G protein-coupled receptors (GPCRs) are essential for the neurogenic control of gastrointestinal (GI) function and are important and emerging therapeutic targets in the gut. Detailed knowledge of both the distribution and functional expression of GPCRs in the enteric nervous system (ENS) is critical toward advancing our understanding of how these receptors contribute to GI function during physiological and pathophysiological states. Equally important, but less well defined, is the complex relationship between receptor expression, ligand binding, signaling, and trafficking within enteric neurons. Neuronal GPCRs are internalized following exposure to agonists and under pathological conditions, such as intestinal inflammation. However, the relationship between the intracellular distribution of GPCRs and their signaling outputs in this setting remains a “black box”. This review will briefly summarize current knowledge of agonist-evoked GPCR trafficking and location-specific signaling in the ENS and identifies key areas where future research could be focused. Greater understanding of the cellular and molecular mechanisms involved in regulating GPCR signaling in the ENS will provide new insights into GI function and may open novel avenues for therapeutic targeting of GPCRs for the treatment of digestive disorders.

INTRODUCTION G protein-coupled receptors (GPCRs) are dynamic signaling proteins capable of sensing and responding to different physiological stimuli and to diverse extracellular ligands, such as neurotransmitters, hormones, and environmental or dietary cues. GPCRs are ubiquitously expressed by both neuronal and nonneuronal cells in the gastrointestinal (GI) tract, where their signaling outputs modulate a broad range of processes, including motility, barrier function, secretion, and immune responses (41). Although a suite of GPCRs function synergistically to achieve physiological change, it is now appreciated that the complex signaling mediated by individual GPCR subtypes is a multifaceted process influenced by the intracellular localization of the GPCR itself and the properties of the ligand, as well as by the necessity for precise interactions between the GPCR and effector proteins to elicit downstream signaling responses (15, 34, 49). GPCRs are involved in a wide array of physiological and pathophysiological processes and are the target of more than 30% of current therapeutic drugs (18). However, the progression of GPCR-targeted compounds from the laboratory to the clinic has been limited by high attrition rates, often caused by a lack of efficacy. This is generally attributed to the lack of receptor subtype specificity and adverse on-target side-effect liabilities. Thus, to overcome these challenges, there remains an unmet need for reexamination of fundamental mechanisms underlying GPCR signaling in physiologically relevant systems. This is particularly important in the enteric nervous system (ENS), as current and emerging concepts of GPCR signaling, such as biased agonism, allosteric modulation, and the “location bias” of cell surface and intracellular receptors remain largely unexplored. Stimulated GPCRs activate and dissociate from heterotrimeric G proteins and can become rapidly phosphorylated by GPCR kinases (GRKs) to enhance interactions with β-arrestin adaptor proteins and trafficking machinery for clathrin- and dynamin-mediated internalization. Established receptor signaling paradigms hypothesize that internalization facilitates signal termination by targeting receptors into the degradative lysosomal pathway or by disassembling the ligand-receptor-effector complex in early endosomes to promote recruitment of receptors back to the cell surface via recycling endosomes (15). However, there is increasing evidence that internalized receptors can remain associated with their cognate G proteins and can initiate signaling processes that are temporally and spatially distinct from cell surface events, thus broadening our understanding of how receptors function (6, 7, 22, 50). This is highly consistent with distinct classes of receptor tyrosine kinase (RTK) families, such as the insulin and EGF receptors, which have been studied in explicit detail to demonstrate that most, if not all, signaling is transduced from intracellular RTKs associated with vesicles or endosomes (3). Moreover, recent evidence demonstrates that a preexisting pool of intracellular GPCRs can be activated by certain drugs, such as morphine and metoprolol, which can diffuse across the plasma membrane (21, 47). Activation of GPCRs located within the intracellular membranes can generate unique signaling profiles, which then define the ensuing responses by cells and tissues (49). The importance of postendocytic or intracellular receptor signaling by GPCRs for the control of GI function is unknown. Agonist-evoked internalization of GPCRs in enteric neurons was first demonstrated more than 20 years ago (5, 46), and the current understanding of this process is briefly discussed below. However, the functional importance of many of these trafficking events remains poorly defined. This review explores some of the key molecular and cellular consequences of receptor endocytosis and trafficking and their potential relationship to chronic conditions of the gut. Moreover, a summary of currently available approaches to define the importance of GPCR internalization for neuronal signaling and GI function is provided. A better understanding of these fundamental processes is essential for development of novel and more effective therapeutic interventions for the treatment of digestive diseases and disorders.

