The type 2 cytokines interleukin (IL)-4, IL-5, IL-9 and IL-13 play critical roles in stimulating innate and adaptive immune responses required for resistance to helminth infection and promotion of allergic inflammation, metabolic homeostasis and tissue repair1–3. Group 2 innate lymphoid cells (ILC2s) are a potent source of type 2 cytokines and while significant advances have been made in understanding the cytokine milieu that promotes ILC2 responses4–9, there are fundamental gaps in knowledge regarding how ILC2 responses are regulated by other stimuli. In this report, we demonstrate that ILC2s in the gastrointestinal tract co-localize with cholinergic neurons that express the neuropeptide neuromedin U (NMU)10,11. In contrast to other hematopoietic cells, ILC2s selectively express the NMU receptor 1 (NMUR1). In vitro stimulation of ILC2s with NMU induced rapid cell activation, proliferation and secretion of type 2 cytokines IL-5, IL-9 and IL-13 that was dependent on cell-intrinsic expression of NMUR1 and Gαq protein. In vivo administration of NMU triggered potent type 2 cytokine responses characterized by ILC2 activation, proliferation and eosinophil recruitment that was associated with accelerated expulsion of the gastrointestinal nematode Nippostrongylus brasiliensis or induction of lung inflammation. Conversely, worm burden was higher in Nmur1−/− mice compared to control mice. Further, use of gene-deficient mice and adoptive cell transfer experiments revealed that ILC2s were necessary and sufficient to mount NMU-elicited type 2 cytokine responses. Together, these data indicate that the NMU-NMUR1 neuronal signaling circuit provides a selective and previously unrecognized mechanism through which the enteric nervous system and innate immune system integrate to promote rapid type 2 cytokine responses that can induce anti-microbial, inflammatory and tissue-protective type 2 responses at mucosal sites.

Despite the discovery of heterotrimeric αβγ G proteins ∼25 years ago, their selective perturbation by cell-permeable inhibitors remains a fundamental challenge. Here we report that the plant-derived depsipeptide FR900359 (FR) is ideally suited to this task. Using a multifaceted approach we systematically characterize FR as a selective inhibitor of Gq/11/14 over all other mammalian Gα isoforms and elaborate its molecular mechanism of action. We also use FR to investigate whether inhibition of Gq proteins is an effective post-receptor strategy to target oncogenic signalling, using melanoma as a model system. FR suppresses many of the hallmark features that are central to the malignancy of melanoma cells, thereby providing new opportunities for therapeutic intervention. Just as pertussis toxin is used extensively to probe and inhibit the signalling of Gi/o proteins, we anticipate that FR will at least be its equivalent for investigating the biological relevance of Gq.

Some 865 genes in man encode G-protein-coupled receptors (GPCRs). The heterotrimeric guanine nucleotide-binding proteins (G-proteins) function to transduce signals from this vast panoply of receptors to effector systems including ion channels and enzymes that alter the rate of production, release or degradation of intracellular second messengers. However, it was not until the 1970s that the existence of such transducing proteins was even seriously suggested. Combinations of bacterial toxins that mediate their effects via covalent modification of the a-subunit of certain G-proteins and mutant cell lines that fail to generate cyclic AMP in response to agonists because they either fail to express or express a malfunctional G-protein allowed their identification and purification. Subsequent to initial cloning efforts, cloning by homology has defined the human G-proteins to derive from 35 genes, 16 encoding a-subunits, five b and 14 g. All function as guanine nucleotide exchange on-off switches and are mechanistically similar to other proteins that are enzymic GTPases. Although not readily accepted initially, it is now well established that b/g complexes mediate as least as many functions as the asubunits. The generation of chimeras between different a-subunits defined the role of different sections of the primary/secondary sequence and crystal structures and cocrystals with interacting proteins have given detailed understanding of their molecular structure and basis of function. Finally, further modifications of such chimeras have generated a range of G-protein a-subunits with greater promiscuity to interact across GPCR classes and initiated the use of such modified G-proteins in drug discovery programmes.

Background-We previously identified the G-protein-coupled receptor Mas, encoded by the Mas proto-oncogene, as an endogenous receptor for the heptapeptide angiotensin-(1-7); however, the receptor is also suggested to be involved in actions of angiotensin II. We therefore tested whether this could be mediated indirectly through an interaction with the angiotensin II type 1 receptor, AT 1 . Methods and Results-In transfected mammalian cells, Mas was not activated by angiotensin II; however, AT 1 receptor-mediated, angiotensin II-induced production of inositol phosphates and mobilization of intracellular Ca 2ϩ was diminished by 50% after coexpression of Mas, despite a concomitant increase in angiotensin II binding capacity. Mas and the AT 1 receptor formed a constitutive hetero-oligomeric complex that was unaffected by the presence of agonists or antagonists of the 2 receptors. In vivo, Mas acts as an antagonist of the AT 1 receptor; mice lacking the Mas gene show enhanced angiotensin II-mediated vasoconstriction in mesenteric microvessels.Conclusions-These results demonstrate that Mas can hetero-oligomerize with the AT 1 receptor and by so doing inhibit the actions of angiotensin II. This is a novel demonstration that a G-protein-coupled receptor acts as a physiological antagonist of a previously characterized receptor. Consequently, the AT 1 -Mas complex could be of great importance as a target for pharmacological intervention in cardiovascular diseases.

