dataesr/scientific-paragraphs-categorization huggingface dataset:
The pleiotropic signaling and ligand-dependent bias of GPCRs pose a grand challenge in human biology to map the differential activation of specific G proteins. The IUPHAR/BPS Guide to Pharmacology (GtP) database contains reference data from expert curation of literature  (1). GtP couplings covers 253 GPCRs and the four G protein families. The G protein families have been classified into "primary" and "secondary" transducers without quantitative values. Recently, the Inoue group determined the first large-scale quantitative coupling profiles of 148 GPCRs to the Gq wildtype and 10 G protein chimeras employing a TGF-α shedding assay (2) (NTS1 and TRH1 added herein making it 150 receptors). Those chimeras consist of a Gq backbone in which the six most C-terminal Ga residues -a part of the H5 domain inserting to the intracellular receptor cavity -have been replaced to represent all 16 human G proteins (five of which have identical sequences to other G proteins, see below).
In a paper accompanying the present analysis , the Bouvier group quantified the couplings of 100 GPCRs to 12 G proteins: Gs, Gi1, Gi2, Go (GoA and GoB isoforms), Gz, Gq, G11, G14, G15, G12 and G13 but not Golf (couples mainly to olfactory receptors), Gi3 and Gt1-2 (Transducin, couples to rhodopsin (visual) receptors) and Ggust (Gustducin, couples to taste receptors)  (3). The authors used novel enhanced bystander bioluminescence resonance energy transfer biosensors that allow to monitor G protein activation (G protein Effector Membrane Translocation assay; GEMTA) without need to modify the G protein subunits (except for Gs) or the receptors. Here, we analyze the GtP, Inoue and Bouvier coupling datasets to determine confident couplings supported by at least two independent sources, including novel couplings discovered jointly by the two latter sources.
We establish a scalable protocol to normalize quantitative G protein couplings, combine Emax and EC50 into a common log(Emax/EC50) (4) value and aggregate subtypes to allow comparisons across G protein families. On this basis, we develop a unified map of GPCR-G protein couplings that is also made available in the GPCRdb database hub  (5) , describe GPCR-G protein selectivity across an unprecedented number of receptors and coupling data points and reveal correlated co-couplings.
Each profiling study doubled the receptors' average number of G protein family couplings To obtain an overview of the coverages of GPCR-G protein coupling sources, we compared all couplings reported by both the Bouvier (3) and Inoue (2) groups and annotated in the Guide to Pharmacology database (GtP (1)) (Fig. 1a ). This shows that the three sources together comprise couplings for 265 (67%) out of the 398 non-olfactory GPCRs and that 70 of these receptors are present in all datasets. The Bouvier and Inoue datasets have collectively quantified individual G protein couplings of 178 receptors using one assay, whereas the remaining 87 receptors have so far only been annotated in GtP on the G protein family level from a multitude of publications and assays (Fig. 1a ). To allow the comparison of coupling densities and distributions across datasets, we selected the Emax threshold (1.
4 standard deviations above basal) that gives the best agreement between the Bouvier and Inoue dataset. We believe that this is the best possible means to estimate what is correct data (rather than false negative/positive couplings), as large-scale information about what G proteins and GPCRs coupling in nature is not available. This cut-off is more stringent the minimum of 3% signal above basal used in Inoue's original report  (2). We also aggregated G protein subtype couplings of the families (see Methods). This reveals that while GtP covers the largest number of receptors they have relatively few couplings -62% of all GPCR-G protein family pairs are 'non-couplers' (or not tested or annotated) compared to 35% in the Bouvier and 25% in the Inoue dataset (average of 1. 5, 2. 6 and 3. 0 G protein families per GPCR, respectively; Fig.
1b ). In particular G12/13 couplings are underrepresented in GtP where they account for 3% of GPCR-G protein pair datapoints compared to 13% in Bouvier and 17% in Inoue. The Bouvier and Inoue datasets share a more similar overall distribution of couplings, expect for Gs coupling which is twice as frequent within the latter dataset (16% compared to 8%). This demonstrates that the two first systematic coupling profiling studies have substantially expanded the known GPCR-G protein 'couplome' and that their assay platforms are amenable to high-throughput profiling also for the G12/13 family for which robust activation assays appeared only recently (6-8).
