Department of Experimental Medical Science, Lund University, Lund, Sweden.
Int Immunopharmacol. 2013 Jan;15(1):121-8. doi: 10.1016/j.intimp.2012.11.012. Epub 2012 Nov 29.
The kinin B1 receptor (B1R) is a G protein-coupled receptor with pro-inflammatory activity that is latent in healthy tissues but induced by tissue insult. Here, we investigated if B1R homo-oligomerization is a possible mechanism regulating the presentation of this receptor at the level of maturation and trafficking to the cell surface. To this end, we used HEK293 cells stably expressing N-terminal FLAG and HA epitope-tagged wild-type human B1R and an N-terminal receptor fragment, B1stop135, which terminates at the C-terminal end of the third transmembrane domain and has previously been shown to oligomerize with B1R. Receptors were monitored by immunoblotting and immunoprecipitation, receptor function by agonist binding and agonist-promoted phosphoinositide hydrolysis, and receptor trafficking by confocal immunofluorescence microscopy. When expressed alone, B1R is core N-glycosylated and forms oligomers localized intracellularly and on the cell surface. B1stop135 also exists as core N-glycosylated oligomers but is localized exclusively intracellularly. When co-expressed, B1stop135 prevents specifically B1R homo-oligomerization by forming nonfunctional B1R-B1stop135 hetero-oligomers, retains B1R intracellularly at least in part in the endoplasmatic reticulum (ER), increases calnexin binding to the receptor, and increases receptor degradation. We conclude that B1R homo-oligomerization is necessary for B1R maturation and trafficking to the cell surface. Modulating this mechanism may be a novel therapeutic avenue in inflammatory disease.
激肽 B1 受体（B1R）是一种具有促炎活性的 G 蛋白偶联受体，在健康组织中处于潜伏状态，但在组织损伤时被诱导。在这里，我们研究了 B1R 同源寡聚化是否是一种可能的机制，用于调节该受体在成熟和向细胞表面转运水平上的表达。为此，我们使用稳定表达 N 端 FLAG 和 HA 表位标签的野生型人 B1R 和 N 端受体片段 B1stop135 的 HEK293 细胞，该片段在第三个跨膜域的 C 端末端终止，先前已显示与 B1R 寡聚化。通过免疫印迹和免疫沉淀监测受体，通过激动剂结合和激动剂促进的磷酸肌醇水解监测受体功能，通过共聚焦免疫荧光显微镜监测受体转运。当单独表达时，B1R 是核心 N 糖基化的，并形成定位于细胞内和细胞表面的寡聚体。B1stop135 也作为核心 N 糖基化的寡聚体存在，但仅定位于细胞内。当共表达时，B1stop135 通过形成非功能性的 B1R-B1stop135 异源寡聚体特异性地阻止 B1R 同源寡聚化，至少部分地将 B1R 保留在细胞内的内质网（ER）中，增加钙连蛋白与受体的结合，并增加受体降解。我们得出结论，B1R 同源寡聚化是 B1R 成熟和向细胞表面转运所必需的。调节这种机制可能是炎症性疾病的一种新的治疗途径。
