以螢光劑修飾之 RGS 發展新的 Gα 蛋白質相關疾病之藥物篩選策略

Abstract

異三元體 G-蛋白質系統由 α、β、γ 三個次單元體所組成，參與將細胞外訊息藉由 G-蛋白質偶合受體 (G-protein-coupled receptor) 傳遞到細胞內的過程。RGS (regulator of G protein signaling) 是一種 GAP 蛋白質 (GTPase-accelerating proteins)，可和 Gα 次單元體作用並活化其本身之 GTPase 活性，在 G-protein 系統中可加速關閉訊息的傳遞。在近幾年的研究中，RGS 蛋白質的重要性逐漸提高，特別是在心血管及神經系統方面，因此許多製藥學的研究主要都是集中在影響 RGS 的 GAP 活性上，期望能降低藥物的副作用及加強其使用效率。所以需要能大量進行藥物篩選的方法來偵測 GAP 活性的效率。 本實驗中，使用 Lucifer yellow 此螢光劑標定之 RGS4-box (RGS4 之功能性區域)，即時監測其與 Gαi1 之間的作用。在所建立之 96 孔微量滴定盤分析結果顯示，Gα 處於活化態時，可觀測到約 17% 的螢光上升，確定以此螢光試劑作為篩選的方法是可產生專一且足夠的訊號。

Heterotimeric G-protein system consists of Gα and Gβγ subunits, and it involves in the signal transferring and amplification from GPCR (G-protein-coupled receptor) to cell inside. RGS (regulator of G protein signaling) protein functions as a GAP (GTPase-accelerating protein) by interacting and stimulating the intrinsic GTPase activity of specific Gα subunit and consequently accelerates the termination of the signal transition in G-protein system. Recently, the importance of RGS proteins is escalating, especially in the cardiovascular and nerve systems; many pharmacological studies currently focus on the GAP activity of RGS that potentially can reduce side effects or enhance drug efficiency. The need to have a high throughput drug screening method to detect GAP activity efficiencies becomes obvious. Here, we report a method that use a fluorescent probe, lucifer yellow, modified RGS4-box (the GAP functional domain of RGS4) to real-time monitor its interaction with Gαi1 protein. Our results show that a 17% fluorescence increase upon Gα activation can be reported by this fluorescent probe and the amplitude of this signal change is practically significant enough for reagent screening purpose when it is performed in a 96-well Reader setup.