Clinical Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Thyroid. 2011 Aug;21(8):907-12. doi: 10.1089/thy.2011.0025. Epub 2011 Jul 11.
Subclinical hypothyroidism (SHT), characterized by normal thyroid hormone levels maintained by elevated thyrotropin (TSH), predisposes patients to health problems as they age. Some cases arise from mutations of the TSH receptor (TSHR) that confer TSH resistance. This resistance might be circumvented by TSHR agonists with different modes of binding compared with TSH. We hypothesized that the recently discovered small-molecule TSHR agonist C2, with its unique mode of receptor binding, would activate mutant TSHRs associated with SHT, facilitating their study.
HEK-EM293 cells transiently expressing TSHR variants-wild-type TSHR or mutants C41S, L252P, L467P, or C600R-were analyzed for TSH or C2-induced cyclic adenosine monophosphate (cAMP) signaling to establish C2 as a mutant TSHR agonist. These cells were also pretreated with TSH or C2 to characterize each mutant receptor's ability to maintain and desensitize cAMP signaling.
We showed that C2 could activate the TSH-unresponsive TSHR ectodomain mutants C41S and L252P but had no effect on the serpentine mutant L467P. We found that TSH and C2 could acutely activate the serpentine mutant C600R. Preincubation with C2 caused persistent cAMP signaling and receptor desensitization in wild-type TSHR and cAMP signal persistence with no detectable desensitization in the cases of C41S and L252P.
The small-molecule agonist C2 is a useful pharmacological tool for the study of mutant TSHRs. It revealed that some naturally occurring TSH-insensitive mutants can mediate induction of cAMP elevation upon stimulation with C2 and that this signal is differentially maintained within cells.
亚临床甲状腺功能减退症（SHT）的特征是甲状腺激素水平正常，但促甲状腺激素（TSH）升高，随着年龄的增长，患者易出现健康问题。有些病例是由于 TSH 受体（TSHR）的突变引起的，这些突变赋予了 TSH 抵抗性。与 TSH 相比，具有不同结合模式的 TSHR 激动剂可能会绕过这种抵抗。我们假设最近发现的小分子 TSHR 激动剂 C2，具有独特的受体结合模式，将激活与 SHT 相关的突变 TSHR，从而促进对其的研究。
用 TSH 或 C2 预处理瞬时表达 TSHR 变体（野生型 TSHR 或突变体 C41S、L252P、L467P 或 C600R）的 HEK-EM293 细胞，分析 TSH 或 C2 诱导的环磷酸腺苷（cAMP）信号，以确定 C2 是一种突变 TSHR 激动剂。还对这些细胞进行预处理，以表征每个突变受体维持和脱敏 cAMP 信号的能力。
我们表明，C2 可以激活 TSH 无反应的 TSHR 胞外结构域突变体 C41S 和 L252P，但对蛇形突变体 L467P 没有影响。我们发现，TSH 和 C2 可以急性激活蛇形突变体 C600R。C2 预孵育导致野生型 TSHR 中的持续 cAMP 信号和受体脱敏，而在 C41S 和 L252P 的情况下，持续存在 cAMP 信号而无可检测到的脱敏。
小分子激动剂 C2 是研究突变 TSHR 的有用药理学工具。它表明，一些天然存在的 TSH 不敏感突变体在受到 C2 刺激时可以介导 cAMP 升高的诱导，并且这种信号在细胞内以不同的方式维持。