There has been much speculation regarding the functional relevance of G protein-coupled receptor heterodimers, primarily because demonstrating their existence in vivo has proven to be a considerable challenge. Here we show that the opioid agonist ligand 6-guanidinonaltrindole (6-GNTI) has the unique property of selectively activating only opioid receptor heterodimers but not homomers. Importantly, 6-GNTI is an analgesic, thereby demonstrating that opioid receptor heterodimers are indeed functionally relevant in vivo. However, 6-GNTI induces analgesia only when it is administered in the spinal cord but not in the brain, suggesting that the organization of heterodimers is tissue-specific. This study demonstrates a proof of concept for tissue-selective drug targeting based on G protein-coupled receptor heterodimerization. Importantly, targeting opioid heterodimers could provide an approach toward the design of analgesic drugs with reduced side effects.opioid

G protein-independent, arrestin-dependent signaling is a paradigm that broadens the signaling scope of G protein-coupled receptors (GPCRs) beyond G proteins for numerous biological processes. However, arrestin signaling in the collective absence of functional G proteins has never been demonstrated. Here we achieve a state of “zero functional G” at the cellular level using HEK293 cells depleted by CRISPR/Cas9 technology of the Gs/q/12 families of Gα proteins, along with pertussis toxin-mediated inactivation of Gi/o. Together with HEK293 cells lacking β-arrestins (“zero arrestin”), we systematically dissect G protein- from arrestin-driven signaling outcomes for a broad set of GPCRs. We use biochemical, biophysical, label-free whole-cell biosensing and ERK phosphorylation to identify four salient features for all receptors at “zero functional G”: arrestin recruitment and internalization, but—unexpectedly—complete failure to activate ERK and whole-cell responses. These findings change our understanding of how GPCRs function and in particular of how they activate ERK1/2.

(4) disclose previously undetected features of GPCR behavior. Significant impact of DMR is therefore anticipated in the emerging areas of systems biology and systems pharmacology but also for the discovery of mechanistically novel drugs.3

Members of the muscarinic acetylcholine receptor family (M1-M5) are known to be involved in a great number of important central and peripheral physiological and pathophysiological processes. Because of the overlapping expression patterns of the M1-M5 muscarinic receptor subtypes and the lack of ligands endowed with sufficient subtype selectivity, the precise physiological functions of the individual receptor subtypes remain to be elucidated. To explore the physiological roles of the M2 muscarinic receptor, we have generated mice lacking functional M2 receptors by using targeted mutagenesis in mouse embryonic stem cells. The resulting mutant mice were analyzed in several behavioral and pharmacologic tests. These studies showed that the M2 muscarinic receptor subtype, besides its well documented involvement in the regulation of heart rate, plays a key role in mediating muscarinic receptor-dependent movement and temperature control as well as antinociceptive responses, three of the most prominent central muscarinic effects. These results offer a rational basis for the development of novel muscarinic drugs.Muscarinic acetylcholine receptors are known to regulate numerous fundamental physiological processes, including the muscarinic actions of acetylcholine on peripheral effector tissues and a multitude of central sensory, vegetative, and motor functions (1-4). In addition, disturbances in central muscarinic neurotransmission have been implicated in a variety of pathophysiological conditions, including Alzheimer's and Parkinson's diseases (1-4).Molecular cloning studies have revealed the existence of five molecularly distinct muscarinic receptor subtypes referred to as M1-M5 (5-7). The M1-M5 receptors are prototypical members of the superfamily of G protein-coupled receptors. Although the odd-numbered muscarinic receptor subtypes (M1, M3, and M5) are selectively linked to G q͞11 proteins, the even-numbered receptors (M2 and M4) are preferentially coupled to G proteins of the G i͞o family (5-7).The M1-M4 receptors are widely expressed throughout the central nervous system and the body periphery (6,(8)(9)(10). Studies with subtype-selective antibodies and in situ mRNA hybridization experiments have shown that most brain regions express several different muscarinic receptor subtypes (8-10). Based on this observation, it has been extremely difficult to assign specific central functions to individual muscarinic receptor subtypes.In addition, the lack of muscarinic agonists and antagonists with pronounced subtype selectivity also has represented a major limitation in studying the physiological roles of the M1-M5 receptors (5-7). This problem is accentuated further in the case of in vivo studies in which the actual concentrations of drugs at their sites of action are difficult to determine because of pharmacokinetic factors.In the body periphery, muscarinic receptors mediate the well known functions of acetylcholine at parasympathetically innervated effector organs, including contraction of smooth muscle, stimulatio...