Given the importance of GPCR signaling in controlling physiological functions, multiple investigations over several decades have sought to fully appreciate how these transmembrane receptors control cellular activity at the signal transduction level. From a functional receptor pharmacological approach, controllers of these receptors were originally designed to exert either a simple positive effect (increasing the activity of downstream signaling systems, e.g., adenylate cyclase) or by inhibiting this activity by occupying the receptor and antagonizing the positive actions of stimulatory ligands. Therapeutic agents were classified as simple agonists (stimulatory) or antagonists (inhibitory) based on the concept that receptors could exist predominantly in two distinct states, i.e., inactive and active. Over the next thirty years, intense research largely confirmed this ‘two-state’ GPCR model [11,12,13,14,15,16,17]. Using specific site-directed mutagenesis of key residues in GPCRs [18], it was demonstrated that GPCRs indeed likely exist in a spontaneous equilibrium between two conformations, i.e., active (R*) and inactive (R). The active conformation is naturally stabilized by agonist binding or, in these numerous experiments, by residue mutation that serves to relax intramolecular constraints [18,19,20,21,22,23]. In this initial functional model, GPCRs transmit signals through their capacity to act as guanine nucleotide exchange factors for heterotrimeric guanine nucleotide-binding proteins (G proteins) in response to stimulatory ligand binding (or via constitutively activating point mutagenesis). Ligand-mediated G protein activation is initiated through conformational rearrangement of the heptahelical GPCR core and juxtamembrane loop regions, eventually catalyzing the exchange of GDP for GTP on the receptor-associated Gα subunit [24,25,26,27,28]. Guanine nucleotide exchange (GDP for GTP) then initiates the dissociation of the heterotrimeric G protein from the GPCR, followed by the dissociation of the G protein heterotrimer releasing free GTP-bound α and βγ subcomplexes. These two signaling components can stimulate, inhibit or physically recruit multiple downstream signal transduction effectors, e.g., adenylyl cyclase (AC), phospholipase C (PLC), GPCR kinases (GRKs) or GRK-interacting proteins [29]. In this manner, the heterotrimeric G protein can transmit information to the intracellular milieu about the qualitative and quantitative nature of specific extracellular stimuli [30,31].

There are 16 Gα, 5 Gβ and 13 Gγ subunits in humans, which allows many different aggregations and signaling outputs [32]. The responses range from activation to inhibition, and G proteins are classified based on the downstream function of the alpha (α) subunit into four families, Gαs, Gαi/o, Gαq/11, and Gα12/13. Gαs activates adenylyl cyclase, which then converts ATP into the second messenger cyclic adenosine monophosphate (cAMP) and activates the cAMP-dependent pathway. Conversely, Gαi/o decreases cAMP levels [33]. Apart from the classical G protein signaling, multiple lines of research have validated the existence of multiple non-G protein signaling activities, such as β-arrestin [34,35,36], proto-oncogene tyrosine-protein kinase Src (c-Src) [37,38,39] and the ADP-ribosylation factor GTPase activating protein-2 (GIT2) [40,41,42]. Thus, it is now clear that GPCR signaling is more nuanced and complex than previously thought. The dogma of simple physiological G protein signaling specificity of downstream signaling was broken with the initial demonstration that alternative modes of signaling, e.g., the first being the β-arrestin paradigm, are physiologically relevant and are therapeutically tractable [28,34,35,36,43,44,45,46,47]. β-arrestins were first characterized as negative regulatory proteins for signaling through G proteins and were considered responsible for GPCR internalization and separation from G protein engagement [48,49,50]. β-arrestin has been subsequently shown to serve as a scaffold for a variety of signaling complexes associated with GPCR signaling pathways [28,34,43].

While the field of non-G protein-dependent GPCR signaling has been historically dominated by β-arrestin activity, several other modalities have been demonstrated, including Janus kinase 2 (JAK2) [51], 14-3-3 proteins [52], RGS proteins [53], Proline-rich tyrosine kinase 2 (PYK2) [39,54] and the ADP-ribosylation factor GTPase-activating protein 2 (GIT2) [40,55]. With regards to this last GPCR signaling adaptor, GIT2, several strong contrasts to β-arrestin signaling have been reported, thus making this paradigm an interesting one to compare with the pioneering β-arrestin pathway [2,9,29]. With the specific link to aging paradigms, it has been shown that β-arrestin activation leads to increased DNA damage in stress conditions, degradation of p53, suppression of NF-kB and the promotion of apoptosis [56,57]. Conversely, it appears that GIT2 may represent a natural mechanism to prevent aging-associated molecular and cellular damage. The GPCR-kinase interacting protein (GIT) family of proteins (GIT1 and GIT2) were originally identified as GRK and GPCR interacting proteins [58]. Subsequently, it has been shown that especially GIT2 exerts systemic effects upon a multitude of signaling and physiological systems, including oxidative stress resistance [59], glucose metabolism [60], circadian rhythm regulation [61], mitochondrial activity [41,60], DNA damage repair response [40,62], immunosenescence [61] and gender-specific lifespan regulation [41]. Given this information, it is unsurprising that additional interest in non-G protein-dominated GPCR signaling pathways has been shown with respect to the proposal that signaling paradigms such as β-arrestin and GIT2 may indeed possess specific benefits for the GPCR-mediated interdiction of aging-related disease [35,40,42,63,64,65,66,67]. For both signaling paradigms, significant evidence has shown that these two GPCR signaling modalities converge on the regulation of energy metabolism and DNA damage/repair [34,42,56,57,59,60,61,62,66,68,69,70,71,72,73,74,75].