The Bouvier and Inoue datasets converge for 68% of G protein couplings, including 101 that are novel Given that the Bouvier and Inoue groups used different biosensors and GtP annotates literature reports from very diverse assays, we sought to determine to which extent they report the same couplings for the same GPCRs i. e. , their 70 common receptors (data in tab 'BIG-QualComp' in Spreadsheet S3). We find that all three sources agree on 39 (55%) of Gs, 45 (64%) of Gi/o, 35 (50%) of Gq/11 and 18 (25%) of G12/13 couplings/non-couplings (mean 34 (49%), Fig. 2a ) showing that the agreement varies substantially across the individual G protein families. When instead analyzing the agreement of just the quantitative studies (excluding GtP) and the G protein subtype level (Fig. 2b ), it increases to an overall of 68% and ranging from at least 53% (for G15) up to 81% (for GoA).
This uncovered such Gs-coupling to the α2C-adrenoceptor and cannabinoid CB1-2 receptors, G12/13 coupling to the purinergic P2Y2 receptor and Gi/o-coupling to the β2-adrenoceptor, which however had weak Gz and GoB coupling in the Bouvier study but did not cross the signal threshold (3). Notably, this is only 2% (5/254) of all GtP's GPCR-G protein family pairs. Taken together, these findings serve to quantify the expansion of the GPCR-G protein 'couplome' while also confirming the outstanding accuracy of the expert annotation in the GtP database. Therefore, the large number of new couplings results mainly from that these couplings were not investigated in literature and not inadequate curation.
Comparison across datasets reveal unsupported couplings requiring additional studies While the Bouvier and recent Inoue datasets give an unprecedented mapping of GPCR activation on the single G protein subtype level, the above analyses point to a need to explain their observed differences to establish which couplings to use where datasets do not agree (Fig. 2 ). To this end, we analyzed the 70 common receptors for 'unsupported' and 'missing' couplings defined as being present or absent in only one of the three datasets (i. e. , non-couplings and couplings, respectively, that are proposed by one but not supported by two datasets). For GtP, we consider coupling to a G protein subtype possible if a coupling has been observed from the respective family. For G15, 30% of Bouvier's couplings are unsupported (Fig.
3a ). The Bouvier dataset has a large fraction, 37% of G15 couplings that are new compared to GtP whereas the Inoue dataset has no new couplings and is missing 13% G15 reported in GtP. This indicates that G15 couplings are underrepresented in both literature (which is annotated in GtP) as well as in Inoue's data. The few known couplings for G15 is likely explained by its lack of expression in the cells most used for in vitro experiments, such as HEK293 cells (3) and by the lack or weak (at very high concentration) effect of the Gq/11 inhibitor tools YM-254890 and FR900359, respectively  (9). Furthermore, several of these novel G15 couplings were validated using Ca 2+ assays in the accompanying paper from the Bouvier group  (3). Taken together, these findings suggest that most of the unsupported G15 couplings in the Bouvier dataset are real (true positive) unique couplings.
For Gs and G12, 27% and 24%, respectively of Inoue's couplings are unsupported (not present in Bouvier or GtP) and 29% of Gs and 49% of G12 couplings are new compared to GtP (Fig. 3b ). In contrast, the Bouvier dataset has no and 13% unsupported Gs and G12 couplings, respectively and is missing 7% of Gs and 6% of G12 couplings in GtP. The lower representation of these two G proteins in Bouvier compared to Inoue may be due to the lower assay window (tab 'DataStats' in Spreadsheet S1). Therefore, additional studies would be needed to distinguish the real new Gs couplings in the Inoue dataset from non-couplers in GtP, which are likely many since robust assays and selective inhibitors have long been available for Gs signaling. Furthermore, the Bouvier data contains fewer G12 couplings than the Inoue group (32% relative 50%, overlap is 26%) that are new compared to GtP.
Further studies with alternative assays maintaining a high G12 sensitivity would be desirable to gain independent support to these couplings. For Gq, G11 and G14, 13%, 16% and 16%, respectively of Inoue's couplings are unsupported (not present in Bouvier or GtP). The Bouvier dataset is instead missing 10%, 10% and 7%, respectively of the known couplings in GtP (Fig. 3b ) and has only 1, 0 and 1 unique such couplings, respectively. This could be due to the fact that in many studies, people have equated Calcium signaling with Gq activation underestimating the role of Gi derived bg in the activation of the PLC-IP3-Calcium signaling pathway  (10) (11) (12) (13). Like for Gs and G15 above, further studies would be needed to reach consensus on whether these couplings are closest to the level of known in GtP or reported by Inoue group.
