TY - JOUR
T1 - A luciferase complementation assay system using transferable mouse artificial chromosomes to monitor protein–protein interactions mediated by G protein-coupled receptors
AU - Uno, Narumi
AU - Fujimoto, Tomohito
AU - Komoto, Shinya
AU - Kurosawa, Gene
AU - Sawa, Masaaki
AU - Suzuki, Teruhiko
AU - Kazuki, Yasuhiro
AU - Oshimura, Mitsuo
N1 - Funding Information:
Acknowledgements We thank Nakamura for providing pTNH6-H and p-CAG-T7-F plasmids, and Mitchell Arico from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript. This work was supported in part by the Regional Innovation Strategy Support Program of the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (M.O.).
Funding Information:
Funding The Chromosome Engineering Research Center (CERC) has a collaboration with Carna Biosciences, Inc. related to this project. CERC received funding from Carna Biosciences, Inc.
Funding Information:
We thank Nakamura for providing pTNH6-H and p-CAG-T7-F plasmids, and Mitchell Arico from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript. This work was supported in part by the Regional Innovation Strategy Support Program of the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (M.O.).
Publisher Copyright:
© 2018, The Author(s).
PY - 2018/12/1
Y1 - 2018/12/1
N2 - G protein-coupled receptors (GPCRs) are seven-transmembrane domain receptors that interact with the β-arrestin family, particularly β-arrestin 1 (ARRB1). GPCRs interact with 33% of small molecule drugs. Ligand screening is promising for drug discovery concerning GPCR-related diseases. Luciferase complementation assay (LCA) enables detection of protein–protein complementation via bioluminescence following complementation of N- and C-terminal luciferase fragments (NEluc and CEluc) fused to target proteins, but it is necessary to co-express the two genes. Here, we developed LCAs with mouse artificial chromosomes (MACs) that have unique characteristics such as stable maintenance and a substantial insert-carrying capacity. First, an NEluc-ARRB1 was inserted into MAC4 by Cre-loxP recombination in CHO cells, named ARRB1-MAC4. Second, a parathyroid hormone receptor 2 (PTHR2)-CEluc or prostaglandin EP4 receptor (hEP4)-CEluc were inserted into ARRB1-MAC4, named ARRB1-PTHR2-MAC4 and ARRB1-hEP4-MAC4, respectively, via the sequential integration of multiple vectors (SIM) system. Each MAC was transferred into HEK293 cells by microcell-mediated chromosome transfer (MMCT). LCAs using the established HEK293 cell lines resulted in 35,000 photon counts upon somatostatin stimulation for ARRB1-MAC4 with transient transfection of the somatostatin receptor 2 (SSTR2) expression vector, 1800 photon counts upon parathyroid hormone stimulation for ARRB1-PTHR2-MAC4, and 35,000 photon counts upon prostaglandin E2 stimulation for ARRB1-hEP4-MAC4. These MACs were maintained independently from host chromosomes in CHO and HEK293 cells. HEK293 cells containing ARRB1-PTHR2-MAC4 showed a stable reaction for long-term. Thus, the combination of gene loading by the SIM system into a MAC and an LCA targeting GPCRs provides a novel and useful platform to discover drugs for GPCR-related diseases.
AB - G protein-coupled receptors (GPCRs) are seven-transmembrane domain receptors that interact with the β-arrestin family, particularly β-arrestin 1 (ARRB1). GPCRs interact with 33% of small molecule drugs. Ligand screening is promising for drug discovery concerning GPCR-related diseases. Luciferase complementation assay (LCA) enables detection of protein–protein complementation via bioluminescence following complementation of N- and C-terminal luciferase fragments (NEluc and CEluc) fused to target proteins, but it is necessary to co-express the two genes. Here, we developed LCAs with mouse artificial chromosomes (MACs) that have unique characteristics such as stable maintenance and a substantial insert-carrying capacity. First, an NEluc-ARRB1 was inserted into MAC4 by Cre-loxP recombination in CHO cells, named ARRB1-MAC4. Second, a parathyroid hormone receptor 2 (PTHR2)-CEluc or prostaglandin EP4 receptor (hEP4)-CEluc were inserted into ARRB1-MAC4, named ARRB1-PTHR2-MAC4 and ARRB1-hEP4-MAC4, respectively, via the sequential integration of multiple vectors (SIM) system. Each MAC was transferred into HEK293 cells by microcell-mediated chromosome transfer (MMCT). LCAs using the established HEK293 cell lines resulted in 35,000 photon counts upon somatostatin stimulation for ARRB1-MAC4 with transient transfection of the somatostatin receptor 2 (SSTR2) expression vector, 1800 photon counts upon parathyroid hormone stimulation for ARRB1-PTHR2-MAC4, and 35,000 photon counts upon prostaglandin E2 stimulation for ARRB1-hEP4-MAC4. These MACs were maintained independently from host chromosomes in CHO and HEK293 cells. HEK293 cells containing ARRB1-PTHR2-MAC4 showed a stable reaction for long-term. Thus, the combination of gene loading by the SIM system into a MAC and an LCA targeting GPCRs provides a novel and useful platform to discover drugs for GPCR-related diseases.
UR - http://www.scopus.com/inward/record.url?scp=85051548423&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85051548423&partnerID=8YFLogxK
U2 - 10.1007/s10616-018-0231-7
DO - 10.1007/s10616-018-0231-7
M3 - Article
AN - SCOPUS:85051548423
SN - 0920-9069
VL - 70
SP - 1499
EP - 1508
JO - Cytotechnology
JF - Cytotechnology
IS - 6
ER -