Visualization of agonist-induced sequestration and down-regulation of a green fluorescent protein-tagged β2-adrenergic receptor

Abstract

To date, the visualization of β2-adrenergic receptor (β2AR) trafficking has been largely limited to immunocytochemical analyses of acute internalization events of epitope-tagged receptors in various transfection systems. The development of a β2AR conjugated with green fluorescent protein (β2AR-GFP) provides the opportunity for a more extensive optical analysis of β2AR sequestration, down-regulation, and recycling in cells. Here we demonstrate that stable expression of β2AR-GFP in HeLa cells enables a detailed temporal and spatial analysis of these events. Time- dependent colocalization of β2AR-GFP with rhodamine-labeled transferrin and rhodamine-labeled dextran following agonist exposure demonstrates receptor distribution to early endosomes (sequestration) and lysosomes (down- regulation), respectively. The observed temporal distribution of β2AR-GFP was consistent with measures of receptor sequestration and down-regulation generated by radioligand-receptor binding assays. Cells stimulated with different β-agonists revealed time courses of β2AR-GFP redistribution reflective of the intrinsic activity of each agonist.