Peptides are sustainable alternatives to conventional therapeutics for G protein-coupled receptor (GPCR) linked disorders, promising biocompatible and tailorable next-generation therapeutics for metabolic disorders including type-2 diabetes, as agonists of the glucagon receptor (GCGR) and the glucagon-like peptide-1 receptor (GLP-1R). However, single agonist peptides activating GLP-1R to stimulate insulin secretion also suppress obesity-linked glucagon release. Hence, bioactive peptides cotargeting GCGR and GLP-1R may remediate the blood glucose and fatty acid metabolism imbalance, tackling both diabetes and obesity to supersede current monoagonist therapy. Here, we design and model optimized peptide sequences starting from peptide sequences derived from earlier phage-displayed library screening, identifying those with predicted molecular binding profiles for dual agonism of GCGR and GLP-1R. We derive design rules from extensive molecular dynamics simulations based on peptide-receptor binding. Our newly designed coagonist peptide exhibits improved predicted coupled binding affinity for GCGR and GLP-1R relative to endogenous ligands and could in the future be tested experimentally, which may provide superior glycemic and weight loss control.

Commensal bacteria and/or their products engender beneficial effects to the mammalian gut, including stimulating physiological cellular turnover and enhancing wound healing, without activating overt inflammation. In the present study, we observed commensal bacteria-mediated activation of the noninflammatory extracellular signal-regulated kinase[ERK]/mitogen-activated protein kinase and Akt signaling pathways in gut epithelial cells and delineated a mechanism for this bacterially activated signaling. All tested strains of commensal bacteria induced ERK phosphorylation without stimulating pro-inflammatory phospho-IκB or pro-apoptotic phospho-c-Jun NH(2)-terminal kinase, with Lactobacillus species being most potent. This pattern of signaling activation was recapitulated using the peptide N-formyl-Met-Leu-Phe, a bacterial product known to stimulate signaling events in mammalian phagocytes. Sensing of N-formyl-Met-Leu-Phe by gut epithelial cells occurs via recently characterized formyl peptide receptors located in the plasma membrane. Both commensal bacteria and N-formyl-Met-Leu-Phe application to the apical surface of polarized gut epithelial cells resulted in specific formyl peptide receptor activation. In addition, pretreatment of model epithelia and murine colon with Boc2 (a specific peptide antagonist) or pertussis toxin (a G(i)-protein inhibitor) abolished commensal-mediated ERK phosphorylation. Taken together, these data show that commensal bacteria specifically activate the ERK/mitogen-activated protein kinase pathway in an formyl peptide receptor-dependent manner, delineating a mechanism by which commensal bacteria contribute to cellular signaling in gut epithelia.

Directed evolution of membrane receptors is challenging as the evolved receptor must not only accommodate a non-native ligand, but also maintain the ability to transduce the detection of the new ligand to any associated intracellular components. The G-protein coupled receptor (GPCR) superfamily is the largest group of membrane receptors. As members of the GPCR family detect a wide range of ligands, GPCRs are an incredibly useful starting point for directed evolution of user-defined analytical tools and diagnostics. The aim of this study was to determine if directed evolution of the yeast Ste2p GPCR, which natively detects the α-factor peptide, could yield a GPCR that detects Cystatin C, a human peptide biomarker. We demonstrate a generalizable approach for evolving Ste2p to detect peptide sequences. Because the target peptide differs significantly from α-factor, a single evolutionary step was infeasible. We turned to a substrate walking approach and evolved receptors for a series of chimeric intermediates with increasing similarity to the biomarker. We validate our previous model as a tool for designing optimal chimeric peptide steps. Finally, we demonstrate the clinical utility of yeast-based biosensors by showing specific activation by a C-terminally amidated Cystatin C peptide in commercially sourced human urine. To our knowledge, this is the first directed evolution of a peptide GPCR.

The intestinal hormone and neuromodulator cholecystokinin (CCK) receptors CCK1R and CCK2R act as a signaling hub in brain-gut axis, mediating digestion, emotion, and memory regulation. CCK receptors exhibit distinct preferences for ligands in different posttranslational modification (PTM) states. CCK1R couples to Gs and Gq, whereas CCK2R primarily couples to Gq. Here we report the cryo-electron microscopy (cryo-EM) structures of CCK1R-Gs signaling complexes liganded either by sulfated cholecystokinin octapeptide (CCK-8) or a CCK1R-selective small-molecule SR146131, and CCK2R-Gq complexes stabilized by either sulfated CCK-8 or a CCK2R-selective ligand gastrin-17. Our structures reveal a location-conserved yet charge-distinct pocket discriminating the effects of ligand PTM states on receptor subtype preference, the unique pocket topology underlying selectivity of SR146131 and gastrin-17, the conformational changes in receptor activation, and key residues contributing to G protein subtype specificity, providing multiple structural templates for drug design targeting the brain-gut axis.

