Use of Novel ebBRET Biosensors for Comprehensive Signaling Profiling of One Hundred Therapeutically Relevant Human GPCRs
Bibliographic record
Abstract
Functional selectivity is the ability of a given GPCR to engage multiple signaling pathways, with distinct ligands of the given receptor displaying different efficacies in engaging receptor‐coupled pathways. Full exploitation of functional selectivity in drug development will require an exhaustive description of the effectors that can be engaged by a given receptor, thus revealing receptor‐ and ligand‐specific signaling signatures. Here, we describe the signaling profiles of 100 therapeutically relevant human GPCRs in response to their endogenous (or prototypical) ligands. Profiling was performed with 15 pathway‐selective enhanced bystander bioluminescence resonance energy transfer (ebBRET) biosensors monitoring the activation of specific Gα proteins and βarrestins 1 and 2. The G protein biosensors represent a new generation of BRET‐based sensors that measure the translocation of G protein effectors to the plasma membrane with no need for modifying the G proteins or the receptors. Over 1,500 dose‑response curves were generated, revealing a great diversity in GPCR coupling selectivity. Our data highlight that the Gi family displayed the highest general coupling while Gs and G12/13 families were less frequently engaged by the receptors tested. Certain GPCRs (17%) showed greater selectivity, with coupling restricted to a single G protein subtype or members of the same G protein family. Others showed broader activation profiles; specifically, 39%, 35% and 9% of GPCRs tested coupled to members of two, three and all four G protein families, respectively. Somewhat surprisingly, some receptors showed G protein subtype selectivity among the members of the same family. Of note, over 50 novel GPCR/G protein couplings were uncovered. In addition to highlighting receptor G protein coupling preferences, our data revealed that 78% of GPCRs recruited βarrestin 1 and/or 2. For certain GPCRs, co‑expression of GRK2 augmented (and even exposed) βarrestin engagement. Finally, in addition to the signaling profiling application, we demonstrated the versatility and usefulness of our ebBRET biosensor platform to study constitutive GPCR activity/inverse agonism, ligand‐ and SNP‐induced biased signaling and cross‐talk systems pharmacology. Overall, this work complements and enriches the current body of data on GPCR effector coupling. Moreover, it presents innovative tools allowing to further explore novel GPCR pharmacology. The resources provided in this study, combined to other signaling profiling and omic‐scale datasets, will help deconvolute the complexities of GPCR biology and pharmacology and lead to innovative therapeutic exploitation of 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 | 0.001 | 0.001 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.001 | 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".