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Vol. 273, Issue 1, 322-328, January 2, 1998

Visualization of Agonist-induced Sequestration and Down-regulation of a Green Fluorescent Protein-tagged beta 2-Adrenergic Receptor

Lorena Kallal, Alison W. Gagnon, Raymond B. Penn, and Jeffrey L. Benovic

From the Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

To date, the visualization of beta 2-adrenergic receptor (beta 2AR) 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 2AR conjugated with green fluorescent protein (beta 2AR-GFP) provides the opportunity for a more extensive optical analysis of beta 2AR sequestration, down-regulation, and recycling in cells. Here we demonstrate that stable expression of beta 2AR-GFP in HeLa cells enables a detailed temporal and spatial analysis of these events. Time-dependent colocalization of beta 2AR-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 2AR-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 2AR-GFP redistribution reflective of the intrinsic activity of each agonist.

Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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