Department of Molecular Reproduction, Development and Genetics,Indian Institute of Science, Bangalore, Karnataka, India.
PLoS One. 2012;7(7):e40291. doi: 10.1371/journal.pone.0040291. Epub 2012 Jul 6.
The mechanism by which the hinge regions of glycoprotein hormone receptors couple hormone binding to activation of downstream effecters is not clearly understood. In the present study, agonistic (311.62) and antagonistic (311.87) monoclonal antibodies (MAbs) directed against the TSH receptor extracellular domain were used to elucidate role of the hinge region in receptor activation. MAb 311.62 which identifies the LRR/Cb-2 junction (aa 265-275), increased the affinity of TSHR for the hormone while concomitantly decreasing its efficacy, whereas MAb 311.87 recognizing LRR 7-9 (aa 201-259) acted as a non-competitive inhibitor of Thyroid stimulating hormone (TSH) binding. Binding of MAbs was sensitive to the conformational changes caused by the activating and inactivating mutations and exhibited differential effects on hormone binding and response of these mutants. By studying the effects of these MAbs on truncation and chimeric mutants of thyroid stimulating hormone receptor (TSHR), this study confirms the tethered inverse agonistic role played by the hinge region and maps the interactions between TSHR hinge region and exoloops responsible for maintenance of the receptor in its basal state. Mechanistic studies on the antibody-receptor interactions suggest that MAb 311.87 is an allosteric insurmountable antagonist and inhibits initiation of the hormone induced conformational changes in the hinge region, whereas MAb 311.62 acts as a partial agonist that recognizes a conformational epitope critical for coupling of hormone binding to receptor activation. The hinge region, probably in close proximity with the α-subunit in the hormone-receptor complex, acts as a tunable switch between hormone binding and receptor activation.
糖蛋白激素受体的铰链区将激素结合与下游效应器的激活偶联的机制尚不清楚。在本研究中，使用针对 TSH 受体细胞外结构域的激动剂（311.62）和拮抗剂（311.87）单克隆抗体（MAb）来阐明铰链区在受体激活中的作用。识别 LRR/Cb-2 连接（aa265-275）的 MAb 311.62 增加了 TSHR 对激素的亲和力，同时降低了其效力，而识别 LRR7-9（aa201-259）的 MAb 311.87 作为甲状腺刺激素（TSH）结合的非竞争性抑制剂。MAb 的结合对激活和失活突变引起的构象变化敏感，并对这些突变体的激素结合和反应表现出不同的影响。通过研究这些 MAb 对甲状腺刺激素受体（TSHR）截断和嵌合突变体的影响，本研究证实了铰链区的束缚反向激动剂作用，并绘制了 TSHR 铰链区与外环之间的相互作用图，这些外环负责维持受体处于基础状态。对抗体-受体相互作用的机制研究表明，MAb 311.87 是一种变构不可逾越的拮抗剂，可抑制激素诱导的铰链区构象变化的起始，而 MAb 311.62 作为一种部分激动剂，可识别对激素结合与受体激活偶联至关重要的构象表位。铰链区可能与激素-受体复合物中的α亚基紧密接近，充当激素结合与受体激活之间的可调开关。
