测定使用O-聚糖缺乏的CHO-ldlD细胞显示，O-聚糖的缺失会导致周转率提高，从而导致质膜受体水平降低。另外，所鉴定的O-糖基化位点的突变导致配体结合感受态受体的数量减少，并且激动剂介导的HEK293细胞中环AMP积累的抑制作用减弱。因此，通过选择的GalNAc-T同工型的位点特异性O-糖基化可以在调节细胞表面功能性受体的组成转换和稳态水平的过程中提高GPCR的稳定性。