A β-Arrestin/Green Fluorescent Protein Biosensor for Detecting G Protein-coupled Receptor Activation

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 ␤-arrestin2/green fluorescent protein conjugate (␤arr2-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 ␤arr2-GFP to more than 15 different ligand-activated GPCRs. These data clearly support the common hypothesis that the ␤-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 ␤-arrestins, demonstrate that the cytosol is the predominant reservoir of biologically active ␤-arrestins, and provide the first direct demonstration of the critical importance of G protein-coupled receptor kinase phosphorylation to the biological regulation of ␤-arrestin activity and GPCR signal transduction in living cells. The use of ␤arr2-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.The G protein-coupled receptor (GPCR) 1 superfamily is growing rapidly (1-3), creating many new orphan receptors whose properties remain undefined (4 -6). 2 Currently characterized GPCRs display many distinct pharmacologies. For example, they interact with a vast array of ligands and generate intracellular signals by multiple second messenger pathways (4,8,9). Based on work with rhodopsin and the ␤ 2 -adrenergic receptor (␤ 2 AR), it has been postulated that members of the GPCR superfamily desensitize via a common mechanism involving the arresting proteins visual arrestin, ␤-arrestin1 and ␤-arrestin2 (10 -13). However, mainly due to the inherent difficulties of examining the interaction of the components mediating desensitization in their native environment or the need for purified reconstituted systems, this has not been clearly established for many GPCRs. Biochemical studies indicate that arrestins regulate GPCR signal transduction (desensitization) by binding agonist-activated receptors that have been phosphorylated by G protein-coupled receptor kinases (GRKs) (12). While the functional source of arrestin molecules targeted to receptors remains unknown, it is apparent that arrestin binding terminates signaling by interdicting receptor interaction with G proteins (12).To characterize the interaction between ␤-arrestin and different GPCRs and to assess the contribution of GRKs to this process, we examined using confocal microscopy how a green fluorescent protein...

Identification of Drosophila Neuropeptide Receptors by G Protein-coupled Receptors-β-Arrestin2 Interactions

“…Such ligands signal well but are very poor at inducing phosphorylation of the activated receptor. In order to study such receptors in the ␤arr2-GFP translocation assay, it has proven useful to overexpress a GRK to enhance receptor phosphorylation, thereby facilitating arrestin recruitment (12,38 observed that most did not require additional GRK activity to display specific responses. However, that same strategy did prove useful to help de-orphan CG13803 and CG8985.…”

“…Some experiments included overexpression of a G protein receptor kinase (GRK2). This was achieved using HEK-293 cells that had GRK2 stably integrated into the genome (12), or by including GRK2 in transfections at a ratio of 5:1:1 (GPCR:␤arr2-GFP:GRK2). To study calcium responses, stable lines expressing different receptors were generated and selected for resistance to hygromycin B (13).…”

“…Within minutes of exposure to specific ligands, the majority of the fluorescence translocates to become associated with the membrane. On that basis, we have suggested its use as a generalized method for GPCR de-orphaning (12).…”

Comparative Studies of Molecular Mechanisms of Dopamine D2 and D3 Receptors for the Activation of Extracellular Signal-regulated Kinase

“…Plasmids-Wild-type human dopamine D 2 receptor (short splice variant, D 2S R; long splice variant, D 2L R) and D 3 receptor (D 3 R) subtypes in mammalian expression vector pCMV5, G protein-coupled receptor kinases (GRKs) 2 and 3, ␤-arrestins 1 and 2, ␤-arrestin 1-GFP, ␤-arrestin 2-GFP, V53D-␤-arrestin 1, and K44A-dynamin I have been described in our previous studies (14,(15)(16)(17)(18). The pEGFP-N1 expression plasmid encoding GFP-tagged ERK2 (19) ERK Measurement-After transfection, cells were cultured in 6-well plates.…”

Constitutive arrestin-mediated desensitization of a human vasopressin receptor mutant associated with nephrogenic diabetes insipidus

“…The increased affinity of activated receptors for arrestins results in the observable translocation of arrestins from the cell cytosol to the plasma membrane, frequently followed by clathrin-coated pit internalization of the arrestin-bound receptors (5)(6)(7)(8)(9). Arrestin translocation has been visualized by using fusion proteins of green fluorescent protein (GFP) homologues and an arrestin family member (visual arrestin, ␤arrestin1 and ␤arres-tin2) for numerous GPCRs including the angiotensin, neurokinin, thyrotropin-releasing hormone, and human vasopressin type II receptors (V2R) (5,6,(10)(11)(12).…”