THE ENTERIC NERVOUS SYSTEM: AN INTRINSIC COORDINATOR OF GASTROINTESTINAL FUNCTION The GI tract is intrinsically innervated by the enteric nervous system (ENS). It consists of the myenteric and submucosal plexuses, which are primarily involved in the reflex regulation of motility and secretomotor/vasodilator functions, respectively. The circuitry of the ENS mediates coordinated reflex responses by the GI tract to mechanical or chemical stimuli independently of central input, although the central nervous system (CNS) has a modulatory influence. Several comprehensive reviews describe the structure and function of the ENS, to which the reader is directed (12, 13). The ENS contains a wide range of GPCRs and their respective endogenous ligands, many of which are also expressed in the CNS. There are key features that make the ENS ideal for examining GPCR trafficking and signaling. The ENS is a comparatively simple and well-defined system: enteric neurons can be readily examined in situ within whole-mount preparations; the neurochemical coding, function, and connectivity of most neurons has been characterized; and the physiological consequences of neuronal activation can be experimentally determined in both isolated tissues and in live animals. It should also be noted that the intestine is one of the few routinely available sources of viable human neurons, making it an invaluable resource for preclinical studies (31, 51).

GPCR ENDOCYTOSIS AND TRAFFICKING IN THE ENS The initial observations that exposure of enteric neurons to exogenous agonists resulted in the movement of receptor immunoreactivity from the cell surface to endosomes were first described in the mid-1990s (5, 16, 46). Since these original reports, only a limited number of studies have examined the kinetics, mechanisms, and consequences of GPCR trafficking and associated signaling in enteric neurons. Furthermore, these studies have focused on very few receptors, largely because of the limited availability of antibodies to adequately and specifically detect these GPCRs. Most research has focused on the initial events that occur upon agonist activation of GPCRs at the cell surface and have delineated the pathways through which GPCRs are trafficked and sorted. These studies demonstrated the subsequent recruitment of key regulatory proteins (29), the clathrin and dynamin dependence of receptor endocytosis (16, 32, 35, 36, 45, 46), and the movement of both receptors and bound ligands to early endosomes (5, 16, 35). The subsequent postendocytic sorting events that occur have been examined in some detail, including the targeting of receptors to recycling endosomes or to lysosomes (29, 35, 37), the requirement for endosomal endopeptidase activity, and acidic pH for effective GPCR recycling (16, 35, 36, 55), and the kinetics of GPCR recycling and resensitization under different conditions (29, 35). A summary of currently available tools for examining GPCR endocytosis and signaling in the ENS, their targets, and specific examples of their use are presented in Fig. 1 and Supplemental Table S1 (deposited in Figshare: https://doi.org/10.26180/5c20e75aa9824). Fig. 1.Chemical and genetic approaches to examining G protein-coupled receptor (GPCR) trafficking and signaling in the enteric nervous system (ENS). The black text labels identify key proteins or organelles involved in the internalization and trafficking of GPCRs. The red text labels indicate tools that may be used to selectively inhibit each of these targets to prevent trafficking of GPCRs and to modulate their signaling in enteric neurons. Ryngo 1-23 (blue text and arrow) can selectively activate dynamin and dynamin-dependent processes. Specific examples of where these tools have been used for the study of enteric neurons or intestinal function are provided in Supplemental Table S1 (deposited in Figshare: https://doi.org/10.26180/5c20e75aa9824). AP2, adaptor protein 2; CCP, clathrin-coated pit; GRK, GPCR kinases; KO, knockout mouse; V-H+-ATPase, vacuolar-type H+-ATPase. Download figureDownload PowerPoint

With very few exceptions, all trafficking studies have used relatively nonphysiological stimuli to evoke endocytosis. These stimulus methods generally involve exogenous application of agonists, are unfocused, and use potentially supraphysiological or saturating concentrations of agonist. Furthermore, the duration of agonist exposure most likely does not reflect the events that occur under physiological conditions. More physiologically relevant studies have examined GPCR trafficking in response to release of endogenous neuropeptides by electrical stimulation (9, 20, 33), application of high K+ (16, 36, 37), or by mechanical activation of enteric reflexes (9, 45). Endocytosis has been used to study functional coupling between cell types. For example, internalization of the neurokinin 1 receptor (NK 1 R) occurs in interstitial cells of Cajal (ICC) in response to activation of the ENS (20). Similarly, internalization of labeled peptide agonists into specific cells within tissues has been used as a surrogate measure to define sites of GPCR expression, particularly when detection with antibodies is problematic. This approach has provided evidence for NK 1 R expression by intestinal smooth muscle, confirming pharmacological evidence (44). Uptake of a fluorescently labeled neurokinin 3 receptor (NK 3 R) agonist (senktide) has also been used to characterize NK 3 R distribution in the guinea pig ileum (23).

