Mechanical stimulus alters conformation of type 1 parathyroid hormone receptor in bone cells.
Mechanical stimulus alters conformation of type 1 parathyroid hormone receptor in bone cells.
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DOI：
10.1152/ajpcell.00549.2008
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发表时间：
2009-06
期刊：
影响因子：
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通讯作者：
Yan-Liang Zhang;J. Frangos;M. Chachisvilis
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文献类型：
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作者：
Yan-Liang Zhang;J. Frangos;M. Chachisvilis
骨细胞将机械刺激转化为细胞内生化反应的分子机制尚未建立。有证据表明，机械刺激与甲状旁腺激素PTH（1-34）协同作用，介导骨生长。利用皮秒时间分辨荧光显微镜和G蛋白偶联受体构象敏感的荧光共振能量转移（FRET），我们研究甲状旁腺激素1型受体（PTH 1 R）的构象转变。1)构建了含有分子内FRET对的基因工程PTH 1 R传感器，其能够检测单个细胞中PTH 1 R的构象活性。2)PTH 1 R的配体依赖性构象变化的性质取决于配体的类型：用PTH（1-34）刺激导致以FRET效率降低为特征的构象转变，而NH（2）末端截短的配体PTH（3-34）刺激以更高的FRET效率为特征的构象转变。3)用流体剪切应力（FSS）刺激小鼠前成骨细胞（MC 3 T3-E1）导致MC 3 T3-E1细胞中PTH 1 R构象平衡的显著变化，表明质膜的机械扰动导致PTH 1 R的配体非依赖性反应。由机械应力诱导的构象转变的特征在于FRET效率的增加，类似于由NH（2）-末端截短的配体PTH（3-34）诱导的那些。在流动介质中存在PTH（1-34）时，对FSS刺激的反应受到抑制。这些结果表明，FSS可以调节PTH（1-34）配体的作用。4)使用苯甲醇或胆固醇提取的质膜流化也导致以FRET水平增加为特征的构象转变。因此，我们认为PTH 1 R参与介导MC 3 T3-E1细胞中的初级机械化学信号转导。
The molecular mechanisms by which bone cells transduce mechanical stimuli into intracellular biochemical responses have yet to be established. There is evidence that mechanical stimulation acts synergistically with parathyroid hormone PTH(1-34) in mediating bone growth. Using picosecond time-resolved fluorescence microscopy and G protein-coupled receptor conformation-sensitive fluorescence resonance energy transfer (FRET), we investigated conformational transitions in parathyroid hormone type 1 receptor (PTH1R). 1) A genetically engineered PTH1R sensor containing an intramolecular FRET pair was constructed that enabled detection of conformational activity of PTH1R in single cells. 2) The nature of ligand-dependent conformational change of PTH1R depends on the type of ligand: stimulation with the PTH(1-34) leads to conformational transitions characterized by decrease in FRET efficiency while NH(2)-terminal truncated ligand PTH(3-34) stimulates conformational transitions characterized by higher FRET efficiencies. 3) Stimulation of murine preosteoblastic cells (MC3T3-E1) with fluid shear stress (FSS) leads to significant changes in conformational equilibrium of the PTH1R in MC3T3-E1 cells, suggesting that mechanical perturbation of the plasma membrane leads to ligand-independent response of the PTH1R. Conformational transitions induced by mechanical stress were characterized by an increase in FRET efficiency, similar to those induced by the NH(2)-terminal truncated ligand PTH(3-34). The response to the FSS stimulation was inhibited in the presence of PTH(1-34) in the flow medium. These results indicate that the FSS can modulate the action of the PTH(1-34) ligand. 4) Plasma membrane fluidization using benzyl alcohol or cholesterol extraction also leads to conformational transitions characterized by increased FRET levels. We therefore suggest that PTH1R is involved in mediating primary mechanochemical signal transduction in MC3T3-E1 cells.