Institute of Science and Technology Austria (ISTA), Am Campus 1, 3400, Klosterneuburg, Austria.
Nat Commun. 2022 Aug 15;13(1):4728. doi: 10.1038/s41467-022-32390-1.
G protein-coupled receptors (GPCRs) regulate processes ranging from immune responses to neuronal signaling. However, ligands for many GPCRs remain unknown, suffer from off-target effects or have poor bioavailability. Additionally, dissecting cell type-specific responses is challenging when the same GPCR is expressed on different cells within a tissue. Here, we overcome these limitations by engineering DREADD-based GPCR chimeras that bind clozapine-N-oxide and mimic a GPCR-of-interest. We show that chimeric DREADD-β2AR triggers responses comparable to β2AR on second messenger and kinase activity, post-translational modifications, and protein-protein interactions. Moreover, we successfully recapitulate β2AR-mediated filopodia formation in microglia, an immune cell capable of driving central nervous system inflammation. When dissecting microglial inflammation, we included two additional DREADD-based chimeras mimicking microglia-enriched GPR65 and GPR109A. DREADD-β2AR and DREADD-GPR65 modulate the inflammatory response with high similarity to endogenous β2AR, while DREADD-GPR109A shows no impact. Our DREADD-based approach allows investigation of cell type-dependent pathways without known endogenous ligands.
G 蛋白偶联受体（GPCRs）调节的过程范围从免疫反应到神经元信号传递。然而，许多 GPCR 的配体仍然未知，存在脱靶效应或生物利用度差。此外，当同一 GPCR 在组织内的不同细胞中表达时，解析细胞类型特异性反应具有挑战性。在这里，我们通过工程化基于 DREADD 的 GPCR 嵌合体来克服这些限制，该嵌合体结合氯氮平-N-氧化物并模拟感兴趣的 GPCR。我们表明，嵌合 DREADD-β2AR 触发的反应与第二信使和激酶活性、翻译后修饰和蛋白-蛋白相互作用上的β2AR 相当。此外，我们成功地在小神经胶质细胞中再现了β2AR 介导的丝状伪足形成，小神经胶质细胞是一种能够驱动中枢神经系统炎症的免疫细胞。在剖析小胶质细胞炎症时，我们包括了另外两种模拟富含小胶质细胞的 GPR65 和 GPR109A 的基于 DREADD 的嵌合体。DREADD-β2AR 和 DREADD-GPR65 以与内源性β2AR 高度相似的方式调节炎症反应，而 DREADD-GPR109A 则没有影响。我们基于 DREADD 的方法允许在没有已知内源性配体的情况下研究依赖于细胞类型的途径。
