Red de Apoyo a la Investigación, National University of Mexico-Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico.
Department of Reproductive Biology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico.
J Clin Endocrinol Metab. 2021 Jan 23;106(2):e534-e550. doi: 10.1210/clinem/dgaa782.
Follicle-stimulating hormone (FSH) plays an essential role in gonadal function. Loss-of-function mutations in the follicle-stimulating hormone receptor (FSHR) are an infrequent cause of primary ovarian failure.
To analyze the molecular physiopathogenesis of a novel mutation in the FSHR identified in a woman with primary ovarian failure, employing in vitro and in silico approaches, and to compare the features of this dysfunctional receptor with those shown by the trafficking-defective D408Y FSHR mutant.
Sanger sequencing of the FSHR cDNA was applied to identify the novel mutation. FSH-stimulated cyclic adenosine monophosphate (cAMP) production, ERK1/2 phosphorylation, and desensitization were tested in HEK293 cells. Receptor expression was analyzed by immunoblotting, receptor-binding assays, and flow cytometry. Molecular dynamics simulations were performed to determine the in silico behavior of the mutant FSHRs.
A novel missense mutation (I423T) in the second transmembrane domain of the FSHR was identified in a woman with normal pubertal development but primary amenorrhea. The I423T mutation slightly impaired plasma membrane expression of the mature form of the receptor and severely impacted on cAMP/protein kinase A signaling but much less on β-arrestin-dependent ERK1/2 phosphorylation. Meanwhile, the D408Y mutation severely affected membrane expression, with most of the FSH receptor located intracellularly, and both signal readouts tested. Molecular dynamics simulations revealed important functional disruptions in both mutant FSHRs, mainly the loss of interhelical connectivity in the D408Y FSHR.
Concurrently, these data indicate that conformational differences during the inactive and active states account for the distinct expression levels, differential signaling, and phenotypic expression of the I423T and D408Y mutant FSHRs.
卵泡刺激素（FSH）在性腺功能中起着至关重要的作用。卵泡刺激素受体（FSHR）的功能丧失性突变是原发性卵巢功能衰竭的一个罕见原因。
运用体外和计算机模拟方法分析在一名原发性卵巢功能衰竭女性中发现的 FSHR 新型突变的分子病理生理学，并将这种功能失调的受体与表现出转运缺陷的 D408Y FSHR 突变体的特征进行比较。
应用 Sanger 测序对 FSHR cDNA 进行测序，以鉴定新型突变。在 HEK293 细胞中测试 FSH 刺激的环磷酸腺苷（cAMP）产生、ERK1/2 磷酸化和脱敏。通过免疫印迹、受体结合测定和流式细胞术分析受体表达。进行分子动力学模拟以确定突变 FSHR 的计算机行为。
在一名青春期正常发育但原发性闭经的女性中发现 FSHR 的第二个跨膜结构域中的新型错义突变（I423T）。I423T 突变轻微损害成熟形式的受体的质膜表达，严重影响 cAMP/蛋白激酶 A 信号传导，但对β-arrestin 依赖性 ERK1/2 磷酸化的影响较小。同时，D408Y 突变严重影响膜表达，大部分 FSH 受体位于细胞内，两种信号转导均受到影响。分子动力学模拟揭示了这两种突变 FSHR 中的重要功能障碍，主要是 D408Y FSHR 中螺旋间连接的丧失。
同时，这些数据表明，在无活性和活性状态下的构象差异解释了 I423T 和 D408Y 突变 FSHR 的不同表达水平、信号转导差异和表型表达。
