Visualization of Agonist-induced Sequestration and Down-regulation of a Green Fluorescent Protein-tagged β2-Adrenergic Receptor*
- From the Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
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.
Footnotes
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↵* This work was supported in part by National Institutes of Health Grants GM44944 and GM47417.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ Established investigator of the American Heart Association. To whom correspondence should be addressed: Kimmel Cancer Inst., Thomas Jefferson University, 233 South 10th St., Philadelphia, PA 19107. Tel.: 215-503-4607; Fax: 215-923-1098; E-mail:benovic{at}lac.jci.tju.edu.
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↵1 The abbreviations used are: β2AR, β2-adrenergic receptor; GFP, green fluorescent protein; PCR, polymerase chain reaction; PBS, phosphate-buffered saline.
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- Received May 22, 1997.
- Revision received October 22, 1997.
- The American Society for Biochemistry and Molecular Biology, Inc.
