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

To date, the visualization of beta(2)-adrenergic receptor (beta(2)AR) trafficking has been largely limited to immunocytochemical analyses of acute internalization events of epitope-tagged receptors in various transfection systems, The development of a beta(2)AR conjugated with green fluorescent protein (beta(2)AR-GFP) provides the opportunity for a more extensive optical analysis of beta(2)AR sequestration, down-regulation, and recycling in cells. Here we demonstrate that stable expression of beta(2)AR-GFP in HeLa cells enables a detailed temporal and spatial analysis of these events, Time dependent colocalization of beta(2)AR-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 beta(2)AR-GFP was consistent with measures of receptor sequestration and down-regulation generated by radioligand-receptor binding assays. Cells stimulated with different beta-agonists revealed time courses of beta(2)AR-GFP redistribution reflective of the intrinsic activity of each agonist.