Notably, although Inoue used the wildtype protein for Gq it has a similar or higher frequency of unsupported couplings compared to all other measured G proteins (Gq: 13% vs. Gi1: 7%, Gi2: 9%, GoA: 7%, GoB: 6%, Gz: 13%, G11: 16%, G14: 16%, G15: and G13: 17%), except Gs and G12 (addressed above). In all, this suggests that the use of chimeric G proteins carrying over of Gq coupling to the other G proteins is not a main cause of Inoue's unsupported couplings. To explain the reason to the unsupported couplings, beyond those addressed above for G15, Gs and G12, we investigated their potencies and efficacies (rows 106-110 in tab 'BIG-QualComp' in Spreadsheet S3). We find that unsupported couplings in the Bouvier and Inoue datasets have average pEC50 values that are 0. 4 and 1. 5 log units -2. 5-fold and 32-fold, respectively -lower than the supported couplings.
Furthermore, their corresponding average Emax values are 18% and 15% lower. This points to the possibility that part of differences across datasets is due to weak couplings which can be difficult to distinguish from basal levels, although the lack of support by GtP may suggest that some couplings are false positives. Both possibilities may be addressed by future datasets or by optimization or development of the profiling platforms. However, until further data becomes available, we recommend a requirement of two independent studies in support of a GPCR-G protein coupling. This is the criterion used herein for the novel couplings and in our online coupling atlas in the GPCRdb database  (5). Together these data highlight the importance of large-scale G protein coupling profiling studies using complementary approaches to establish a robust atlas of GPCR coupling selectivity.
Coupling map reveals 73% promiscuity within and 49% selectivity across protein families To enable quantitative correlation of the Bouvier, Inoue and future couplings, we established a data processing protocol giving the highest similarity and correlation of coupling measurements across datasets (Methods). This uses log transformed EC50 values and minimum-maximum normalized Emax values combined into a unified log(Emax/EC50) value and an aggregation of G proteins onto families based on the maximum subtype values (Fig. 4a ). Based on this protocol, we created a common coupling map, integrating the Bouvier, Inoue and GtP datasets, which is shown in Fig. 4b  and made available in the GPCRdb database hub  (5). This unified coupling map establishes that it is possible to obtain comparable quantitative values despite the differences between biosensors and enables quantitative cross-study comparisons herein and in future studies from the field.
To gain insight into their levels of coupling selectivity, we intersected the G protein profiles of all receptors and counted the number of coupling partners for GPCRs and G proteins (panels a, b and c, respectively in Fig. 5 ). On the G protein family level (topmost in Fig. 5 ), our analysis spans 90 receptors with data only in GtP and 166 GPCRs with data from the Bouvier and/or Inoue groups -totaling 256 receptors. We require couplings from the Bouvier and Inoue groups to be supported by a second dataset (GtP couplings are already typically supported by several publications). We find that a GPCR couples to on average 1. 7 G protein families distributed as 126 single-, 83 double-, 34 triple-and 13 all-family activating receptors (49%, 32%, 13% and 5%, respectively, Fig. 5b ). The share of fully selective (single-family activating) receptors differs largely across G protein families spanning from 6% for G12/13 to 22% for Gq/11, 26% for Gs and up to 40% for Gi/o (tab 'Fig_5' in Spreadsheet S5).
Within each G protein family (rows 2-5 in Fig. 5 ), on average 73% of GPCRs promiscuously activate all its members (Gq/11: 73%, G12/13: 66%, Gi/o: 75% and Gs: 80%). In contrast, activation of only one subtype of a G protein family is only observed for 11 Gs, 4 Gz, 1 G14, 10 G15, 5 G12 and 10 G13-coupled receptors (Fig. 4  or Spreadsheet S5). Most other receptors are activated by a subset of G proteins in each family. Strikingly, P2Y1,4 (G15), P2Y14 (Gz) and GPR55 (G13) are fully selective also when considering G proteins from all families i. e. , they only couple to a single of the 16 human G proteins (1. 4*SD cut-off applied, 6 Gs-coupling receptors are left out, as Golf has so far only been tested by the Inoue group). However, the three purinergic receptors have additional couplings although not supported by a second dataset (P2Y4 and P2Y14) or above the 1.
