Introduction
The ~800 human G protein-coupled receptors
(GPCRs ) transduce sensory inputs and systemic signals into
appropriate cellular responses in numerous physiological processes. They
recognize a vast diversity of signals from photons, tastants and odors
to ions, neurotransmitters, hormones, and cytokines (Armstrong et al.,
2020; Wacker, Stevens & Roth, 2017). Even though GPCRs represent the
primary target of 34% of FDA-approved drugs, more than 220
non-olfactory GPCRs have disease associations which are as yet untapped
in clinical research (Hauser, Attwood, Rask-Andersen, Schioth &
Gloriam, 2017; Sriram & Insel, 2018). Despite the diversity of ligands
and physiological roles of GPCRs, these cell surface receptors share a
conserved molecular fold and intracellular transducers. Agonist binding
stabilizes active conformations of the receptor, facilitating the
binding of one or more cytosolic transducer proteins. These include the
heterotrimeric G proteins consisting of α, β and γ subunits
that dissociate to α and βγ upon activation by the receptor. G proteins
comprise 16 distinct α subunits and are divided into four families based
on homology and associated downstream signaling pathways:
Gs (Gs and Golf),
Gi/o (Gi1, Gi2,
Gi3, Go, Gt1,Gt2, Gt3, and Gz),
Gq/11 (Gq, G11,
G14 and G15) and G12/13(G12 and G13). Moreover, there are 5
different β and 12 γ subunit types, resulting in a vast number of
possible heterotrimeric G protein combinations (Hillenbrand, Schori,
Schoppe & Plückthun, 2015; Masuho, Skamangas, Muntean & Martemyanov,
2021; Milligan & Kostenis, 2006; Olsen et al., 2020).
Activated GPCRs are also bound and phosphorylated at multiple Ser and
Thr residues by one or more of the seven different GPCR kinases
(GRKs ) or effector kinases (e.g. PKA). Receptor phosphorylation
by GRKs is a key functional determinant for the binding ofarrestin proteins (4 subtypes), which can ‘arrest’ signaling by
blocking G protein coupling and facilitating receptor internalization
although phosphorylation-independent arrestin interactions have also
been described (Eichel et al., 2018). Arrestins are membrane-located
scaffold proteins that recruit and/or assemble other proteins that
signal (Ahn, Shenoy, Luttrell & Lefkowitz, 2020). Interestingly,
evidence published over the last decade suggests that the pattern and/or
distribution of phosphorylated sites on the receptor plays a major role
in governing the binding mode and cellular functions of receptor-bound
arrestin (Ostermaier, Schertler & Standfuss, 2014). Ultimately, the
functional interplay between G protein, GRK, other kinases, arrestin and
other interaction partners at the GPCR shapes the outcome of receptor
signaling in space and time (Gutkind & Kostenis, 2018; Kenakin, 2019).
However, the molecular mechanisms underlying these complex and variable
interactions remain far from fully understood (Smith et al., 2021;
Thomsen et al., 2016).
‘Biased signaling’ (also known as agonist-directed trafficking and
leading to ‘functionally selective’ response), is ligand-dependent
activation of certain pathways (defined in the next section) over
others. The concept is rooted in a natural allosteric behavior of
receptors by distinct conformations interacting differently with ligands
and cellular transducers at varying stoichiometries and durations. This
has allowed endogenous agonists to fine tune their signaling at receptor
subtypes throughout evolution. Alternative endogenous agonists directing
signaling have been observed for multiple receptors e.g., chemokine
(Kohout, Nicholas, Perry, Reinhart, Junger & Struthers, 2004), opioid
(Gomes et al., 2020), PACAP (Spengler et al., 1993), protease-activated
(Hollenberg et al., 2014), serotonin (Schmid, Raehal & Bohn, 2008) and
PTH (Dean, Vilardaga, Potts & Gardella, 2008) receptors.
‘Biased signaling’ first became evident with numerous reports
of aberrations in agonist potency ratios in the mid 1980’s following the
advent of assays that measured separate effects from G proteins and
other cellular transducers (Roth & Chuang, 1987). Although many of
these papers made observations that were compatible with what we now
know as bias, relative differences were still difficult to discern from
different levels of assay amplification. The general acceptance of
biased signaling came with evidence that the order of potency of ligands
could be different for different pathways engaged by a single receptor
(Spengler et al., 1993) or inversion of the ligand modality (Azzi et
al., 2003; Baker, Hall & Hill, 2003). The first and most frequently
studied bias has been that between G proteins and arrestins, while more
recent studies have compared G protein families and even subtypes
belonging to the same G protein family. An early theory proposed that
the bias is caused by the stabilization of different receptor active
states by agonists (Kenakin & Morgan, 1989; Roth & Chuang, 1987). The
allosteric communication between the ligand and G protein has been shown
to be reciprocal, as G protein pre-coupling can potentiate agonist
binding (De Lean, Stadel & Lefkowitz, 1980; Lefkowitz, Mullikin &
Caron, 1976; Maguire, Van Arsdale & Gliman, 1976) and has been
explained on the molecular structure level by conformational selection
(Galandrin, Oligny-Longpre & Bouvier, 2007; Kenakin, 1995; Smith,
Lefkowitz & Rajagopal, 2018). An activated receptor state has also been
linked to a high affinity binding state for arrestin (Gurevich &
Benovic, 1997). However, it is still unclear what the precise
relationship between conformation and signaling is – at least at the
level of detail required to predict such outcomes.
