ABSTRACT
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.
Subject(s)
Genes, Reporter , Receptors, G-Protein-Coupled/metabolism , B-Lymphocytes/metabolism , Cell Line, Tumor , Cyclic AMP Response Element-Binding Protein/genetics , Cyclic AMP Response Element-Binding Protein/metabolism , Gene Expression , Glucagon-Like Peptide-1 Receptor , High-Throughput Screening Assays , Humans , Ligands , Luciferases/genetics , Luciferases/metabolism , Plasmids , Receptors, Adrenergic, beta-2/genetics , Receptors, Adrenergic, beta-2/metabolism , Receptors, G-Protein-Coupled/genetics , Receptors, Glucagon/genetics , Receptors, Glucagon/metabolism , Receptors, Histamine H1/genetics , Receptors, Histamine H1/metabolism , Receptors, Interleukin-8A/genetics , Receptors, Interleukin-8A/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
RECQL1 and RECQL5 as well as BLM reportedly interact with TOP3alpha whose defect is lethal for the cell. Therefore in this study, we characterized recql5/recql1/blm triple mutants from DT40 cells to determine whether the triple mutants show a top3alpha disrupted cell-like phenotype. The triple mutants are viable. Moreover, both blm/recql1 and recql5/blm cells, and recql5/recql1/blm cells grew slightly slower than blm cells, that is, triple mutant cells grew almost the same rate as either of the double mutant cells. The blm cells showed sensitivity to methyl methanesulfonate (MMS) and ultraviolet light (UV), about a 10-fold increase in sister chromatid exchange (SCE), and about a 3-fold increase in damage-induced mitotic chiasma compared to wild-type cells. The triple mutants showed the same sensitivity to MMS or UV and the same frequency of damage-induced mitotic chiasma compared to those of blm cells, indicating that unlike BLM, RECQL1 and RECQL5 play a little role in the repair of or tolerance to DNA damages. However, recql5/blm cells showed higher frequency of SCE than blm cells, whereas the RECQL1 gene disruption had no effect on SCE in blm cells and even in recql5/blm cells.
Subject(s)
Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/physiology , DNA Helicases/genetics , DNA Helicases/physiology , DNA Topoisomerases, Type I/metabolism , DNA Topoisomerases, Type I/physiology , RecQ Helicases/genetics , RecQ Helicases/physiology , Adenosine Triphosphatases/metabolism , Animals , Cells, Cultured , Chickens , DNA Damage/physiology , DNA Helicases/metabolism , Methyl Methanesulfonate/pharmacology , Models, Biological , Mutant Proteins/metabolism , Mutant Proteins/physiology , Protein Binding , Protein Interaction Domains and Motifs/genetics , Protein Interaction Domains and Motifs/physiology , RecQ Helicases/metabolism , Sister Chromatid Exchange/radiation effects , Ultraviolet RaysABSTRACT
Human RECQL1 and RECQL5 belong to the RecQ family that includes Bloom syndrome, Werner syndrome, and Rothmund-Thomson syndrome causative genes. Cells derived from individuals suffering from these syndromes show significant levels of genomic instability. However, neither RECQL1 nor RECQL5 has been related to a disease, and nothing is known about the functions of RecQL1 and RecQL5. We generated here RECQL1(-/-), RECQL5(-/-), RECQL1(-/-)/RECQL5(-/-), RECQL1(-/-)/BLM(-/-), and RECQL5(-/-)/BLM(-/-) cells from chicken B-lymphocyte line DT40 cells. Although BLM(-/-) DT40 cells showed a slow-growth phenotype, a higher sensitivity to methyl methanesulfonate than the wild type, and an approximately 10-fold increase in the frequency of sister chromatid exchange (SCE) compared to wild-type cells, RECQL1(-/-), RECQL5(-/-), and RECQL1(-/-)/RECQL5(-/-) cells showed no significant difference from the wild-type cells in growth, sensitivity to DNA-damaging agents, and the frequency of SCE. However, both RECQL1(-/-)/BLM(-/-) and RECQL5(-/-)/BLM(-/-) cells grew more slowly than BLM(-/-) cells because of the increase in the population of dead cells, indicating that RecQL1 and RecQL5 are somehow involved in cell viability under the BLM function-impaired condition. Surprisingly, RECQL5(-/-)/BLM(-/-) cells showed a higher frequency of SCE than BLM(-/-) cells, indicating that RecQL5 suppresses SCE under the BLM function-impaired condition.