Class B1 G protein-coupled receptors (GPCRs) are important targets for many diseases, including cancer, diabetes, and heart disease. All the approved drugs for this receptor family are peptides that mimic the endogenous activating hormones. An understanding of how agonists bind and activate class B1 GPCRs is fundamental for the development of therapeutic small molecules. We combined supervised molecular dynamics (SuMD) and classic molecular dynamics (cMD) simulations to study the binding of the calcitonin gene-related peptide (CGRP) to the CGRP receptor (CGRPR). We also evaluated the association and dissociation of the antagonist telcagepant from the extracellular domain (ECD) of CGRPR and the water network perturbation upon binding. This study, which represents the first example of dynamic docking of a class B1 GPCR peptide, delivers insights on several aspects of ligand binding to CGRPR, expanding understanding of the role of the ECD and the receptor-activity modifying protein 1 (RAMP1) on agonist selectivity.

Esophageal cancer-related gene 4 (Ecrg4) encodes a hormone-like peptide that is believed to be involved in a variety of physiological phenomena, including tumour suppression. Recent progress in the study of Ecrg4 has shown that Ecrg4 is a proinflammatory factor and induces the expression of several cytokines and chemokines in macrophages/microglia. However, the detailed molecular mechanisms of Ecrg4 signalling, especially the Ecrg4 receptors, remain poorly understood. Here, using retrovirus-mediated expression cloning, we identified lectin-like oxidised low-density lipoprotein receptor-1 (LOX-1) as a membrane protein that binds amino acid residues 71-132 of Ecrg4 (Ecrg4(71-132)). Moreover, in addition to LOX-1, several scavenger receptors, such as Scarf1, Cd36 and Stabilin-1, facilitated the efficient internalisation of Ecrg4(71-132) into cells. A broad competitive inhibitor of scavenger receptors, polyinosinic acid, reduced both the binding of Ecrg4(71-132) and the activation of NF-κB in microglia. This activation was dependent on MyD88, an adaptor protein that recruits signalling proteins to Toll-like receptors (TLRs), with the consequent induction of various immune responses. These data suggest that multiple scavenger receptors recognise Ecrg4(71-132) and transduce its signals, together with TLRs, in microglia.

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors and are important human drug targets. Of the 826 human GPCRs, 118 of them recognize endogenous peptide or protein ligands, and 30 of the 118 are targeted by approved drug molecules, including the very high-profile class B glucagon-like peptide 1 receptor. In this review, we analyze the 21 experimentally determined three-dimensional structures of the known peptide-binding GPCRs in relation to the endogenous peptides and drug molecules that modulate their cell signaling processes. Our integrated analyses reveal that half of the marketed drugs and most of the drugs in clinical trials that interact with peptide GPCRs are small molecules with a wide range of binding modes distinct from those of large peptide ligands. As we continue to collect additional data on these receptors from orthogonal approaches, including nuclear magnetic resonance and electron microscopy, we are beginning to understand how these receptors interact with their ligands at the molecular level and how improving the pharmacology of GPCR signal transduction requires us to study these receptors using multiple biophysical techniques.

The detection of invasive pathogens is critical for host immune defense. Cell surface receptors play a key role in the recognition of diverse microbe-associated molecules, triggering leukocyte recruitment, phagocytosis, release of antimicrobial compounds, and cytokine production. The intense evolutionary forces acting on innate immune receptor genes have contributed to their rapid diversification across plants and animals. However, the functional consequences of immune receptor divergence are often unclear. Formyl peptide receptors (FPRs) comprise a family of animal G protein-coupled receptors which are activated in response to a variety of ligands including formylated bacterial peptides, pathogen virulence factors, and host-derived antimicrobial peptides. FPR activation in turn promotes inflammatory signaling and leukocyte migration to sites of infection. Here we investigate patterns of gene loss, diversification, and ligand recognition among FPRs in primates and carnivores. We find that FPR1, which plays a critical role in innate immune defense in humans, has been lost in New World primates. Amino acid variation in FPR1 and FPR2 among primates and carnivores is consistent with a history of repeated positive selection acting on extracellular domains involved in ligand recognition. To assess the consequences of FPR divergence on bacterial ligand interactions, we measured binding between primate FPRs and the FPR agonist Staphylococcus aureus enterotoxin B, as well as S. aureus FLIPr-like, an FPR inhibitor. We found that few rapidly evolving sites in primate FPRs are sufficient to modulate recognition of bacterial proteins, demonstrating how natural selection may serve to tune FPR activation in response to diverse microbial ligands.