Discussion Given that researchers are now faced with three large G protein coupling datasets varying in coverage and couplings, our first objective was to establish a common normalization protocol making quantified values comparable across data sources and a map of all couplings and their experimental support. Our meta-dataset analysis of receptors included in all three sources, revealed 101 novel couplings supported by both the Bouvier and Inoue datasets demonstrating the value of dedicated large scale profiling studies. Furthermore, it revealed a large number of yet unsupported couplings, especially for the Gs, G12 and G15 subtypes (Spreadsheet S3) -warranting the generation of additional supporting datasets. The choice of protein systems and biosensors for such future studies should take into account the relative strengths and limitations of the profiling platforms used (3), while future studies could benefit from other recently available biosensors (7, 8, 14) (Gabg sensors first described in  (15) (16) (17) (18) (19) (20) ) to widen the approaches used to establish consensus couplings.
Moreover, tissue-specific couplings  (21) , spatiotemporal-dependent interactions and unproductive couplings  (22)  as opposed to actual down-stream signaling events  (23)  need to be considered when inferring coupling profiles to physiology and pathophysiology. In all, while our analyses point to a need for further studies to confirm couplings detected by a single biosensor, they establish a sizeable reference dataset of supported quantitative couplings suitable for any study. Previous analyses of GPCR-G protein selectivity have all been based on a single dataset from the GtP (24) database or the Inoue (2) or Bouvier (3) laboratories, whereas the analysis herein combined these datasets to establish couplings supported by two studies (including GtP annotations of multiple literature reports) for a total of 256 GPCRs.
G proteins that belong to the same family can have diverse functional outcomes pertaining to effector engagement selectivity and kinetic profiles  (26, 27) , suggesting that bias within a G protein family may have physiological and/or therapeutic implications  (28). The observation that many GPCRs can couple to more than one family of G proteins but yet can show selectivity between members of a same family, opens important questions concerning the structural determinants of such multifaceted selectivity profiles. In all, our cross-dataset analysis has established a protocol and reference set aiding GPCR-G protein coupling studies. The selectivity profiles are the most comprehensive to date spanning 256 receptors with a very diverse activation of a single to all G protein pathways, and presented in a dedicated online atlas of G protein couplings integrated in the GPCRdb database hub  (5).
Study design The primary objective of this study was to generate a unified map of G protein couplings (Fig. 4 ) across the three available large datasets from the Bouvier (3) and Inoue (2) groups, and the Guide to Pharmacology database (1), respectively. To do this, we identified the Emax standard deviation cut-off, quantitative normalization protocol and aggregation of G proteins into families giving the best possible agreement between the GPCR-G protein couplings from the Bouvier and Inoue groups. This analysis also involved assessing the agreement of the Bouvier and Inoue group datasets and determining the number of high-confidence novel couplings supported by these two datasets but not reported in the Guide to Pharmacology database. Furthermore, it included a benchmarking of techniques to determine which biosensor produces the most reproducible qualitative coupling determination (coupling vs.
non-coupling) for each G protein family. To this end, we compared the three datasets to pinpoint, for each dataset and G protein family, the fraction of couplings supported by another dataset. The scientific analyses based on the map feature the most comprehensive analysis to date of GPCR-G protein selectivity. This was done by intersecting the 11 G proteins tested by both Bouvier and Inoue, within and across their families, with respect to the common and unique receptors that they couple to. Finally, the receptor profiles were also used to classify G proteins and to determine any co-correlation between different G protein subtypes and families to GPCRs.
Coupling datasets Updated Spreadsheets containing the pEC50, Emax, basal signals and standard deviation (SD) values were supplied by Asuka Inoue. Basal signals, spontaneous AP-TGF-a elease (in % of total AP-TGF-a expression) were recalculated from raw data of the previous coupling-profiling campaign  (2)  and their SD values were computed from independent experiments (n ≥ 3). This file contains the neurotensin 1 (NTS1) and Thyrotropin-releasing hormone (TRH1) receptors not included in the previous publication  (2). In the Inoue dataset, the protease-activated receptors PAR3-4 had negative pEC50 values (concentration of mU/ml because the ligand, thrombin, was supplied with its enzymatic activity). For the easiness of integration into the coupling map, we added a value of 10 to their pEC50 values.
