Abstract
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.
Highlights
G-protein coupled receptors (GPCRs) represent the largest family of eukaryotic membrane proteins [1]
Localization of Untagged Fluorescent Proteins (XFPs) Do our set of untagged fluorescent proteins contain any internal signals for trafficking to the plasma membrane? We developed a transient transfection system using an olfactory placode cell line (OP 6) to test the fluorescent proteins expressed under a CMV promoter (Figure S1 A)
We have determined that some of the most useful fluorescent proteins do not affect the mouse β2 adrenergic receptor (β2AR) trafficking and function when fused to its carboxyl terminus, consistent with the previous studies of human β2AR::Green fluorescent protein (GFP) [11,12,13]
Summary
G-protein coupled receptors (GPCRs) represent the largest family of eukaryotic membrane proteins [1]. The GPCR superfamily is natively expressed in many cell types; 90% of GPCRs are expressed in the brain. They are activated by almost every known neurotransmitter, peptide, and chemokine [2]. Mutations to GPCRs are known to cause a cascade of neurological and neurodegenerative diseases [3,4]. Common to all GPCRs is their seven transmembrane structure: seven alpha helical transmembrane domains connected by intra and extracellular loops with an extracellular amino terminus and an intracellular carboxyl terminus. The intracellular regions regulate G-protein binding, while ligand-binding properties exist in the extracellular and transmembrane domains [5]. GPCR’s are the target of over 50% of therapeutic drugs, and a quarter of the top marketed drugs target GPCRs [6]
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