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Requirement of atypical N-glycosylation motif for the surface expression and the intracellular signaling

表面表达和细胞内信号转导对非典型 N-糖基化基序的要求

基本信息

批准号：
23770138
负责人：
YASUDA Daisuke
金额：
$ 2.66万
依托单位：
Akita University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Young Scientists (B)
财政年份：
2012
资助国家：
日本
起止时间：
2012 至 2013
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-23770138/
关键词：
N 型糖鎖修飾 Sequon 配列 GPR109A STT3 シグナル伝達形質膜発現 Sequon N-glycosylation Cell signaling STT3B Nicotinic acid GPCR N型糖鎖修飾

项目摘要

We currently found that GPR109A, which is known as a nicotinic acid (niacin) receptor, is modified withN-glycan, even through lacking the sequon motif in the extracellular regions. Here, we demonstrate that Asn17-X-Cys^<19>, termed non-sequon motif, within the N-terminus of this receptor contributes to the N-glycosylation of GPR109A. This modification is indispensable for GPR109A because non-glycosylated GPR109A mutants, GPR109A/N17A and GPR109A/C19A, showed impaired cell surface expression, although GPR109A/C19S and GPR109A/C19T mutants, in which receiveN-glycosylation at Asn^<17>, exhibited similar expression to wild-type receptor. We further suggest that the oligosaccharyltransferase catalytic subunit STT3B, but not STT3A isoform, is implicated in theN-glycan conjugation on the non-sequon motif of GPR109A. Intriguingly, defect in the di-cysteine in the non-sequon motif, Cys^<18>-Cys^<19>, resulted in the impairment of the Gi-mediated signaling via GPR109A, e.g., nicotinic acid-elicited reduction of cAMP synthesis and elevation of intracellular [Ca^<2+>]. Since this deficiency was not compensated by the replacement of Cys19 to Ser or Thr, the impaired function of the di-cysteine defective GPR109A is not due to the lack ofN-glycan.

我们目前发现 GPR109A（称为烟酸）受体被 N-聚糖修饰，即使在细胞外区域缺乏序列肽基序。在这里，我们证明该受体 N 末端的 Asn17-X-Cys^<19>（称为非序列子基序）有助于 GPR109A 的 N-糖基化。这种修饰对于GPR109A是必不可少的，因为非糖基化的GPR109A突变体GPR109A/N17A和GPR109A/C19A显示受损的细胞表面表达，尽管在Asn^17处接受N-糖基化的GPR109A/C19S和GPR109A/C19T突变体表现出与野生型受体相似的表达。我们进一步表明，寡糖基转移酶催化亚基 STT3B（而非 STT3A 同工型）参与 GPR109A 非序列子基序上的 N-聚糖缀合。有趣的是，非序列子基序 Cys^<18>-Cys^<19> 中二半胱氨酸的缺陷导致通过 GPR109A 的 Gi 介导的信号传导受损，例如烟酸引起 cAMP 合成减少和细胞内 [Ca^2+] 升高。由于这种缺陷不能通过将 Cys19 替换为 Ser 或 Thr 来补偿，因此双半胱氨酸缺陷型 GPR109A 的功能受损并不是由于缺乏 N-聚糖。