Terminology summary
Balanced ligand/signaling: Near equal signaling through compared pathways. In the context of pathway-biased ligands/signaling, the reference ligand is a balanced ligand.
Pathway-biased ligand/signaling: Ligand/signaling that is biased relative to a balanced reference ligand and therefore bears the meaning that signaling is preferentially through a given pathway.

Physiology-biased ligands/signaling (using a principal endogenous agonist reference)

When using the principal endogenous agonist as a reference ligand, the statement that a tested ligand is biased carries the meaning that its transducer pathway engagement differs from the physiological one (Table 1). Whether this entails a response through a single isolated pathway or not will therefore depend on the extent to which the physiological ligand engages different transducer pathways.
Recommendation 4: A study seeking to explore to what extent a tested ligand differs from the natural signaling of a receptor system should use the principal endogenous agonist as reference ligand. Furthermore, we recommend that all bias studies include the principal endogenous agonist (unless it has too low efficacy in a pathway), as this facilitates subsequent comparison across studies and laboratories. The principal endogenous agonist is defined as the one which is most active and available in the highest concentration in the same tissues as the receptor. The principal and secondary endogenous agonists of GPCRs are curated by experts coordinated by the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (IUPHAR, https://www.guidetopharmacology.org/nciuphar.jsp) and available in the BPS/IUPHAR Guide to Pharmacology database (Armstrong et al., 2020). In cases where it is not clear which endogenous agonist represents the principal version, we recommend using the endogenous agonist with the highest potency (as efficacy is not annotated in this database) or efficacy in the given assay and system (if tested). As a note, a secondary endogenous agonist can be biased relative to the principal endogenous agonist.
Reason: By using an endogenous agonist as reference, the study better approximates how the ligand tested for bias can alter a receptor’s presumed physiological signal. This is valuable if it is, for example desired to steer signaling away from a pathway that is associated with an unwanted response, such as an adverse drug effect, or wanting to design a probe to determine a specific pathway’s effect in the first place.
Disclaimer: Physiology-bias cannot be determined in cases when no endogenous ligand is known (an ‘orphan’ receptor). Furthermore, it cannot be quantified when the endogenous agonist(s) do not have sufficient efficacy in the studied pathways to ensure robust detection.