G protein-coupled receptor (GPCR) family members can sense an extraordinary variety of biomolecules to activate intracellular signalling cascades that modulate key aspects of cell physiology. Apart from their crucial role in maintaining cell homeostasis, these critical sensory and modulatory properties have made GPCRs the most successful drug target class to date. However, establishing direct links between receptor activation of specific intracellular partners and individual physiological outcomes is still an ongoing challenge. By studying this receptor signalling complexity at increasing resolution through the development of novel biosensors and high-throughput techniques, a growing number of studies are revealing how receptor function can be diversified in a spatial, temporal or cell-specific manner. This mini-review will introduce recent examples of this context-dependent receptor signalling and discuss how it can impact our understanding of receptor function in health and disease, and contribute to the search of more selective, efficacious and safer GPCR drug candidates.
GPCR signalling constitutes a central mechanism allowing cells to sense and adapt to changes in their environment. This signalling can originate from receptors responding to a wide variety of cues including light, odours, ions, small molecules, or peptidic hormones. The key role of GPCRs as privileged entry points for the modulation of cell function has also made them the most successful drug target class in the clinic [1–3]. However, although the fundamental role of GPCR signalling in the regulation of cell physiology has been pharmacologically exploited for decades, researchers are still characterising the complex intracellular signalling processes that are elicited upon receptor activation.
Systematic analyses of intracellular coupling across receptors have revealed how, upon activation, single GPCRs often engage with multiple intracellular signal transducers to regulate cell responses [4]. Dissecting which receptor signalling pathways contribute to specific cell responses can dramatically impact our understanding of GPCR biology by: (i) clarifying how changes in signalling pathway composition in different physiological and pathological conditions can impact cell function; (ii) explaining existing variation in therapeutic responses between drugs displaying differential signalling patterns upon binding to the same receptor; and (iii) guiding the rational selection of biased agonists capable of selectively modifying disease-related pathways [5]. Therefore, determining how the activation of different receptor partners contributes to specific cell responses has been a key aim of the GPCR research community [6]. These efforts have been boosted by the development of multiple biosensors [7] and high-throughput assays [8,9] that allow analysing the GPCR signal transduction process with unprecedented detail. Remarkably, an increasing number of studies exploiting these technologies have started revealing the significance of context when it comes to interpreting GPCR signalling effects [10]. In particular, they have highlighted the importance of examining receptor function at a subcellular, temporal, and cell-type specific resolution [11–13].
Initial indications of the importance of GPCR function away from the plasma membrane originated from observations of second messenger signalling mediated by internalised receptors [14]. This included evidence on how GPCRs, like the thyroid-stimulating hormone receptor, could be internalised into pre-Golgi compartments in primary cells together with G protein αs (Gαs) subunits and adenylyl cyclase [15]. Cyclic adenosine monophosphate (cAMP) production patterns and cytoskeleton remodelling could be altered by impairing this internalisation, highlighting the functional role of receptor signalling from intracellular compartments. Subsequent studies with the β2-adrenoceptor revealed that endosome-based cAMP signalling is key to initiate full transcriptional responses downstream of this prototypical GPCR [16]. This led the authors to propose that spatial encoding of receptor signalling can diversify receptor function. In this way, the same ligand could activate different receptor-mediated responses depending on subcellular location giving rise to ‘location-biased signalling’. Interestingly, recent work has described another instance of β2-adrenoceptor location bias, as endosomal signalling from these receptors has been implicated in their capacity to activate extracellular signal-regulated kinase (ERK) [17]. ERK activity seems to be initiated exclusively from endosomes — and not the plasma membrane — and depends on receptor coupling to the long, but not the short, Gαs splice variant (Figure 1A). This observation is particularly relevant for GPCR pathophysiology, as mutations in splicing factors observed in myelodysplastic syndrome selectively increase the expression of long Gαs and drive abnormal signalling through ERK. In this way, dysregulated location bias could be a source of pathological signalling in this condition.
Figure 1.
(A) Spatial segregation of receptors can lead to compartment-specific coupling and signalling. For instance, recent work has suggested that, apart from the established cAMP signalling of the β2-adrenoceptor (ADRB2) from the plasma membrane, ERK signalling could be associated to endosomal receptor signalling specifically via the long Gαs splice variant (Gαs-L). (B) Differences in residence time between endogenous ligands and drugs have been related to observations of temporal bias. This is the case of dopamine and antipsychotic drug aripiprazole at the dopamine D2 receptor (DRD2) and their capacity to promote G protein (GαoB) vs. β-arrestin 2 (β-arr2) coupling. (C) Cell or tissue-specific expression of different isoforms of the same receptor can diversify signalling outputs. In the case of chemokine receptor CXCR3, the different capacities of isoforms A and B to respond to endogenous ligands like CXCL10 mean that variation in isoform expression between cell types could lead to cell-specific β-arrestin 2 coupling.
Examples of location bias have also been observed for receptor populations that do not depend on internalisation. In the case of the metabotropic glutamate receptor 5, activation of intracellular receptor pools in rat hippocampal neurons showed location-specific effects on Ca 2+ signalling and long-term depression [18]. For β1-adrenoceptors, authors observed cAMP signalling originating from the Golgi that was independent of plasma membrane receptor activation or internalisation [19]. These studies also highlighted an aspect of location-specific signalling that is crucial for GPCR pharmacology: if receptors are capable of signalling from intracellular compartments independently from internalisation, their ligands must be able to access such compartments by crossing the plasma membrane. In the case of β1-adrenoceptors, the authors observed that endogenous and exogenous ligands may do so by different mechanisms. While norepinephrine can cross the plasma membrane via the organic cation transporter 3 (OCT3), other exogenous drugs can do so by virtue of being hydrophobic and passively diffusing through the membrane. This leads to the interesting observation that different drugs like β-blockers could access different receptor pools depending on their physicochemical properties. This, in turn, would result in variation in the overall signalling effects of different β-blockers depending on the subcellular locations they are able to access. Such differences in location-dependent signalling could be behind the divergent therapeutic efficacies among β-blockers in the clinic. Furthermore, these observations also point to the possibility of selecting drug candidates according to their capacity to access receptors in different cellular compartments as a way of rationally modulating location-specific GPCR responses [20].
Further studies analysing how receptor signalling from specific locations can contribute to different therapeutic and disease-related phenotypes have exemplified how this idea could be exploited for rational drug design. In the case of β1-adrenoceptors, preventing activation of the receptor in the Golgi by inhibiting norepinephrine entry into the cell through OCT3 could reduce cardiac myocyte hypertrophy in heart failure [21]. For opioid receptors, the endosomal receptor pool could potentially act as a new target for the management of pain [22]. In particular, δ-opioid receptor agonists activating the endosomal receptor pool have been shown to provide a sustained inhibition of nociception in inflammatory conditions [23]. Interestingly, further studies on the δ-opioid receptor using different conformational biosensors have revealed that the receptor can display location-specific conformations upon activation with the same ligand that could lead to compartment-specific receptor coupling [24]. This highlights the importance of investigating ligand mediated GPCR activation in diverse subcellular contexts to fully characterise and exploit therapeut
