Green fluorescent protein fused to peptide agonists of two dissimilar G protein‐coupled receptors: novel ligands of the bradykinin B<sub>2</sub> (rhodopsin family) receptor and parathyroid hormone <scp>PTH</scp><sub>1</sub> (secretin family) receptor
Bibliographic record
Abstract
We hypothesized that peptide hormone sequences that stimulate and internalize G protein-coupled receptors (GPCRs) could be prolonged with a functional protein cargo. To verify this, we have selected two widely different pairs of peptide hormones and GPCRs that nevertheless share agonist-induced arrestin-mediated internalization. For the parathyroid hormone (PTH) PTH1 receptor (PTH1R) and the bradykinin (BK) B2 receptor (B2R), we have designed fusion proteins of the agonists PTH1-34 and maximakinin (MK, a BK homologue) with the enhanced green fluorescent protein (EGFP), thus producing candidate high molecular weight ligands. According to docking models of each hormone to its receptor, EGFP was fused either at the N-terminus (MK) or C-terminus (PTH1-34) of the ligand; the last construction is also secretable due to inclusion of the preproinsulin signal peptide and has been produced as a conditioned medium. EGFP-MK has been produced as a lysate of transfected cells. Using an enzyme-linked immunosorbent assay (ELISA) for GFP, average concentrations of 1.5 and 1670 nmol/L, respectively, of ligand were found in these preparations. The functional properties and potential of these analogs for imaging receptor-expressing cells were examined. Microscopic and cytofluorometric evidence of specific binding and internalization of both fusion proteins was obtained using recipient HEK 293a cells that expressed the cognate recombinant receptor. Endosomal colocalization studies were conducted (Rab5, Rab7, β-arrestin1). Evidence of agonist signaling was obtained (expression of c-Fos, cyclic AMP responsive element (CRE) reporter gene for PTH1-34-EGFP). The constructs PTH1-34-EGFP and EGFP-MK represent bona fide agonists that support the feasibility of transporting protein cargoes inside cells using GPCRs.
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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 | 0.001 | 0.001 |
| 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".