Centre for Neuroendocrinology, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa.
Department of Immunology, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa.
Endocrinology. 2021 Dec 1;162(12). doi: 10.1210/endocr/bqab134.
Mutations in G protein-coupled receptors (GPCRs) underlie numerous diseases. Many cause receptor misfolding and failure to reach the cell surface. Pharmacological chaperones are cell-permeant small molecules that engage nascent mutant GPCRs in the endoplasmic reticulum, stabilizing folding and "rescuing" cell surface expression. We previously demonstrated rescue of cell surface expression of luteinizing hormone receptor mutants by an allosteric agonist. Here we demonstrate that a similar approach can be employed to rescue mutant follicle-stimulating hormone receptors (FSHRs) with poor cell surface expression using a small-molecule FSHR agonist, CAN1404. Seventeen FSHR mutations described in patients with reproductive dysfunction were expressed in HEK 293T cells, and cell surface expression was determined by enzyme-linked immunosorbent assay of epitope-tagged FSHRs before/after treatment with CAN1404. Cell surface expression was severely reduced to ≤18% of wild-type (WT) for 11, modestly reduced to 66% to 84% of WT for 4, and not reduced for 2. Of the 11 with severely reduced cell surface expression, restoration to ≥57% of WT levels was achieved for 6 by treatment with 1 µM CAN1404 for 24 h, and a corresponding increase in FSH-induced signaling was observed for 4 of these, indicating restored functionality. Therefore, CAN1404 acts as a pharmacological chaperone and can rescue cell surface expression and function of certain mutant FSHRs with severely reduced cell surface expression. These findings aid in advancing the understanding of the effects of genetic mutations on GPCR function and provide a proof of therapeutic principle for FSHR pharmacological chaperones.
G 蛋白偶联受体（GPCR）突变是许多疾病的基础。许多突变导致受体错误折叠，无法到达细胞表面。药理学伴侣是可穿透细胞质的小分子，可与内质网中新生的突变 GPCR 结合，稳定折叠并“挽救”细胞表面表达。我们之前证明了通过变构激动剂挽救黄体生成素受体突变体的细胞表面表达。在这里，我们证明了使用小分子 FSHR 激动剂 CAN1404 可以采用类似的方法挽救具有不良细胞表面表达的突变卵泡刺激素受体（FSHR）。我们在 HEK 293T 细胞中表达了 17 种在生殖功能障碍患者中描述的 FSHR 突变体，并通过酶联免疫吸附试验测定了表位标记的 FSHR 之前/之后用 CAN1404 处理后的细胞表面表达。细胞表面表达严重降低至≤WT 的 11%，适度降低至 WT 的 66%至 84%，对于 4 种，未降低。在严重降低细胞表面表达的 11 种中，通过用 1 µM CAN1404 处理 24 小时，6 种可恢复至≥WT 水平的 57%，并且观察到其中 4 种的 FSH 诱导信号增加，表明功能恢复。因此，CAN1404 作为一种药理学伴侣，可以挽救某些细胞表面表达严重降低的突变 FSHR 的细胞表面表达和功能。这些发现有助于深入了解遗传突变对 GPCR 功能的影响，并为 FSHR 药理学伴侣提供治疗原理的证明。
