A β-Arrestin/Green Fluorescent Protein Biosensor for Detecting G Protein-coupled Receptor Activation*
- From the Howard Hughes Medical Institute Laboratories and Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710
Abstract
G protein-coupled receptors (GPCR) represent the single most important drug targets for medical therapy, and information from genome sequencing and genomic data bases has substantially accelerated their discovery. The lack of a systematic approach either to identify the function of a new GPCR or to associate it with a cognate ligand has added to the growing number of orphan receptors. In this work we provide a novel approach to this problem using a β-arrestin2/green fluorescent protein conjugate (βarr2-GFP). It provides a real-time and single cell based assay to monitor GPCR activation and GPCR-G protein-coupled receptor kinase or GPCR-arrestin interactions. Confocal microscopy demonstrates the translocation of βarr2-GFP to more than 15 different ligand-activated GPCRs. These data clearly support the common hypothesis that the β-arrestin binding of an activated receptor is a convergent step of GPCR signaling, increase by 5-fold the number of GPCRs known to interact with β-arrestins, demonstrate that the cytosol is the predominant reservoir of biologically active β-arrestins, and provide the first direct demonstration of the critical importance of G protein-coupled receptor kinase phosphorylation to the biological regulation of β-arrestin activity and GPCR signal transduction in living cells. The use of βarr2-GFP as a biosensor to recognize the activation of pharmacologically distinct GPCRs should accelerate the identification of orphan receptors and permit the optical study of their signal transduction biology intractable to ordinary biochemical methods.
Footnotes
-
↵* This work was supported by National Institutes of Health Grant NS 19576, a Bristol Myers Squibb Unrestricted Grant Award, and an unrestricted grant from Zeneca Pharmaceuticals (to M. G. C.) and National Institutes of Health Grant HL 03422 (to L. S. B.).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.
-
↵‡ Recipient of a Michael Smith Postdoctoral Fellowship from the Medical Research Council, Canada. Present address: The John P. Robarts Inst., P. O. Box 5015, 100 Perth Dr., London, Ontario N6A 5K8, Canada.
-
↵§ To whom correspondence should be addressed: Duke University Medical Center, Box 3287, Durham, NC 27710. Tel.: 919-684-5433; Fax: 919-681-8641; E-mail: caron002{at}mc.duke.edu.
-
↵1 The abbreviations used are: GPCR, G protein-coupled receptor; GFP, green fluorescent protein; βarr2-GFP, β-arrestin2 green fluorescent protein conjugate; GRK, G protein-coupled receptor kinase; β2AR, β2-adrenergic receptor.
-
↵2 On the World Wide Web atreceptor.mgh.harvard.edu/ GCRDHOME.html.
-
- Received August 21, 1997.
