Division of Endocrinology and Carcinoid and Neuroendocrine Tumor Center, Cedars-Sinai Medical Center, B-131, 8700 Beverly Boulevard, Los Angeles, California 90048, USA.
J Mol Endocrinol. 2012 Jul 25;49(2):69-78. doi: 10.1530/JME-12-0051. Print 2012 Oct.
We have previously demonstrated that a homozygous inactivating P86S mutation of the glucagon receptor (GCGR) causes a novel human disease of hyperglucagonemia, pancreatic α-cell hyperplasia, and pancreatic neuroendocrine tumors (Mahvash disease). The mechanisms for the decreased activity of the P86S mutant (P86S) are abnormal receptor localization to the endoplasmic reticulum (ER) and defective interaction with glucagon. To search for targeted therapies for Mahvash disease, we examined whether P86S can be trafficked to the plasma membrane by pharmacological chaperones and whether novel glucagon analogs restore effective receptor interaction. We used enhanced green fluorescent protein-tagged P86S stably expressed in HEK 293 cells to allow fluorescence imaging and western blotting and molecular modeling to design novel glucagon analogs in which alanine 19 was replaced with serine or asparagine. Incubation at 27 °C largely restored normal plasma membrane localization and normal processing of P86S but osmotic chaperones had no effects. The ER stressors thapsigargin and curcumin partially rescued P86S. The lipophilic GCGR antagonist L-168,049 also partially rescued P86S, so did Cpd 13 and 15 to a smaller degree. The rescued P86S led to more glucagon-stimulated cAMP production and was internalized by glucagon. Compared with the native glucagon, the novel glucagon analogs failed to stimulate more cAMP production by P86S. We conclude that the mutant GCGR is partially rescued by several pharmacological chaperones and our data provide proof-of-principle evidence that Mahvash disease can be potentially treated with pharmacological chaperones. The novel glucagon analogs, however, failed to interact with P86S more effectively.
我们之前已经证明，胰高血糖素受体（GCGR）的纯合失活 P86S 突变会导致一种新型人类高胰高血糖素血症、胰腺α细胞增生和胰腺神经内分泌肿瘤（Mahvash 病）疾病。P86S 突变（P86S）活性降低的机制是异常的受体向内质网（ER）的定位和与胰高血糖素的相互作用缺陷。为了寻找治疗 Mahvash 病的靶向疗法，我们研究了 P86S 是否可以通过药理学伴侣转运到质膜，以及新型胰高血糖素类似物是否可以恢复有效的受体相互作用。我们使用稳定表达于 HEK 293 细胞中的增强型绿色荧光蛋白标记的 P86S 进行荧光成像和 Western blot 分析，并进行分子建模以设计其中丙氨酸 19 被丝氨酸或天冬酰胺取代的新型胰高血糖素类似物。在 27°C 孵育可在很大程度上恢复 P86S 的正常质膜定位和正常加工，但渗透伴侣没有影响。内质网应激物他普西龙和姜黄素部分挽救了 P86S。亲脂性 GCGR 拮抗剂 L-168,049 也部分挽救了 P86S，CPd 13 和 15 则较小程度地挽救了 P86S。挽救的 P86S 导致更多的胰高血糖素刺激 cAMP 产生，并被胰高血糖素内化。与天然胰高血糖素相比，新型胰高血糖素类似物未能通过 P86S 刺激更多的 cAMP 产生。我们得出结论，几种药理学伴侣部分挽救了突变的 GCGR，我们的数据提供了初步证据，表明 Mahvash 病可以用药理学伴侣治疗。然而，新型胰高血糖素类似物未能更有效地与 P86S 相互作用。
