Biased GPCR signaling: Possible mechanisms and inherent limitations

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Abstract

G protein-coupled receptors (GPCRs) are targeted by about a third of clinically used drugs. Many GPCRs couple to more than one type of heterotrimeric G proteins, become phosphorylated by any of several different GRKs, and then bind one or more types of arrestin. Thus, classical therapeutically active drugs simultaneously initiate several branches of signaling, some of which are beneficial, whereas others result in unwanted on-target side effects. The development of novel compounds to selectively channel the signaling into the desired direction has the potential to become a breakthrough in health care. However, there are natural and technological hurdles that must be overcome. The fact that most GPCRs are subject to homologous desensitization, where the active receptor couples to G proteins, is phosphorylated by GRKs, and then binds arrestins, suggest that in most cases the GPCR conformations that facilitate their interactions with these three classes of binding partners significantly overlap. Thus, while partner-specific conformations might exist, they are likely low-probability states. GPCRs are inherently flexible, which suggests that complete bias is highly unlikely to be feasible: in the conformational ensemble induced by any ligand, there would be some conformations facilitating receptor coupling to unwanted partners. Things are further complicated by the fact that virtually every cell expresses numerous G proteins, several GRK subtypes, and two non-visual arrestins with distinct signaling capabilities. Finally, novel screening methods for measuring ligand bias must be devised, as the existing methods are not specific for one particular branch of signaling.

Introduction

The classical paradigm of G protein-coupled receptor (GPCR) signaling posited that GPCRs communicate with the cell via heterotrimeric G proteins, and their signaling is terminated by receptor phosphorylation followed by arrestin binding (reviewed in (Carman & Benovic, 1998)). However, in the last two decades, numerous studies have suggested that receptor-bound arrestins initiate a second round of signaling, which is quite different from the G protein-mediated one (reviewed in (Gurevich & Gurevich, 2006a; Peterson & Luttrell, 2017)). This phenomenon suggested the idea that GPCR signaling can be biased towards either G proteins or arrestins by using ligands that promote receptor interaction with one type of signal transducer but not with the other. Although the first reported examples of bias of a ligand or a GPCR described differential activation of different G protein subtypes (Eason, Jacinto, & Liggett, 1994; Spengler et al., 1993), in recent years discussions of GPCR signaling bias have mostly focused on the G protein-arrestin dichotomy (DeWire & Violin, 2011; Violin & Lefkowitz, 2007; Whalen, Rajagopal, & Lefkowitz, 2011; Wisler, Xiao, Thomsen, & Lefkowitz, 2014). In many cases, it has been proposed that one signaling branch is responsible for the therapeutic effects of drugs, while the other mediates unwanted side effects. Therefore, the idea that biased drugs will be therapeutically superior to the traditional unbiased ones has become popular (extensively reviewed in (DeWire & Violin, 2011; Khoury, Clément, & Laporte, 2014; Luttrell, Maudsley, & Bohn, 2015; Rankovic, Brust, & Bohn, 2016; Whalen et al., 2011; Wisler et al., 2014)). For a number of pathological conditions, drugs that might specifically direct GPCR signaling to G protein- or arrestin-mediated pathways have been identified, and their beneficial effects have been demonstrated (reviewed in (Whalen et al., 2011)). Here we consider the issue of GPCR signaling bias from a structural and mechanistic perspective, discussing the possibilities opened by this approach and its inherent limitations. We do not attempt to provide a comprehensive review of all reported cases where signaling bias has been documented, as this has already been done in the excellent reviews cited above.

Section snippets

Conformational heterogeneity of GPCRs

The classical view of GPCRs considered these receptors as on-off switches that oscillate between two conformations, active and inactive. Agonists were believed to shift the equilibrium towards the active conformation, inverse agonists – towards the inactive one, thereby suppressing constitutive activity, whereas neutral antagonists occupy the ligand binding site without affecting the conformational equilibrium. The classical extended ternary complex model of GPCR signaling (Samama, Cotecchia,

Structures of GPCR complexes with signal transducers

The first solved structure of a GPCR bound to a cognate G protein (β2AR complex with Gs (Rasmussen et al., 2011)) was followed by several GPCR-Gs structures in which the receptor-G protein interfaces looked remarkably similar (Carpenter, Nehmé, Warne, Leslie, & Tate, 2016; Liang et al., 2017; Zhang et al., 2017). These structures confirmed the activation-dependent outward movement of the cytoplasmic ends of the transmembrane helices V and VI earlier described in rhodopsin (Farrens, Altenbach,

What does the mechanism of GPCR desensitization tell us about biased signaling?

