Yu Run, Wawrowsky Kolja, Zhou Cuiqi
Division of Endocrinology, Cedars-Sinai Medical Center, Los Angeles, California, USA.
Endocrinol Nutr. 2011 Jun-Jul;58(6):258-66. doi: 10.1016/j.endonu.2011.04.002. Epub 2011 Jun 15.
To elucidate the pathogenetic mechanisms of a mutant P86S glucagon receptor (GCGR) in causing a novel human disease (Mahvash disease).
Enhanced green fluorescent protein (EGFP)-tagged WT and P86S GCGR were expressed in HEK 293 or H1299 cells either transiently or stably. Receptor localization and internalization, and cell apoptosis were studied by fluorescence microscopy, and calcium signaling by Rhod-3 labeling. Gene expression was assayed by RT-PCR or Western blot. Cell fate was determined by live cell imaging.
Unlike WT GCGR, P86S was partially localized to the plasma membrane and partially in the cytoplasm as previously reported and did not undergo internalization upon glucagon treatment. P86S did not elicit calcium response after treatment with 1 μM glucagon. Cells transiently expressing P86S exhibited more apoptosis than those expressing WT GCGR (18.3% vs 2.1%, P<0.05) but the X-box binding protein 1 mRNA cleavage, a marker of endoplasmic reticulum (ER) stress, was not evident, suggesting that the apoptosis did not result from ER stress. Cells stably expressing P86S did not exhibit apoptosis and a quarter of them harbored a novel inclusion body-like circular structure that was marked by P86S and ER residential proteins. These circular ER bodies were not seen in cells expressing WT GCGR or transiently expressing P86S and were not affected by treatment with proteasome inhibitor or microtubule depolymerizer, suggesting that they do not represent aggresome structures. The circular ER bodies could fuse and split to form new bodies.
The naturally-occurring P86S mutant GCGR exhibits abnormal receptor internalization and calcium mobilization, and causes apoptosis. The novel dynamic circular ER bodies may be adaptive in nature to nullify the toxic effects on P86S. These findings provide further insights into the pathogenetic mechanisms of Mahvash disease.
阐明突变型P86S胰高血糖素受体（GCGR）引发一种新型人类疾病（马赫瓦什病）的致病机制。
将增强型绿色荧光蛋白（EGFP）标记的野生型和P86S GCGR在HEK 293或H1299细胞中进行瞬时或稳定表达。通过荧光显微镜研究受体的定位与内化以及细胞凋亡情况，通过罗丹明-3标记研究钙信号传导。通过逆转录聚合酶链反应（RT-PCR）或蛋白质免疫印迹法检测基因表达。通过活细胞成像确定细胞命运。
与野生型GCGR不同，如先前报道，P86S部分定位于质膜，部分定位于细胞质，并且在胰高血糖素处理后不会发生内化。用1μM胰高血糖素处理后，P86S未引发钙反应。瞬时表达P86S的细胞比表达野生型GCGR的细胞表现出更多的凋亡（18.3%对2.1%，P<0.05），但内质网（ER）应激标志物X盒结合蛋白1 mRNA的切割并不明显，这表明凋亡并非由ER应激导致。稳定表达P86S的细胞未表现出凋亡，其中四分之一含有一种新型的包涵体样环状结构，该结构以P86S和内质网驻留蛋白为标记。在表达野生型GCGR的细胞或瞬时表达P86S的细胞中未见到这些环状内质网体，并且它们不受蛋白酶体抑制剂或微管解聚剂处理的影响，这表明它们不代表聚集体结构。环状内质网体可以融合并分裂形成新的结构。
天然存在的P86S突变型GCGR表现出异常的受体内化和钙动员，并导致细胞凋亡。新型的动态环状内质网体可能本质上具有适应性，以消除对P86S的毒性作用。这些发现为马赫瓦什病致病机制提供了进一步的见解。
