A luciferase complementation assay system using transferable mouse artificial chromosomes to monitor protein–protein interactions mediated by G protein-coupled receptors
Bibliographic record
Abstract
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.
Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.
How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.002 | 0.001 |
| Meta-epidemiology (narrow) | 0.003 | 0.001 |
| Meta-epidemiology (broad) | 0.002 | 0.002 |
| Bibliometrics | 0.003 | 0.002 |
| Science and technology studies | 0.001 | 0.001 |
| Scholarly communication | 0.001 | 0.001 |
| Open science | 0.002 | 0.001 |
| Research integrity | 0.001 | 0.004 |
| Insufficient payload (model declined to judge) | 0.004 | 0.003 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".