Since their discovery, GPCRs have been considered to be primarily G protein-signaling entities. This knowledge has been demonstrated to be exceptionally successful in allowing the creation of a huge variety of effective pharmacotherapeutics. Hauser et al. evaluated in 2017 that 475 FDA-approved drugs target GPCRs, which is 34% of all FDA-approved drugs [32]. Even with this specific G protein focus, agents have been generated that can control the bias amongst diverse forms of G protein-signaling output [23,33]. In the last decade, our appreciation of GPCR-signaling complexity has been enhanced by the demonstration of simultaneous signaling activities emanating from GPCRs that are either G protein-based or controlled by non-G protein-signaling adaptors. Even with just the primary consideration of G protein activation, it is evident that the receptor conformations for G protein activation are different between specific G protein pools and that synthetic and naturally occurring ligands can selectively facilitate the formation of different receptor coupling conformations [23,34]. Multiple distinct forms of agonist ligands for a single GPCR type have now been discovered to only activate a subset of G proteins or a subset of downstream signaling effectors or induce G protein coupling without initiating internalization and desensitization [23,35,36]. Given the successful exploitation of the therapeutic intervention of GPCR-based G protein signaling, it is likely that it will be possible to improve this index even further by exploiting the true complexity and diversity of GPCR signaling [32,37]. Apart from the classical G protein signaling, multiple research lines have pointed towards the presence of non-G protein-based signaling, mainly through β-arrestins [38,39,40]. One of the first studied examples of this novel signaling activity was the β-arrestin-dependent activation of extracellular signal-regulated kinases 1/2 (ERK 1/2) [29,41]. Compared to the rapid and transient manner of G protein signaling [39], β-arrestin-linked pathway activation has a later onset but is sustained over a long period and entrains long-term l cellular transcriptional and proteomic effects [42,43,44,45]. β-arrestin also serves as a negative regulatory protein for signaling through G proteins and is responsible for GPCR internalization [45,46,47]. However, there is still some dispute in the field with respect to the interdependence of G protein and β-arrestin signaling; recently, complete G protein independence could not be proven in a serum-starved in vitro G protein knockout model [41]. Evidence has also been revealed recently that suggests that certain specific forms of orphan receptors (D6R and C5aR2) appear to be able to functionally interact with β-arrestins but not with G proteins [48]. This data reinforces the posit that there are likely a diverse range of receptorsome entities that are prewired to specific independent downstream signaling pathways.

Since 1999, it has become clear that GPCR signaling is more complex, specific and diverse than initially considered in the two-state model [28,49]. One of the factors accounting for this complexity is the ability of receptors to form multistate-signaling complexes or so-called receptorsome structures. These receptorsome structures comprise the receptor itself combined with multiple interacting proteins. These preassembled receptorsomes demonstrate unique pharmacology, signaling, trafficking, desensitization and internalization features [50,51,52,53]. This conditioning of GPCR activity is also influenced by the relative variations of these adaptor proteins in distinct tissues, suggesting the presence of tissue-specific GPCR activity [23,37,54]. Given this growth in GPCR-signaling complexity, it is vital to recognize the unique properties of endogenous or cognate ligands for GPCRs. The proposed cognate ligands of GPCRs attempt to impact every consequence of receptor activation in the same manner, whether desensitization, internalization, trafficking or G protein coupling [43,55]. Hence, these ligands strive to engender an omnipotent efficacy. The ability of a ligand to achieve this at a systemic multi-tissue level is, however, highly unlikely due to tissue-to-tissue variations in the receptor and signaling adaptor expression under the influence of diverse cellular conditions [23,56,57,58,59]. Given the ability for multiple ligands to stimulate the same GPCR, it may be prudent to redefine our conceptualization of endogenous ligand cognation. Indeed, the most accurate definition of a cognate ligand for a specific GPCR could be codified by its ability to most equally regulate the full GPCR-signaling spectrum across a diverse series of tissue/cell settings. Hence, it is likely that receptors and their cognate ligands coevolved to elicit the most physiologically adaptive and effective responses in target cells. With respect to the concept of functional relationships between cognate ligands and their preferred receptors, the aging/stress response paradigm presents an important pathophysiological process that represents perhaps the greatest systemic and coordinated perturbation of cellular signaling in human physiology.

