2012
DOI: 10.1007/978-1-61779-909-9_9
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Measurements of β-Arrestin Recruitment to Activated Seven Transmembrane Receptors Using Enzyme Complementation

Abstract: The recruitment of arrestins to activated 7TMRs results in the activation of alternative signaling pathways, quenching of G-protein activation, and coupling to clathrin-mediated endocytosis. The nearly ubiquitous involvement of arrestin in 7TMR signaling has spurred the development of several methods for monitoring this interaction in mammalian cells. Nonetheless, few maintain the reproducibility and precision necessary for drug discovery applications. Enzyme fragment complementation technology (EFC) is an eme… Show more

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Cited by 45 publications
(64 citation statements)
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“…Next, we investigated the effects of 15 on β 2 AR function in cells by measuring G-protein-mediated cAMP production (28,29) and β-arrestin recruitment to the receptor (29,30). Due to the high signal amplification of the G-protein activation assay compared with β-arrestin recruitment, to achieve comparable signaling outputs between the two, we used endogenously expressed β 2 AR in the reporter cells to measure cAMP production, but we used stably overexpressed β 2 V 2 R for monitoring β-arrestin recruitment.…”
Section: Resultsmentioning
confidence: 99%
“…Next, we investigated the effects of 15 on β 2 AR function in cells by measuring G-protein-mediated cAMP production (28,29) and β-arrestin recruitment to the receptor (29,30). Due to the high signal amplification of the G-protein activation assay compared with β-arrestin recruitment, to achieve comparable signaling outputs between the two, we used endogenously expressed β 2 AR in the reporter cells to measure cAMP production, but we used stably overexpressed β 2 V 2 R for monitoring β-arrestin recruitment.…”
Section: Resultsmentioning
confidence: 99%
“…Potential opioid receptor ligands were identified from an HTS campaign using the PathHunter enzyme complementation assay technology (DiscoveRx) (15). In this system, an N-terminal deletion mutant of β-galactosidase is fused to the C terminus of stably expressed β-arrestin 2 in human osteosarcoma (U2OS) cells.…”
Section: Resultsmentioning
confidence: 99%
“…This assay is based on b-galactosidase enzyme complementation technology in which b-arrestin-2 is fused to an Nterminal deletion mutant of b-galactosidase [enzyme acceptor (EA) protein] and hPTH1R is fused to a smaller weakly binding 42 AA complementing Prolink fragment. Agonist activation of the receptor promotes direct interaction with b-arrestin-2 and the hPTH1R and complementation of the two b-galactosidase fragments to form functional enzyme (McGuinness et al, 2009;Yin et al, 2009;Bassoni et al, 2012). In brief, vials of frozen CHO-K1 cells stably expressing recombinant hPTH1R were thawed in a 37°C water bath for 10 seconds and diluted in prewarmed media, 100-ml aliquots of cell suspension were added to 96-well assay microtiter plates (8,000 cells/ well), and plates were incubated for 48 hours at 37°C (5% CO 2 , 95% RH) before performing assays.…”
Section: Methodsmentioning
confidence: 99%
“…Agonist activation of hPTH1R not only stimulates G s -and G q -signaling, but also stimulates b-arrestin recruitment; b-arrestin-dependent signaling, including activation of ERK1/2, b-arrestin-mediated hPTH1R internalization; and desensitization (Bisello et al, 2002;Gesty-Palmer et al, 2006;Vilardaga et al, 2011). Agonist stimulation of hPTH1R-mediated b-arrestin recruitment in CHO-K1 cells stably expressing recombinant hPTH1R was measured using the DiscoveRX PathFinder assay kit based on the b-galactosidase enzyme complementation platform (Olson and Eglen, 2007;Bassoni et al, 2012). 65, 33, 0.87, 11, 36, 44, 98, and (Fig.…”
Section: Domains Of Pth and Pthrp Mediating Pth1r Signalingmentioning
confidence: 99%