Let's recall the classical paradigm of two-step GPCR desensitization (Carman & Benovic, 1998). It posits that active GPCRs are phosphorylated by GRKs, and then active phosphorylated receptors bind arrestins. The original concept assumed that the same active conformation of the receptor activates G proteins, is recognized and phosphorylated by GRKs, and then binds arrestins (Fig. 1, classical paradigm). Since GRK/arrestin-mediated desensitization has been demonstrated by numerous labs working

Is there G protein-independent arrestin-mediated signaling?

The ERK pathway is the second, after c-Src, signaling mechanism identified as activated via arrestin-dependent scaffolding (Luttrell et al., 2001). It is arguably the best studied arrestin-dependent signaling pathway and often serves as a classic example of arrestin-mediated signaling. ERK is activated by a variety of mechanisms, some of which are GPCR-dependent (Luttrell, 2003), while others involve growth factor receptors (Garrington & Johnson, 1999; McKay & Morrison, 2007), death receptors (

6. Diversity of arrestin and GRK subtypes

Another issue routinely ignored in the discussion of bias is the existence of arrestin isoforms: all vertebrates, including mammals, have two non-visual arrestins (bony fish that underwent an extra whole genome duplication have three) (Gurevich & Gurevich, 2006a; Indrischek, Prohaska, Gurevich, Gurevich, & Stadler, 2017). While they are highly homologous (Attramadal et al., 1992; Indrischek et al., 2017; Sterne-Marr et al., 1993), the fact that vertebrate evolution kept them for millions of

How typical is the ERK pathway for arrestin-dependent signaling?

Arrestins are known to regulate a large and extremely diverse set of signaling pathways (Peterson & Luttrell, 2017). It is hardly conceivable that all these pathways depend in a similar fashion on the arrestin interactions with GPCRs. However, the dependence of a particular signaling function of arrestins on their interactions with GPCRs is rarely studied or discussed. Even though both ERK1/2 and JNK1/2/3 are MAP kinases activated by three-tiered MAP kinase cascades, the modes of activation of

Can changes in the kinetics of G protein activation be confused with arrestin-mediated signaling?

In many cases when ligand bias is discussed, arrestin-mediated signaling is proposed as an alternative to the G protein-dependent signaling. However, it is critical to analyze what was experimentally demonstrated in each study, rather than what was implied or assumed. Let us consider some of the most recent examples. Accessory protein MRAP2 was shown to bias ghrelin receptor GHSR1a towards G proteins (Rouault et al., 2020). In this study, G protein-mediated signaling was measured directly,

The promise and limitations of biased GPCR ligands

Most GPCRs couple to more than one type of G protein and also bind one or more arrestin variant upon phosphorylation by several distinct GRKs. In quite a few cases, some branches of GPCR signaling were shown to be therapeutically beneficial, whereas others caused unwanted on-target side effects. Thus, the development of biased ligands that can initiate certain branches of signaling, but not others, has an obvious value for therapy (reviewed in (Gesty-Palmer & Luttrell, 2011; Luttrell et al.,

Conclusions

Biased GPCR ligands that preferentially direct signaling to particular G proteins, GRKs, and/or arrestins hold promise for better therapeutic outcomes. However, the bias is unlikely to be absolute: other (unwanted) signaling pathways would likely be activated along with the preferred one. Moreover, in the quest to reduce or eliminate the unwanted signaling, the preferred pathway is also likely to be impaired. A huge technical hurdle in the development of biased GPCR-targeting drugs is the issue

Declaration of Competing Interest

The authors declare no conflict of interest.

Acknowledgments

This project was supported by NIH grants RO1 EY011500, R35 GM122491, and Cornelius Vanderbilt Endowed Chair (Vanderbilt University) (VVG), as well as NIH grants RO1 NS065868 and RO1 DA030103 (EVG).

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