A sensitive balance between receptor activation and desensitization is crucial for cellular homeostasis. Like many other GPCR, the human neuropeptide Y2 receptor (hY2R) undergoes ligand dependent activation and internalization into intracellular compartments, followed by recycling to the plasma membrane. This receptor is involved in the pathophysiology of distinct diseases e.g. epilepsy and cancer progression and conveys anorexigenic signals which makes it an interesting and promising anti-obesity target. However, Y2R desensitization was observed after daily treatment with a selective PYY13–36 analog in vivo by a yet unknown mechanism. We studied the desensitization and activatability of recycled Y2R in transiently transfected HEK293 cells as well as in endogenously Y2R expressing SH-SY5Y and SMS-KAN cells. Results were evaluated by one-way ANOVA and Tukey post test. We observed strong desensitization of the Y2R in a second round of stimulation despite its reappearance at the membrane. Already the first activation of the Y2R leads to depletion of the functional cellular Gαi/o protein pool and consequently desensitizes the linked signal transduction pathways, independent of receptor internalization. This desensitization also extends to other Gαi/o-coupled GPCR and can be detected in transfected HEK293 as well as in SH-SY5Y and SMS-KAN cell lines, both expressing the Y2R endogenously. By overexpression of chimeric Gαqi proteins in a model system, activation has been rescued, which identifies a critical role of the G protein status for cellular signaling. Furthermore, Y2R displays strong allosteric coupling to inhibitory G proteins in radioligand binding assays, and loses 10-fold affinity in the G protein-depleted state observed after activation, which can be largely abrogated by overexpression of the Gαi-subunit. The unusually persistent Gαi-signaling of the Y2R leads to a state of cellular desensitization of the inhibitory Gαi-pathway. The strong allosteric effects of the Y2R-Gαi-interaction might be a mechanism that contributes to the burst of Gαi-signaling, but also serves as a mechanism to limit the Y2-mediated signaling after recycling. Thus, the cell is left in a refractory state, preventing further Gαi-signaling of the Y2R itself but also other Gαi/o-coupled receptors by simply controlling the repertoire of downstream effectors.

中文翻译：

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神经肽Y2受体异常持久的Gαi信号耗尽了细胞的Gi / o库，并导致了Gi难治性状态。

受体激活和脱敏之间的敏感平衡对于细胞稳态至关重要。像许多其他GPCR一样，人类神经肽Y2受体（hY2R）经历配体依赖性激活和内化进入细胞内区室，然后再循环至质膜。该受体参与各种疾病的病理生理，例如癫痫和癌症进展，并传达食欲信号，这使其成为有趣且有希望的抗肥胖目标。但是，在每天用选择性PYY13-36类似物体内治疗后，还通过未知的机制观察到Y2R脱敏。我们研究了瞬时转染的HEK293细胞以及内源性表达Y2R的SH-SY5Y和SMS-KAN细胞中回收的Y2R的脱敏和激活性。通过单向方差分析和Tukey后期测试评估结果。我们在第二轮刺激中观察到了Y2R的强烈脱敏，尽管它再次出现在膜上。Y2R的首次激活已经导致功能性细胞Gαi/ o蛋白池耗竭，因此使连接的信号转导途径脱敏，而与受体内在化无关。这种脱敏作用还扩展到其他Gαi/ o偶联GPCR，并且可以在转染的HEK293以及内源性表达Y2R的SH-SY5Y和SMS-KAN细胞系中检测到。通过在模型系统中过表达嵌合Gαqi蛋白，可以挽救激活作用，从而确定G蛋白状态对于细胞信号传导的关键作用。此外，在放射性配体结合测定中，Y2R与抑制性G蛋白表现出很强的变构偶联，在活化后观察到的G蛋白耗尽状态下失去了10倍的亲和力，这在很大程度上可以通过Gαi亚基的过表达而消除。Y2R异常持久的Gαi信号导致抑制性Gαi途径的细胞脱敏状态。Y2R-Gαi相互作用的强别构效应可能是导致Gαi信号爆发的机制，但也可以作为限制循环后Y2介导的信号传导的机制。因此，该细胞处于难治状态，通过简单地控制下游效应子的组成，阻止了Y2R本身以及其他Gαi/ o偶联受体的进一步Gαi信号传递。并在激活后观察到的G蛋白耗尽状态下失去10倍的亲和力，这可以通过Gαi亚基的过表达大大消除。Y2R异常持久的Gαi信号导致抑制性Gαi途径的细胞脱敏状态。Y2R-Gαi相互作用的强别构效应可能是导致Gαi信号爆发的机制，但也可以作为限制循环后Y2介导的信号传导的机制。因此，该细胞处于难治状态，通过简单地控制下游效应子的组成，阻止了Y2R本身以及其他Gαi/ o偶联受体的进一步Gαi信号传递。并在激活后观察到的G蛋白耗尽状态下失去10倍的亲和力，这可以通过Gαi亚基的过表达大大消除。Y2R异常持久的Gαi信号导致抑制性Gαi途径的细胞脱敏状态。Y2R-Gαi相互作用的强别构效应可能是导致Gαi信号爆发的机制，但也可以作为限制循环后Y2介导的信号传导的机制。因此，该细胞处于难治状态，通过简单地控制下游效应子的组成，阻止了Y2R本身以及其他Gαi/ o偶联受体的进一步Gαi信号传递。Y2R异常持久的Gαi信号导致抑制性Gαi途径的细胞脱敏状态。Y2R-Gαi相互作用的强别构效应可能是导致Gαi信号爆发的机制，但也可以作为限制循环后Y2介导的信号传导的机制。因此，该细胞处于难治状态，通过简单地控制下游效应子的组成，阻止了Y2R本身以及其他Gαi/ o偶联受体的进一步Gαi信号传递。Y2R异常持久的Gαi信号导致抑制性Gαi途径的细胞脱敏状态。Y2R-Gαi相互作用的强别构效应可能是导致Gαi信号爆发的机制，但也可以作为限制循环后Y2介导的信号传导的机制。因此，该细胞处于难治状态，通过简单地控制下游效应子的组成，阻止了Y2R本身以及其他Gαi/ o偶联受体的进一步Gαi信号传递。