神经降压素1型受体与内源性配体的信号转导
The signaling signature of the neurotensin type 1 receptor with endogenous ligands
摘要：
人神经降压素1受体（hNTS 1）是一种G蛋白偶联受体，参与许多生理功能，包括镇痛、体温过低和低血压。为了更好地了解哪些信号通路或通路组合与NTS 1活化和功能相关，我们研究了由CHO-K1细胞稳定表达的活化的hNTS 1直接接合G蛋白、激活第二信使级联和募集\b{eta} -抑制蛋白的能力。使用基于BRET的生物传感器，我们发现神经降压素（NT）、NT（8-13）和神经介肽N（NN）激活Gαq-、Gαi1-、GαoA-和Gα13-蛋白信号通路以及\b{eta} -抑制蛋白1和2的募集。使用药理学抑制剂，我们进一步证明了所有三种配体刺激磷酸肌醇的产生和cAMP积累的调节以及ERK 1/2激活。有趣的是，尽管与Gαi1和GαoA功能性偶联，但发现NT在百日咳毒素存在下产生更高水平的cAMP，支持hNTS 1活化以Gα s依赖性方式导致cAMP积累。此外，我们证明了ERK 1/2的完全激活需要通过PTX敏感的Gi/o-c-Src信号通路和Gq下游的PLCb-DAG-PKC-Raf-1依赖性通路的信号传导。最后，基于细胞的表面等离子体共振监测的全细胞集成签名和汇合细胞单层的电阻抗的变化导致三个配体之间相同的表型反应。hNTS 1介导的细胞信号传导网络的表征将有助于加速验证具有改善的治疗/不良反应特征的潜在NTS 1偏向配体。
英文摘要：
The human neurotensin 1 receptor (hNTS1) is a G protein-coupled receptor involved in many physiological functions, including analgesia, hypothermia, and hypotension. To gain a better understanding of which signaling pathways or combination of pathways are linked to NTS1 activation and function, we investigated the ability of activated hNTS1, which was stably expressed by CHO-K1 cells, to directly engage G proteins, activate second messenger cascades and recruit \b{eta}-arrestins. Using BRET-based biosensors, we found that neurotensin (NT), NT(8-13) and neuromedin N (NN) activated the Gαq-, Gαi1-, GαoA-, and Gα13-protein signaling pathways as well as the recruitment of \b{eta}-arrestins 1 and 2. Using pharmacological inhibitors, we further demonstrated that all three ligands stimulated the production of inositol phosphate and modulation of cAMP accumulation along with ERK1/2 activation. Interestingly, despite the functional coupling to Gαi1 and GαoA, NT was found to produce higher levels of cAMP in the presence of pertussis toxin, supporting that hNTS1 activation leads to cAMP accumulation in a Gαs-dependent manner. Additionally, we demonstrated that the full activation of ERK1/2 required signaling through both a PTX-sensitive Gi/o-c-Src signaling pathway and PLCb-DAG-PKC-Raf-1- dependent pathway downstream of Gq. Finally, the whole-cell integrated signatures monitored by the cell-based surface plasmon resonance and changes in the electrical impedance of a confluent cell monolayer led to identical phenotypic responses between the three ligands. The characterization of the hNTS1-mediated cellular signaling network will be helpful to accelerate the validation of potential NTS1 biased ligands with an improved therapeutic/adverse effect profile.
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