GPCR SIGNALING FROM WITHIN ENTERIC NEURONS Although the initial events leading to agonist-evoked receptor endocytosis in enteric neurons are well defined, very little is known about how the plasma membrane is replenished with new or recycled receptors and the signaling events that occur during the trafficking process. Studies of GPCR trafficking in enteric neurons have been limited to a very small number of receptors, due to reliance on availability of adequate antibodies or transgenic mouse strains. At present, only neurokinin (NK 1–3 R), opioid (mu and delta: MOR and DOR, respectively), and somatostatin (SSTR2A) receptor trafficking has been examined in detail and only in laboratory species, with the most comprehensive information available for NK 1 R. These are not necessarily the most important GPCRs for the control of intestinal motility, as endogenous agonists of these receptors are not generally considered to be primary mediators of enteric neurotransmission (13). We are not aware of any studies that have looked at agonist-evoked redistribution of GPCRs in human enteric neurons, although GPCRs have been localized to neurons of the human intestine (26). There has been little correlation between endocytosis and effects on physiological responses, with most examination performed at the single-cell level and focused on desensitization of responses. The complex mechanisms through which motility and secretion are controlled make data interpretation difficult. This is further complicated by the sites of neuronal expression, including presynaptic and postsynaptic locations, and the potential for expression of the same GPCR by multiple cell types within the GI tract [e.g., NK 1 R expression by enteric neurons, ICC, epithelial cells, and intestinal smooth muscle (38, 44)]. Interpretation of in vivo studies of GI function may also be confounded by centrally mediated effects of ligands [e.g., DOR (4)]. With the exception of MOR (46) and SSTR2A (55), studies of GPCR trafficking in the ENS have relied on a limited number of agonists. This is of importance, as different agonists of the same receptor can differ markedly in their ability to induce endocytosis and in their signaling profiles. GRK-dependent phosphorylation of GPCRs is likely to determine the nature of subsequent βArr recruitment and receptor internalization, and all of these are highly ligand-dependent processes (10, 30). The retention of internalized GPCRs in endosomes is similarly dependent on the susceptibility of the associated ligand to degradation by peptidases, with implications for both receptor resensitization and the type and duration of downstream signaling (7, 35, 55). Only a few studies have examined endocytosis evoked by endogenously released agonists, and these have generally used supraphysiological, prolonged, or unfocused stimuli (e.g., depolarization-evoked or mechanically stimulated). We are not aware of any studies of endocytosis in response to focused electrical stimulation (e.g., applied to interganglionic connectives), or to a rapid, transient exposure to an agonist. There is only one report of real-time GPCR trafficking in enteric neurons (9), with all existing data essentially a “snapshot” of the events that are potentially occurring. Moreover, there is no direct correlation between synaptic signaling and receptor trafficking, and, to our knowledge, only one study that examines GPCR endocytosis following a well-characterized physiological event, such as the generation of propagating motor patterns (9). Several endogenous ligands for the same receptor may exist, including those generated through alternative processing of peptide precursors. This raises the potential for distinct GPCR signaling profiles associated with ligand bias (48). For example, differences in SSTR2A recycling following stimulation with the endogenous agonists SST-14 or SST-28 have been demonstrated in myenteric neurons, with the relative susceptibility of these peptides to degradation by endothelin-converting enzyme 1 (ECE-1) identified as a key determinant of endosomal retention time (55). Whether this difference is reflected by corresponding changes to the magnitude, duration, location, or type of SSTR2A signaling has not been examined. Signaling upon direct activation of intracellular GPCRs has not been examined, and almost nothing is known about signaling from endosomes in enteric neurons, or of the importance of GPCR endocytosis to physiological processes regulated by the ENS. Assays of ENS function generally examine indirect outputs, such as the measurement of neurogenic changes in smooth muscle tension. This represents a significant issue, as tools including endocytosis and kinase inhibitors will similarly impact GPCR signaling in all cell types involved in neuromuscular transmission. An additional limitation to the use of existing endocytosis inhibitors is their established effects on neurotransmitter release and uptake (53). An alternative approach may be to compare relative effects of internalizing and noninternalizing agonists of the same GPCR, which improves specificity through selective targeting of cell types that express the receptor of interest (39). However, differences in the pharmacological profiles of these agonists, including partial versus full agonism and relative ability to recruit βArrs, need