Recommendation 9
9a. We recommend that a larger set of known and predicted biased ligands are characterized using recent assays opening for consistent profiling across broad panels of G proteins and arrestins (also with GRKs) (Avet et al., 2020; Olsen et al., 2020).
Reason: When the signal is measured at the transducer (a G protein, GRK or arrestin), there is no downstream signal amplification (although different systems can express receptors and transducers differentially). The upside of this is that signals are more comparable across pathways.
Disclaimer: Amplified signals may be more relevant than those of primary transducers to explain a tissue-level or systemic response. Also, the transducer signal may differ from the true amplified signal (such as second messenger, Figure 3B).
9b. If possible, we recommend to test all or as many as possible transducers for each investigated transducer family. If not all transducer subtypes within a given transducer family (e.g. Gq, G11, G14 and G15 in the Gq/11 family) can be tested, we recommend testing the transducer subtype with the highest activation or recruitment and presence in the given studied cell type. Data to support such selection have, for example been produced for many GPCRs in consistent profiling across broad panels of G proteins and arrestins (Avet et al., 2020; Inoue et al., 2019; Olsen et al., 2020) and integrated in GproteinDb (https://gproteindb.org) and ArrestinDb (https://arrestindb.org), respectively.
Reason: G proteins belonging to the same family may differ in their functional outcome due to unique binding kinetics, cellular expression levels, and engagement of different downstream effectors (Anderson et al., 2020; Avet et al., 2020; Ho & Wong, 2001; Jiang & Bajpayee, 2009; Olsen et al., 2020). Similarly, differential recruitment of the two isoforms of β-arrestin (βarr1 and 2) can translate into distinct functional outcomes with respect to regulatory and signaling paradigms (Ghosh et al., 2019; Srivastava, Gupta, Gupta & Shukla, 2015). If two pathways are compared based on transducer subtypes that represent the maximum and a low activation, respectively, compared to all members of their respective transducer family, this skews the bias comparison of the transducer families (not the specific subtypes). Differential activation or recruitment of transducer family members has been shown both for G protein families (Avet et al., 2020; Inoue et al., 2019; Olsen et al., 2020) and the arrestin family (Avet et al., 2020; Srivastava, Gupta, Gupta & Shukla, 2015).
Bias measured downstream – minimize differential signal amplification and report measured molecules and processes
Whereas bias is often grouped onto pathways represented by the primary receptor-binding transducers (G proteins, GRKs and arrestins), most studies instead measure downstream effector proteins or second messenger molecules which can have a differential signal amplification (also in the same system). Hence, bias values may differ when comparing downstream molecules instead of primary transducers in the same pathways (Figure 3B).