Assays Based on the Agonist Promoted Arrestin Recruitment

Fluorescence and Bioluminescence-Based Protein Translocation Assays

The translocation of P-arrestin to GPCRs is a hallmark of receptor activation that is increasingly used in ligand screening assay. Several assays have been developed, including a fluorescence- based assay that measures the cellular translocation of P-arrestin-green fluorescent protein (GFP) fusion proteins. Whereas P-arrestin-GFP is diffusely distributed in the cytoplasm under basal conditions, upon agonist stimulation, the fusion protein is first translocated to the plasma membrane and then internalized together with the activated receptor [61, 62]. If the changes in P-ARR localization following agonist stimulation are small, data analysis, particularly in a high-throughput setting, may become difficult. A commercially available version of the assay is sold as "Transfluor technology". This assay has been used to identify novel ligands for Drosophila orphan 7TM proteins and to confirm ligand-receptor pairs [63]. The acylation stimulating protein (ASP) has been identified as a ligand for GPR77 (C5L2), using this assay (Table 7.1) [64] . Similar assays have been developed to monitor P-arrestin translocation by bioluminescence resonance energy transfer [65-67].

Protease-Mediated Transcriptional Reporter Gene Assay Another assay based on P-arrestin recruitment and activation of a transcriptional reporter gene has been developed recently [68]. In this assay, named TANGOâ„¢ (invitrogen Corporation, Madison, WI), the receptor is fused at its C-terminal extremity to a protease cleavage site followed by the tetracycline-controlled transactivator (tTA). A second fusion protein of the corresponding protease fused to P-arrestin2 is coexpressed with the receptor fusion protein in a cell line expressing a tTA- dependent reporter gene. Ligand stimulation of the receptor leads to P-arrestin recruitment that induces the cleavage of the transcription factor. The released transcription factor translocates into the nucleus where it activates the transcription of the tTA-dependent luciferase reporter gene. This technology was used to identify the leukocyte chemoattractant chemerin as a ligand for the orphan GPR1 [69].

Enzyme Fragment Complementation This assay relies on the reconstitution of a functional P-galactosidase enzyme from two complementing P-galactosidase mutants, one fused to the receptor of interest, the other to P - arrestin [70] . The commercially available PathHunter technology from DiscoveRx (Fremont, CA) restores P-galactosidase activity by complementing a catalytically inactive form (enzyme acceptor) with a peptide containing the catalytic domain (enzyme donor). P-arrestin recruitment to the receptor and subsequent reconstitution of P -galactosidase activity is detected in lysed cells by measuring chemiluminescence. DiscoveRx is now extending this assay by generating PathHunter cell lines expressing orphan 7TM proteins either alone or in the presence of a non-orphan GPCR.

Collectively, these P - arrestin - dependent approaches are expected to be applied to a wide range of orphan 7TM proteins as the majority of GPCRs recruit P-arrestins [71, 72] . However, exceptions are likely to exist as some receptors may exhibit P - arrestin - independent internalization or interact con-stitutively (in the absence of ligand) with P - arrestin.

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