Data qualities (sigmoidal curves) for the individual GPCR-G protein pairs were manually inspected and concentration-response curves that did not converge nor exceed a threshold (typically, 3% AP-TGF-α release) were regarded as no activity. The Bouvier dataset (n ≥ 3) contained some datapoints that were included or excluded based on dedicated analyses. Firstly, we excluded ligand-promoted responses of overexpressed receptors that were equivalent to those of endogenously expressed receptors (yellow fill in tab 'B-EmEC' in Spreadsheet S1). Secondly, couplings with only approximate Emax and pEC50 values because the dose-response curve did not converge were only included if supported, and not contradicted, by the Inoue and/or GtP datasets (orange fill in tab 'B-EmEC' and analyzed separately in tab 'UnconvergedDRV' in Spreadsheet S1).
Standard deviation cut-off We made a special investigation of couplings that have a full dose-response curve but an Emax less than 2 standard deviations (SDs) from the basal signal (red fill in tab 'B-EmEC' and analyzed separately in tab tabs 'B-<2SDs' and 'I-<2SDs' in Spreadsheet S1). To achieve the best possible separation of putative false and real but weak couplings, we identified the threshold value (a number of SDs from basal signal) that gives the best agreement between the Bouvier and Inoue dataset among the common receptors and G proteins tested by both groups. The obtained cut-off, 1. 4 SDs was applied as a filter to exclude all Bouvier and Inoue Emax and pEC50 values for couplings below this cut-off (columns with '>1. 4SD' in the heading in tabs 'B-EmEC' and 'I-EmEC' in Spreadsheet S1).
As a note, while intra-day measurement error is small for the TGF-α shedding assay (typically, 1-2% AP-TGF-α release), inter-day variability varies widely depending on cell conditions. Since the SD represents inter-day variability, the basal SD cut-off removes more couplings than the SD cut-off of ligand-induced signal or the significance of individual experiments. As a consequence, some of manually annotated couplings in the Inoue dataset are regarded as non-coupling by the basal SD cut-off criteria, including for P2RY2 and P2RY6 that had no couplings above this cut-off.
Generating a subset of comparable GPCR-G protein couplings To enable qualitative comparison of corresponding datapoints in the datasets from the Bouvier (32) and Inoue (2) groups, we used the subset of 70 GPCRs present in both datasets and belonging to the same class, A (removed only two receptors from class B1). The quantitative comparisons focused on a smaller subset of 51 GPCRs tested with the same ligand and excluded non-coupling GPCR-G protein pairs, as they could not be represented by 0 values (due to e. g. , an underrepresentation of couplings in a given datasets and G proteins, see Results). All analyses herein included the 12 G proteins: Gs, Gi1, Gi2, GoA, GoB, Gz, Gq, G11, G14, G15, G12 and G13. Golf and Gi3 could not be analyzed, as they had not been tested by the Bouvier group (Supplementary Table  1 ).
Validation of comparability when Emax or reference agonist differ To get an overview of the distribution of data in the two datasets, we determined the pEC50 and Emax mean, median, min, max, span and standard deviation values and plotted box and whiskers plots for each G protein (Spreadsheet S1). This showed that the Emax values vary much more across the different G proteins than pEC50 values. The Emax variation is largest in the Bouvier data wherein G15 ranges across three orders of magnitude (16 to 1,067). The Bouvier dataset has low means and spans for Gs (42 and 47) and G12 (71 and 101) indicating a narrow assay signal window (low signal-to-noise). We find that minimum-maximum normalization (to 100%) of each G protein across receptors gives a more uniform distribution (SDs for Bouvier: 19-31 and Inoue: 22-26, rightmost plot pair in Spreadsheet S1).
Whereas both studies tested a majority of receptors with their endogenous ligand, surrogate agonists were used for  15 and 4  GPCRs in the data from the Inoue and Bouvier groups, respectively. Although those ligands were selected for their reference character with similar pharmacology to the endogenous ligand, they could introduce differences in a receptor's G protein profile due to ligand-dependent signaling bias. Analysis of GPCR-G protein couplers and non-couplers (tab 'BI' in Spreadsheet S2) shows that on average 74% and 71% agreeing qualitative couplings (i. e. , coupling vs. non-coupling GPCR-G protein pairs) for receptors tested with the same and different agonists, respectively. This is a rather small difference providing confirmation that receptors tested with different ligands can be compared on the qualitative coupling/non-coupling level and be included in the comparison of the different G protein coupling datasets and determination of novel G protein couplings (Fig.