Aging and age-related damage of cellular proteins and nucleic acids are inevitable results of lifelong cellular metabolic activity [40,60,62,76,77,78]. This cellular damage occurs most frequently because of the production of deleterious metabolites, e.g., reactive oxygen species (ROS), as by-products of energy management processes such as mitochondrial oxidative phosphorylation [9,79,80,81]. There are many other sources of aging-related damage, but many lines of evidence have suggested that this process is one of the most potent sources of recurring cellular damage and, ultimately, age-related disease [82,83,84,85,86,87]. This stress-related damage essentially degrades the functionality of active signaling systems as well as reactive cytoprotective cellular systems that exist to combat the metabolically induced cellular damage [2,9,88,89,90,91]. In recent years it has been demonstrated that—as with many other forms of cellular and tissue signaling [90,92,93]—stress response and DNA damage repair processes are strongly controlled and regulated by signaling networks composed of multiple GPCR types [5,9,29,63,64,65,66,73,94,95]. Thus, well-informed therapeutic targeting of GPCRs holds a strong promise for the generation of a broad series of anti-aging therapeutics.

GPCRs represent one of the most important therapeutic targets for controlling disease generation and progression [96,97]. Underlying their importance in the broad range of biological functions, GPCRs are the most structurally diverse family of transmembrane proteins. The superfamily comprises more than 800 proteins, which are grouped based on evolutionary homology and common physiological ligands. Human GPCRs are divided into six major classes, class A (rhodopsin-like), class B1 (secretin receptor-like), class B2 (adhesion receptors), class C (metabotropic glutamate receptor-like) and class F (frizzled-like) subfamilies, as well as the taste 2 sensory receptor subfamily [98]. GPCRs sense a tremendous variety of stimulating entities ranging from photons, ions, and neurotransmitters, to complex hormones and exogenous animal toxins. This nuanced sensory system allows cells to react quickly to diverse endogenous and environmental perturbations [99]. GPCRs are of major interest for drug development due to their regulatory function for a multitude of physiological processes, as well as their accessibility for exogenous ligands. Hauser et al. evaluated in 2017 that 475 FDA-approved drugs target GPCRs, which is 34% of all FDA-approved drugs [96].

As we have previously described, it has been proposed that non-G protein-dependent signaling paradigms may hold specific promise for the amelioration of aging-associated diseases [2,29,42]. Arrestin-dependent signaling is one of the most important and well-characterized of these signaling modalities [34,94,100]. Recent translational research has demonstrated that the β-arrestin-based signaling modality can generate a clinically relevant signaling paradigm [36,46,47,101]. Using a novel, in vivo-biased signaling demonstration, it was found that β-arrestin-GPCR complexes likely possess the ability to elicit a coherently conserved signaling cascade across multiple tissues, distinct from the G protein paradigm, even after a month of continuous drug dosing of the biased agent [46]. Furthermore, and in stark contrast to G protein-sourced signaling that primarily controls acute intermediary cell metabolism events (e.g., intracellular calcium mobilization), β-arrestin-dependent signaling generates a strong transcriptional and translational signaling functionality [102,103,104,105]. This aspect of β-arrestin-dependent signaling, therefore, lends itself to the concept that β-arrestin-biased ligands could be rationally designed to therapeutically regulate complex protein networks that underpin many complex aging-related diseases, e.g., Type II Diabetes Mellitus (T2DM), neurodegeneration and cancer [29,91,106]. The expression levels and signaling activity of β-arrestin have been shown to be involved with metabolic aging conditions such as Alzheimer’s disease [107,108], Parkinson’s’ disease [109,110,111], T2DM [112,113,114], osteoporosis [44,46,115] and schizophrenia [116,117]. In a similar vein, research has also demonstrated that the GPCR adaptor GIT2 can play a pivotal role in metabolic aging conditions and disorders, including neurodegenerative diseases [118,119], T2DM [60] osteoporosis [120,121] and psycho-affective disorders [122,123]. In addition to these two major forms of GPCR signaling adaptors, considerable evidence has been generated to demonstrate the role of GRKs, PYK2 and JAK2 in aging-related conditions associated with metabolic dysfunction [2]. This consistent finding, therefore, suggests that perhaps the association of GPCRs with these non-G protein signal adaptors may create a stress-sensory mechanistic network of receptors that naturally control the severity of these conditions [9]. Hence, we contend that the molecular intersection between cellular damage control and metabolic dysfunction systems plays a pivotal role in regulating the balance of energy regulation and cellular stress responses. While presenting tremendous promise for the future of pharmacotherapy via precision medicine, the multiplicity and importance of β-arrestin and GIT2 in a plethora of physiological processes does raise the possibility of incurring systemic side effects resulting from excessive, or even total β-arrestin or GIT2 bias, at the expense of G protein activity. Hence, an intelligently informed and subtle approach to signaling bias exploitation should be taken. Potentially the development of novel biased ligands targeting the β-arrestin-or GIT2-associated receptorsome currently represents a vital new pharmacotherapeutic domain [40,44,63,124,125].