Pole d'expertise Biotechnologie, Chimie & Biologie, Institut de Recherches Servier, Croissy-sur-Seine, France.
Eurofins Discovery, Celle Levescault, France.
Methods Mol Biol. 2022;2550:189-193. doi: 10.1007/978-1-0716-2593-4_23.
The main step of classical desensitization of a receptor, by mean of its disappearance from the plasma membrane, is its internalization. This is a key factor in the regulation of agonist-mediated signaling pathways, as it most of the time stops the activation of the receptor. Internalization is thus important to evaluate, as a complementary information for a natural ligand or an alternative synthetic agonist. Enzyme fragment complementation is an elegant but delicate way to measure this phenomenon, by fusing two complementary parts of an enzyme to two partners, and to measure the activity of the reconstituted enzyme upon complexation of the partners. In the present chapter, using two parts of β-galactosidase, one fused to the C-terminus of the MT1 receptor, the other to an endosomal protein, one can measure the formation of the complex; thus, the transfer of the receptor to the endosome from which MT1 will be recirculated.
经典脱敏作用的主要步骤是受体从质膜消失，这是其内化的过程。这是调节激动剂介导的信号通路的关键因素，因为它通常会停止受体的激活。因此，内化对于评估天然配体或替代合成激动剂是一个重要的补充信息。酶片段互补是一种优雅但微妙的方法来测量这种现象，通过将酶的两个互补部分融合到两个伴侣上，并在伴侣复合物形成时测量重新构成的酶的活性。在本章中，使用β-半乳糖苷酶的两个部分，一个融合到 MT1 受体的 C 末端，另一个融合到内体蛋白，就可以测量复合物的形成，从而测量受体从内体转移的情况，MT1 将从内体中再循环。
