Functional Analysis of Human D1 and D5 Dopaminergic G Protein-Coupled Receptors: Lessons from Mutagenesis of a Conserved Serine Residue in the Cytosolic End of Transmembrane Region 6
Bibliographic record
Abstract
In mammals, dopamine G protein-coupled receptors (GPCR) are segregated into two categories: D1-like (D1R and D5R) and D2-like (D2R(short), D2R(long), D3R, and D4R) subtypes. D1R and D5R are primarily coupled to stimulatory heterotrimeric GTP-binding proteins (Gs/olf) leading to activation of adenylyl cyclase and production of intracellular cAMP. D1R and D5R share high level of amino acid identity in transmembrane (TM) regions. Yet these two GPCR subtypes display distinct ligand binding and G protein coupling properties. In fact, our studies suggest that functional properties reported for constitutively active mutants of GPCRs (e.g., increased basal activity, higher agonist affinity and intrinsic activity) are also observed in cells expressing wild type D5R when compared with wild type D1R. Herein, we describe an experimental method based on mutagenesis and transfection of human embryonic kidney 293 (HEK293) cells to explore the molecular mechanisms regulating ligand affinity, agonist-independent and dependent activity of D1R and D5R. We will demonstrate how to mutate one conserved residue in the cytosolic end of TM6 of D1R (Ser263) and D5R (Ser287) by modifying two or three nucleotides in the cDNA of human D1-like receptors. Genetically modified D1R and D5R cDNAs are prepared using a polymerase chain reaction method, propagated in E. coli, purified and mutations confirmed by DNA sequencing. Receptor expression constructs are transfected into HEK293 cells cultured in vitro at 37°C in 5% CO(2) environment and used in radioligand binding and whole cAMP assays. In this study, we will test the effect of S263A/G/D and S287A/G/D mutations on ligand binding and DA-dependent activation of D1R and D5R.
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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 | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
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".