A homogeneous enzyme fragment complementation-based beta-arrestin translocation assay for high-throughput screening of G-protein-coupled receptors.
G-protein-coupled receptors (GPCRs) represent one of the largest gene families in the human genome and have long been regarded as valuable targets for small-molecule drugs. The authors describe a new functional assay that directly monitors GPCR activation. It is based on the interaction between beta-arrestin and ligand-activated GPCRs and uses enzyme fragment complementation technology. In this format, a GPCR of interest is fused to a small (approximately 4 kDa), optimized alpha fragment peptide (termed ProLink) derived from beta-galactosidase, and beta-arrestin is fused to an N-terminal deletion mutant of beta-galactosidase (termed the enzyme acceptor [EA]). Upon activation of the receptor, the beta-arrestin-EA fusion protein binds the activated GPCR. This interaction drives enzyme fragment complementation, resulting in an active beta-galactosidase enzyme, and thus GPCR activation can be determined by quantifying beta-galactosidase activity. In this report, the authors demonstrate the utility of this technology to monitor GPCR activation and validate the approach using a Galphai-coupled GPCR, somatostatin receptor 2. Potential application to high-throughput screens in both agonist and antagonist screening modes is exemplified.
Zhao
Xiaoning
X
Lead Discovery Department, Chemistry Research and Discovery, Amgen, Inc., South San Francisco, California 94080, USA. xiaoning@amgen.com
Jones
Adrie
A
Olson
Keith R
KR
Peng
Kun
K
Wehrman
Tom
T
Park
Adam
A
Mallari
Rommel
R
Nebalasca
Danilo
D
Young
Stephen W
SW
Xiao
Shou-Hua
SH
D000818:Animals; D019390:Arrestins / Q000235:genetics / Q000378:metabolism*; D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D006801:Humans; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D017481:Receptors, Somatostatin / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D013004:Somatostatin / Q000235:genetics / Q000378:metabolism; D000071557:beta-Arrestins; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D019390:Arrestins; D010455:Peptides; D043562:Receptors, G-Protein-Coupled; D017481:Receptors, Somatostatin; D011993:Recombinant Fusion Proteins; D000071557:beta-Arrestins; D013004:Somatostatin; C093733:somatostatin receptor 2; D001616:beta-Galactosidase