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).