Molecular Mechanism of Biased Signaling
GPCRs are Nature’s prototypic allosteric proteins designed to bind a ligand that subsequently modifies the interaction of the receptor with another body (i.e. G protein, β-arrestin, etc.); bias is the result of standard probe dependent protein allostery where ‘probes’ (signaling proteins) are affected differentially by agonist receptor activation. The link between biased signaling and standard receptor allostery are evident in the mathematical models used to describe both phenomena. Thus, it can be shown that the standard equations describing receptor allostery and agonism also can be used to describe biased signaling (Kenakin, 2021).
Allosterism is a modification of an immensely complex tertiary protein structure and if more than one entity interacts with the receptor, there are no rules to dictate that the effect of a change in conformation (such as that produced by an allosteric ligand like an agonist) will be unform for the two interactions at different sites (in fact, experimental evidence shows that this rarely if ever is the case). Thus the multiple interrogators of receptor information in the cell cytosol are excellent reporters of different receptor conformational states and this is the source of allosteric heterogeneity.
There are biochemical and biophysical assays that can directly identify agonist-specific receptor states. Biosensors have been employed to detect separate receptor active states (Ghanouni et al, 2001) as in the recent study with the angiotensin II type (Devost et al, 2017). BRET experiments also have been used to identify δ-opioid receptor agonist-selective receptor conformations (Audet et al, 2008).19F-NMR has been used to identify agonist-selective β2-adrenergic receptor agonist-receptor complexes (Liu et al, 2012). Distinctly different receptor active states also have been identified through receptor structure (El Daibani et al, 2023; Wingler et al, 2019; 2020).
Data suggests that Nature has hard wired ‘bias’ into natural neurotransmitters and hormones according to the needs of physiology. While natural agonists are commonly referred to as ‘non-biased’ or ‘balanced’ these actually have ‘Nature’s’ bias with little relation toin vitro assays of varying sensitivity. Terms such as non-biased or balanced have no intrinsic meaning as arbitrarily they are controlled by the sensitivity of in vitro pharmacologic assays. For practical application of bias to therapy, so called ‘non-biased’ or balances’ (natural) agonists only serve as a point of reference for synthetic agonists to demonstrate different signaling profiles in the therapeutic environment. It can be seen that Nature’s bias is exploited in natural systems for fine tuning multi agonist signals for receptors; for example the chemokine receptor CXCR7 has two natural agonists (CCL19, CCL21); one recruits β-arrestin and the other does not (Kohout et al, 2004; Sarma et al, 2023).
Experimentally, once pharmacologists had the means to separately measure agonist mediated receptor activation of different signaling pathways, probe dependence was revealed in the form different signaling patterns for different agonists, i.e. biased signaling. Importantly, natural probe dependent allostery then suggests that the quality of efficacy (mixture of signaling pathways to the cell) would beexpected to differ for a synthetic agonist, i.e.we should not expect synthetic agonists to have the same quality of efficacy as natural agonists. Operating on the premise that biased signaling should be an expected property of a new synthetic agonist, it is useful to consider the methods to detect this molecular property