Therapeutic exploitation of biased signaling could lead to
safer or more efficacious drug therapies. Several studies have outlined
disease-relevant pathways for future therapeutic targeting (Urban et
al., 2007; Whalen, Rajagopal & Lefkowitz, 2011) or retrospective
cross-screening yielding biased ligands predicted to yield potentially
useful phenotypes in therapy (Che, Dwivedi-Agnihotri, Shukla & Roth,
2021; Galandrin, Oligny-Longpre & Bouvier, 2007; Kenakin, 2019; Urban
et al., 2007; Whalen, Rajagopal & Lefkowitz, 2011). Biased signaling is
presently a very active area for pharmacological research, as it might
provide a means to elicit signaling profiles differing from the ones
caused by natural hormones and neurotransmitters, thus imparting
different qualities of efficacy (mixtures of cellular signaling) to
therapeutic systems (Che, Dwivedi-Agnihotri, Shukla & Roth, 2021;
Kenakin, 2021; Urban et al., 2007).
However, the advance of the field is currently hampered by confusing
terminology and inconsistent interpretations of results due to a lack of
commonly agreed guidelines for reporting bias and the underlying
experiments e.g., what has really been measured. Here, we
provide recommendations for the terminology to use or avoid and the
minimum requirements to conclude, report and quantify bias in a
reproducible fashion. The recommendations are supported by the
authoritative organization for pharmacological nomenclature, the
International Union of Basic and Clinical Pharmacology
(https://iuphar.org), and the COST Action CA18133 ERNEST (European
Research NEtwork on Signal Transduction) (Sommer et al., 2020). These
are not recommendations for how to perform experiments, but to adopt a
common terminology facilitating consistent reporting, joint
understanding of what has been done and what were the results, and more
comparable research data. A clearer understanding of biased signaling
may also improve the challenging translation of in vitro findings
to disease-relevant in vivo models.
Pathway definition and
modulation
A GPCR pathway is here defined by a transducer protein, or family
thereof, binding intracellularly to the receptor and eliciting a
distinct cellular downstream signaling cascade, trafficking or
internalization. Based on present knowledge, this includes the four G
protein families – i.e., the Gs, Gi/o,
Gq/11, G12/13 pathways. It also includes
the arrestin and GPCR kinase (GRK) families which are recruited to and
bind activated GPCRs, even when G proteins are pharmacologically
inhibited or when Gα proteins are partially or entirely
genetically ablated (Grundmann et al., 2018; Hunton et al., 2005;
Sauliere et al., 2012; Wehbi, Stevenson, Feinstein, Calero, Romero &
Vilardaga, 2013). For example, GRK4-6 functions do not appear to require
either G proteins or arrestins, as they are not recruited by
Gβγ but anchored to the plasma membrane via polybasic
domains and lipid modification (Komolov & Benovic, 2018). Importantly,
functionally relevant bias can also occur across different members of
the same protein family and pathway. 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).
In addition, there are proteins that are modulators of receptors,
transducers and effectors and can influence signaling indirectly. For
example, receptor activity-modulating proteins (RAMPs) bind to receptors
and can alter G protein and/or arrestin binding (Hay & Pioszak, 2016).
In the case of the calcitonin and calcitonin receptor-like receptor,
different receptor-RAMP complexes produce distinct pharmacological
responses and are therefore considered as separate receptor subtypes:
one calcitonin, two adrenomedullin and three amylin receptors (Hay,
Garelja, Poyner & Walker, 2018). Similarly, the cannabinoid CB1
receptor can bind to Cannabinoid Receptor Interacting Protein 1a
(CRIP1a) yielding distinct pharmacology (Oliver, Hughes, Puckett, Chen,
Lowther & Howlett, 2020). GPCRs are also substrates for second
messenger-activated kinases such as the cAMP-dependent kinase (PKA),
protein kinase C (PKC) and the Casein Kinase (CK) with each producing
different effects on receptor signaling and trafficking (Bouvier,
Leeb-Lundberg, Benovic, Caron & Lefkowitz, 1987; Hausdorff, Bouvier,
O’Dowd, Irons, Caron & Lefkowitz, 1989; Tobin, Totty, Sterlin &
Nahorski, 1997). It should be noted that, similarly to
second-messenger-driven kinases, GRK can also catalyze the
phosphorylation of many non-receptor substrates (Gurevich, Tesmer,
Mushegian & Gurevich, 2012). Additionally, numerous downstream
intracellular effectors modulate pathway responses as scaffolding
proteins e.g., kinases, PDZ proteins) (Bockaert, Fagni, Dumuis & Marin,
2004; Kenakin, 2019; Maurice, Guillaume, Benleulmi-Chaachoua, Daulat,
Kamal & Jockers, 2011). The regulator of G protein signaling (RGS)
proteins selectively modulate G protein subtypes and differentially
alter G protein signal strength (Hollinger & Hepler, 2002; Neubig &
Siderovski, 2002). Furthermore, GRK2 and GRK3 have a regulator of G
protein signaling (RGS) homology domain (RH) binding to
Gq/11 to inhibit signaling, and a PH domain that can
bind to Gβγ to inhibit its signaling while inducing
recruitment of GRK to the receptors (Carman et al., 1999; DebBurman,
Ptasienski, Benovic & Hosey, 1996; Ribas et al., 2007). Therefore, it
is clear that multiple proteins can influence the ability of a receptor
to interact with a transducer.