Real-time imaging of green fluorescent protein-tagged β 2-adrenergic receptor distribution in living cells

Abstract

In an attempt to investigate the subcellular trafficking of β 2-adrenergic receptor (β 2AR) in living cells, we performed real-time imaging of β 2AR tagged with green fluorescent protein (GFP). We transiently transfected a chimera construct of β 2AR and GFP (β 2AR-GFP) into HEK 293 cells, primary cultured rat hippocampal neurons and cortical neuronal cells, and then compared the dynamic changes in subcellular localization of β 2AR-GFP in these live cells. In the absence of ligands, β 2AR-GFP fluorescence was detected predominantly on the plasma membrane in HEK 293 cells as well as on the surface of cell somata and dendrites in cortical neuronal cells. In contrast, in hippocampal neurons, β 2AR-GFP was diffusely distributed not only on the surface of cells but in the whole cell somata and dendrites. In HEK 293 cells, cortical neuronal cells and cortical glial cells, time-lapse images showed the rapid appearance of a punctate distribution pattern that became more numerous over the 15-min course of agonist exposure. Semiquantitative analysis revealed the time-course internalization of β 2AR-GFP in a single living cell. In hippocampal neurons, β 2AR-GFP distribution became scattered both in cell somata and dendrites following agonist exposure. Three-dimensional analysis of time-lapse images revealed a significant portion of β 2AR-GFP was distributed in endosomal compartments, along with Alexa 546-labeled transferrin, in all types of cells. Our results demonstrate spatial and temporal redistribution pattern of β 2AR in living non-neuronal cells and neuronal cells.