This complexity and diversity of GPCR signaling also reveals the necessity to change our definition of agonists and antagonists originally based on the two-state model [18,19,20,21,22]. Ligands, to varying degrees, likely stabilize these different active conformational states and thereby initiate diverse signaling pathways. This concept is known as biased agonism. Verifying and understanding this mechanism will likely help to develop functionally selective drugs, which activate beneficial downstream pathways and suppress adverse side effects [23,24].

It is evident that an in-depth understanding of the effective G protein-coupling capacity of GPCRs has been effective for the development of GPCR-based therapeutics [60]. The G protein-centric focus of GPCR signaling was expanded by the discovery that β-arrestins—originally thought of just as terminators of G protein signaling [61,62]—can also act as productive signaling effectors [49]. Further research has demonstrated that the realm of GPCR signaling is far more complex and diverse than initially imagined [2,37,43]. This signaling diversity arises from several factors associated with subcellular localization, specific post-translational modification states of the receptor and, perhaps most importantly, the ability of the receptor to exist in multiple receptorsome-signaling states. In multistate signaling GPCR models, specific agonists likely possess the ability to activate distinct active receptorsomes by exposing different intracellular regions involved in coupling separate G protein pools, initially demonstrated for the β2-adrenergic receptor antagonist ICI-118-551 [63,64], and β-arrestin signaling [45,49]. It is becoming more evident each year that agonist-selective receptor signaling, targeting a subset of the possible response profiles, may represent an opportunity to develop drugs that are more precise and could also have an increased efficacy.

To assist the capacity to investigate these GPCR receptorsomes, there have been considerable advances in the accuracy and selectivity of proteomics-focused mass spectrometers that can investigate the specific protein stoichiometries in these complexes. This increase of protein detection sensitivity has enabled the experimental transition from whole-tissue/cell investigation to allow an analysis of protein–protein interactions at a subcellular level, i.e., interactomics [65]. The dynamic investigation of how multiprotein complexes alter in quantity and quality in response to drug exposure has enabled the examination of the subcellular network functionality of therapeutic agents [66,67,68,69]. The importance of GPCR interactomics lies in the posit that the local context of protein associations/interactions is often more strongly linked to the biological activity of a certain form of signaling pathway or disease process, as opposed to simple global cellular or tissue protein expression levels [68,70].

The canonical model of GPCR signaling, either through G proteins or non-G protein adaptors, has been largely considered to emanate from the plasma membrane localized receptors. One potential mechanism contributing to the diversity and specificity of GPCR signaling is through membrane trafficking and alternative residential and signaling sites of the receptor [71,72,73]. Until recently, the cell surface plasma membrane trafficking of GPCRs was considered as a mechanism to control the sensitivity to an extracellular stimulus by changing the receptor level through ligand-mediated endocytosis or reduced trafficking to the plasma membrane of newly synthesized receptors [74]. It has recently been demonstrated that GPCRs can signal from intracellular membranes such as endosomes, mitochondria, the endoplasmic reticulum, the Golgi apparatus and the nucleus [75,76]. This shift in signaling location was first identified on Gαs-coupled receptors such as the parathyroid receptor, thyroid-stimulating hormone receptor and the β2 adrenergic receptor, where cyclic adenosine monophosphate production was still evident after endocytosis [77]. Given the subcellular variations of GPCR adaptor protein expression, it is highly likely that the subcellular location of GPCR receptorsomes can assist in defining specific ensembles of adaptor-directed specific signaling activity [43].