to be accounted for when interpreting data. Potential for Location-Biased Signaling to Influence ENS Function Under Pathophysiological Conditions Intracellular GPCRs can drive unique signaling events and may play a role in pathophysiology or represent a distinct therapeutic entity. As outlined earlier, the potential for the targeting of endosomal GPCR signaling for analgesia has been described (24, 54). Despite recent significant advances in our understanding of the importance and unique role of endosomal signaling, postendocytic GPCR signaling in the ENS remains largely unexamined (7, 35). The metalloendopeptidase ECE-1 is a major regulator of endosomal signaling by neuropeptide receptors (42). ECE-1-dependent degradation of neuropeptides, including substance P (SP), within the acidic microenvironment of endosomes effectively disrupts signaling endosomes, thereby terminating their signaling and enabling GPCR recycling. This mechanism also operates in the ENS, where ECE-1 inhibition blocks recycling of internalized NK 1 R and SSTR2A (7, 35, 55) and prolongs MAPK signaling through stabilization of SP-NK 1 R-βArr-MAPK-containing signalosomes (7, 35). The functional consequences of sustained endosomal retention of activated GPCRs are poorly defined in the ENS. Endosomal signaling may be of greatest importance under pathophysiological conditions. Significant NK 1 R internalization occurs in myenteric neurons of the diseased or inflamed colon (28, 36), and ECE-1 expression is downregulated (36). These changes are predicted to lead to sustained endosomal signaling by NK 1 R, although this has not been specifically examined. Prolonged signaling by internalized NK 1 R promotes neuronal death (7) and diminishes resensitization of subsequent responses to SP by preventing replenishment of cell surface NK 1 R (35). The related peptidase ECE-2 degrades enkephalins, influencing DOR trafficking and signaling (17). DOR is also internalized in myenteric neurons of the acutely inflamed colon (9). Thus, a range of endosomal enzymes are likely to control the kinetics of neuropeptide receptor recycling and determine the type and duration of signaling that ensues upon receptor activation and internalization. The trafficking and subcellular distribution of GPCRs in enteric neurons may be altered following prolonged exposure to agonists. Acute administration of morphine does not normally promote MOR internalization in myenteric neurons (46). However, morphine stimulates MOR endocytosis in myenteric neurons following chronic exposure (32). This change was attributed to an associated increase in the expression and redistribution of dynamin. In contrast, chronic exposure to another opiate, fentanyl, had no effect on dynamin expression and did not mediate subsequent morphine-induced MOR internalization (2). These data suggest that the reported effects of chronic agonist exposure are ligand-specific. Kang et al. (25) demonstrated that expression of the scaffolding protein βArr2 was significantly decreased in the ileum, but not the colon, following chronic morphine exposure. βArrs have been identified as key regulators of MOR signaling in the ENS, with region-specific effects on tolerance development (14). Although βArr2 deletion significantly attenuates the constipating effects of opiates (40), no studies have directly examined the effect of βArr2 deletion on MOR trafficking and signaling in the ENS. It is likely that trafficking of MOR will be influenced by interaction with βArr2, based on evidence that the recycling rate of internalized NK 1 R is significantly enhanced in enteric neurons of βArr2 knockout mice (36). “Location bias” has been described for endogenous opioid peptides and their synthetic derivatives, which can activate MOR and DOR at the cell surface to promote internalization of activated receptors into endosomes. In contrast, clinically important opioid drugs, such as morphine, can cross the plasma membrane to directly activate MOR in the Golgi network (47). Thus, activation of a single GPCR subtype can drive three distinct downstream signaling pathways within the same cell (i.e., plasma membrane-, endosomal membrane-, and intracellular membrane-derived). This has significant mechanistic implications for our current understanding of how GPCRs control neuronal excitability and coordinate physiological processes, including motility and secretion. It is unknown whether morphine can similarly activate Golgi-resident MOR in the ENS, leading to unique signaling and functional effects in the gut, but this is of clear therapeutic interest. Similar location bias has been demonstrated for the β 1 adrenergic receptor and mGluR5, with distinct signaling generated from the cell surface and the Golgi apparatus and nuclear membrane, respectively (21, 52). It is possible that several GPCRs, including those that are important for the regulation of enteric neuron function, also exhibit location bias. Whether GPCR signaling in discrete subcellular sites occurs in the ENS and can be selectively targeted in a location-specific manner is unknown. These studies suggest that pharmacological targeting of GPCR signaling at specific subcellular locales may prove to be an effective and novel therapeutic approach for the treatment of digestive diseases. An overview of potential locations and modes of GPCR activation and trafficking in the ENS is presented in Fig. 2. Fig. 2.An overview of key concepts in G protein-coupled receptor (GPCR) trafficking and signaling in the enteric nervous system (ENS). 