A reporter assay for G-protein-coupled receptors using a B-cell line suitable for stable episomal expression.
We have established a cAMP response element (CRE)-mediated reporter assay system for G-protein-coupled receptors (GPCRs) using an oriP-based estrogen-inducible expression vector and the B-cell line (GBC53 or GBCC71) that expresses EBNA-1 and is adapted to serum-free culture. GBC53 harbors a GAL4-ER expression unit and a CRE-luciferase gene in the genome, and GBCC71 also harbors expression units for two chimeric Galphas proteins (Gs/q and Gs/i). Introduction of a GPCR expression plasmid into GBC53 or GBCC71 creates polyclonal stable transformants in 2 weeks, and these are easily expanded and used for assays after induction of the GPCR expression. Using GBC53, we detected ligand-dependent signals of Gs-coupled GPCRs such as glucagon-like peptide 1 receptor (GLP1R) and beta2 adrenergic receptor (beta2AR) with high sensitivity. Interestingly, we also detected constitutive activity of beta2AR. Using GBCC71, we detected ligand-dependent signals of Gq- or Gi-coupled GPCRs such as H1 histamine receptor and CXCR1 chemokine receptor in addition to Gs-coupled GPCRs. An agonist, antagonist, or inverse agonist was successfully evaluated in this system. We succeeded in constructing a 384-well high-throughput screening (HTS) system for GLP1R. This system enabled us to easily and rapidly make a large number of efficient GPCR assay systems suitable for HTS as well as ligand hunting of orphan GPCRs.
Saeki
Satoshi
S
Drug Discovery Research Laboratories, Research Division, Kyowa Hakko Kirin, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8731, Japan.
Kunitomo
Hirofumi
H
Narita
Yoshiyasu
Y
Mimura
Hideki
H
Nishi
Tatsunari
T
Sasaki
Katsutoshi
K
D001402:B-Lymphocytes / Q000378:metabolism; D045744:Cell Line, Tumor; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter; D000067757:Glucagon-Like Peptide-1 Receptor; D057166:High-Throughput Screening Assays; D006801:Humans; D008024:Ligands; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D018027:Receptors, Glucagon / Q000235:genetics / Q000378:metabolism; D011969:Receptors, Histamine H1 / Q000235:genetics / Q000378:metabolism; D023062:Receptors, Interleukin-8A / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D017362:Cyclic AMP Response Element-Binding Protein; C000593833:GLP1R protein, human; D000067757:Glucagon-Like Peptide-1 Receptor; D008024:Ligands; D018343:Receptors, Adrenergic, beta-2; D043562:Receptors, G-Protein-Coupled; D018027:Receptors, Glucagon; D011969:Receptors, Histamine H1; D023062:Receptors, Interleukin-8A; D011993:Recombinant Fusion Proteins; D008156:Luciferases