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.