β2 Adrenergic Receptor Fluorescent Protein Fusions Traffic to the Plasma Membrane and Retain Functionality

Green fluorescent protein (GFP) has proven useful for the study of protein interactions and dynamics for the last twenty years. A variety of new fluorescent proteins have been developed that expand the use of available excitation spectra. We have undertaken an analysis of seven of the most useful fluorescent proteins (XFPs), Cerulean (and mCerulean3), Teal, GFP, Venus, mCherry and TagRFP657, as fusions to the archetypal G-protein coupled receptor, the β2 adrenergic receptor (β2AR). We have characterized these β2AR::XFP fusions in respect to membrane trafficking and G-protein activation. We noticed that in the mouse neural cell line, OP 6, that membrane bound β2AR::XFP fusions robustly localized in the filopodia identical to gap::XFP fusions. All β2AR::XFP fusions show responses indistinguishable from each other and the non-fused form after isoprenaline exposure. Our results provide a platform by which G-protein coupled receptors can be dissected for their functionality.近二十年来，绿色荧光蛋白（GFP）已被广泛应用于蛋白质相互作用与动态变化的相关研究。目前已有多种新型荧光蛋白被开发出来，拓展了现有激发光谱的应用边界。本研究针对七种应用最为广泛的荧光蛋白（XFPs）——包括Cerulean（及mCerulean3）、Teal、GFP、Venus、mCherry与TagRFP657——开展分析，将其与典型的G蛋白偶联受体（G-protein coupled receptor）β2肾上腺素能受体（β2AR）构建融合蛋白。我们对这些β2AR::XFP融合蛋白的膜运输与G蛋白激活特性进行了系统表征。在小鼠神经细胞系OP6中，研究人员观察到膜结合型β2AR::XFP融合蛋白可高效定位于丝状伪足，其定位模式与gap::XFP融合蛋白完全一致。所有β2AR::XFP融合蛋白在经异丙肾上腺素刺激后，均展现出彼此无显著差异、且与未融合形式一致的响应特性。本研究结果为G蛋白偶联受体的功能解析提供了可靠的研究平台。