Recommendations 10
10a. Measure at similar pathway levels: We recommend measuring bias at the same or similar depth of compared pathways. Where the readout for one pathway is measured further downstream than for another, one should take special care by noting potential amplification effects.
10b. Report measured molecules: To provide clarity on what has been measured, we recommend that the pathways are not only referred to by the GPCR-binding transducer, but additionally by the downstream molecule or molecule pair that was measured to generate the signals in the experiments.
10c. Report measured processes: As the same molecule can be involved in related but distinct steps of the signaling, we also recommend reporting the measured process. An example of separate processes involving the same (hetero)protein is G protein receptor-binding versus subunit dissociation. A non-exhaustive list of terms to distinguish such signaling processes is visualized in Figure 3A and tabulated in Table 5 along with example assay principles. It is not sufficient to describe an assay or experiment by the detection method, e.g., a ‘BRET assay’, as the same detection technique can be used to measure different molecules and processes, such as GPCR-G protein binding/coupling and Gα-Gβγdissociation. Furthermore, any receptor, transducer and effector modifications must be explicitly defined (i.e. tags, chimeras, etc.).
10d. Consider pathway convergence: Several pathways intercept each other or converge, meaning that one measured molecule can have been activated and inhibited through multiple primary transducers simultaneously. For example, extracellular signal-regulated kinase (ERK) proteins can be activated by all four G protein families (Jain, Watson, Vasudevan & Saini, 2018) and this process is shaped in space and time by arrestins and GRKs (Eichel, Jullie & von Zastrow, 2016; Gutkind & Kostenis, 2018; Wehbi, Stevenson, Feinstein, Calero, Romero & Vilardaga, 2013). For this reason, studies reporting bias at downstream effectors or second messengers need to carefully consider whether multiple pathways could have contributed to the effect and, if so, interpret findings in light of their relative strength for the given receptor and ligand. For example, calcium, PKC or DAG measurements should not be equated exclusively with Gq activation. Indeed, different receptor transducers, including Gβγreleased from Gαi can also lead to the generation of these second messengers and activation of this kinase (Dorn, Oswald, McCluskey, Kuhel & Liggett, 1997). Therefore, the specific type of measurement should be provided. When pathways are truly inseparable and their contributions cannot be dissected using upstream assays, the bias may be considered a type of ‘effector bias’ (instead of pathway-bias) accounting for the net pathway contributions.

Kinetics and choosing measurement time points

Recommendation 11: The time points refer to data collection times, which should be carefully selected based on kinetic parameters and residence time, where known. For instance, comparing non-equilibrium readings with equilibrium reading due to different binding kinetics or type of biological responses (ion flux vs. reporter gene) can be a major confounding factor (Klein Herenbrink et al., 2016). When possible, complete time courses are preferred and could be quantified by onset kinetics, e.g. time constant tau (τ) or time to reach half maximal response amplitude (Hoare, Pierre, Moya & Larson, 2018). When a single time point is chosen, it should be controlled to ensure that it measures maximum effect (peak), or otherwise the physiologically most relevant time point. This would often be the same across pathways but could differ if different biological processes are measured that occur at different time scales. In all cases, the reasons for the time selected should be reported. See also (Lane, May, Parton, Sexton & Christopoulos, 2017).