1. Enteric neurons express GPCRs that are activated and signal at the cell surface. 2. Activated GPCRs internalize into endosomes through a GRK-, βArr-, dynamin- and clathrin-dependent process. This internalization triggers intracellular signaling cascades that are spatially, temporally, and functionally distinct from those initiated at the cell surface. Internalized GPCRs are either recycled back to the surface (e.g., mu opioid receptor) or are targeted to lysosomes for degradation (e.g., delta opioid receptor). 3. Drugs can be transported or diffuse through the plasma membrane to directly activate intracellular GPCRs located on the Golgi apparatus and nuclear membrane to promote distinct intracellular signaling events. 4. The kinetics and nature of localized GPCR trafficking and signaling in peripheral terminals may differ to that which occurs at the soma and proximal neurites, consistent with the polarized and highly differentiated morphology of enteric neurons. Download figureDownload PowerPoint



FUTURE OPPORTUNITIES FOR UNDERSTANDING GI PHYSIOLOGY AND ASSISTING DRUG DISCOVERY At present, there are no definitive studies that correlate the subcellular location of a receptor in an enteric neuron with associated receptor-mediated functional outcomes. Moreover, studies involving physiologically equivalent or relevant stimuli are lacking. This includes the use of appropriate agonist concentrations and exposure durations. Furthermore, there is very limited information about GPCR endocytosis in myenteric neurons after the in vivo administration of agonists (37, 46). Information of this nature is highly important and directly relevant to the design of more effective GPCR-targeted therapeutics. Although there have been recent significant technical and conceptual advances in GPCR biology, the fundamental science associated with these new concepts has not been effectively translated to the neurogastroenterology field. In our opinion, this represents a major impediment to both our basic understanding of GI physiology and to effective drug discovery efforts. The core questions regarding GPCR signaling remain: what needs to be targeted, when should this be targeted, and where should this be targeted? Our recent studies in acute somatic pain models indicate that receptor internalization and endosomal signaling are associated with unique functional effects, and that intracellular GPCRs can be specifically targeted pharmacologically to suppress nociceptive responses (24, 54). It is likely that signaling from endosomes within enteric neurons can similarly result in distinct functional outcomes, although this remains to be determined. Furthermore, the concept of location bias, where drugs (e.g., morphine) can directly activate intracellular GPCRs independently of the cell surface represents a paradigm shift in our understanding of how GPCRs may regulate neuronal function. This has the potential to significantly impact how future drugs may be developed and targeted for the treatment of digestive disease. The correlation between function, signaling, and receptor trafficking is relatively unknown. Moreover, the link between downstream signaling events, such as βArr-mediated MAPK activation, with the functional effects of GPCR activation is poorly defined. Studies focusing on MOR would be a logical starting point, given the evidence for unique regulation of MOR signaling in the ENS by βArr2 and the continued interest in developing biased opioid analgesics with reduced constipating actions (8, 27). Although it is established that βArr2 regulates morphine-induced constipation (40, 43), there is very limited information regarding the importance of βArr2 for MOR trafficking and cellular signaling under these same conditions (11). Recent studies indicate that G protein-biased MOR agonists are likely to retain limiting side-effects, such as constipation (1) and respiratory depression (19). A comprehensive understanding of the unique nature of MOR signaling in myenteric neurons of the colon would account for potential system bias and greatly inform development of better opioid analgesics with reduced GI-associated liabilities. Summary The ENS is an ideal system for examining GPCR trafficking and signaling in a native setting. However, as outlined in this review, there are clear limitations to our understanding of the postendocytic processing of GPCRs and the existence and functional consequences of location-specific signaling. Moreover, current conceptual and technological advances in GPCR biology and pharmacology have not been adequately translated to the neurogastroenterology field. This has significant implications for both our basic understanding of GI physiology and for future drug discovery efforts.

GRANTS This study was funded by National Health and Medical Research Council Australia Grants 1049730, 1083480, and 1121029 (to D. P. Poole) and by the Australian Research Council Centre of Excellence in Convergent Bio-Nano Science & Technology (to N. A. Veldhuis), and Takeda Pharmaceuticals (to D. P. Poole and N. A. Veldhuis).

DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors.