Visualization of agonist-induced sequestration and down-regulation of a green fluorescent protein-tagged...

To date, the visualization of beta2-adrenergic receptor (beta2AR) trafficking has been largely limited to immunocytochemical analyses of acute internalization events of epitope-tagged receptors in various transfection systems. The development of a beta2AR conjugated with green fluorescent protein (beta2AR-GFP) provides the opportunity for a more extensive optical analysis of beta2AR sequestration, down-regulation, and recycling in cells. Here we demonstrate that stable expression of beta2AR-GFP in HeLa cells enables a detailed temporal and spatial analysis of these events. Time-dependent colocalization of beta2AR-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 beta2AR-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 beta2AR-GFP redistribution reflective of the intrinsic activity of each agonist.