Endothelin system comprises three endogenous 21-amino-acid peptide ligands endothelin-1, -2, and -3 (ET-1/2/3), and two G protein-coupled receptor (GPCR) subtypes-endothelin receptor A (ETAR) and B (ETBR). Since ET-1, the first endothelin, was identified in 1988 as one of the most potent endothelial cell-derived vasoconstrictor peptides with long-lasting actions, the endothelin system has attracted extensive attention due to its critical role in vasoregulation and close relevance in cardiovascular-related diseases. Here we present three cryo-electron microscopy structures of ETAR and ETBR bound to ET-1 and ETBR bound to the selective peptide IRL1620. These structures reveal a highly conserved recognition mode of ET-1 and characterize the ligand selectivity by ETRs. They also present several conformation features of the active ETRs, thus revealing a specific activation mechanism. Together, these findings deepen our understanding of endothelin system regulation and offer an opportunity to design selective drugs targeting specific ETR subtypes.

Natriuretic peptides (NPs) exert their actions through three membrane-bound receptors, which are known as NP receptors (NPRs: NPR-A, NPR-B and NPR-C). In this work we examined the expression of three NPRs in rat retinal ganglion cells (GCs), retrogradely labeled and intracellularly dye-injected, by double immunofluorescence labeling. In vertical sections, almost all GCs, retrogradely labeled by cholera toxin B, were stained by antibodies against the three NPRs. The labeling for three NPRs was observed mainly on the membranes of the somata of GCs, whereas the staining for NPR-A was also seen in the cytoplasm. Moreover, with tangential sections, almost all cells located in the ganglion cell layer were NPR-A, B, C immunoreactive. By combining with intracellular injection of Neurobiotin into GCs in whole mount retinas that enables to identify ON-, OFF- and ON-OFF-types of GCs according to arborization of their dendrites in the inner plexiform layer, we further demonstrated that NPRs were expressed in these major types of GCs.

Adipokinetic hormone (AKH) is a highly researched insect neuropeptide that induces the mobilization of carbohydrates and lipids from the fat body at times of high physical activity, such as flight and locomotion. As a naturally occurring ligand, AKH has undergone quite a number of amino acid changes throughout evolution, and in some insect species multiple AKHs are present. AKH acts by binding to a rhodopsin-like G protein-coupled receptor, which is related to the vertebrate gonadotropin-releasing hormone receptors. In the current study, we have cloned AKH receptors (AKHRs) from seven different species, covering a wide phylogenetic range of insect orders: the fruit fly, Drosophila melanogaster, and the yellow fever mosquito, Aedes aegypti (Diptera); the red flour beetle, Tribolium castaneum, and the large pine weevil, Hylobius abietis (Coleoptera); the honeybee, Apis mellifera (Hymenoptera); the pea aphid, Acyrthosiphon pisum (Hemiptera); and the desert locust, Schistocerca gregaria (Orthoptera). The agonistic activity of different insect AKHs, including the respective endogenous AKHs, at these receptors was tested with a bioluminescence-based assay in Chinese hamster ovary cells. All receptors were activated by their endogenous ligand in the nanomolar range. Based on our data, we can refute the previously formulated hypothesis that a functional AKH signaling system is absent in the beneficial species, Apis mellifera. Furthermore, our data also suggest that some of the investigated AKH receptors, such as the mosquito AKHR, are more selective for the endogenous (conspecific) ligand, while others, such as the locust AKHR, are more promiscuous and can be activated by AKHs from many other insects. This information will be of high importance when further analyzing the potential use of AKHRs as targets for developing novel pest control agents.

The oxytocin/vasopressin signalling system is conserved across the animal kingdom. In insects, the role of oxytocin-type (inotocin) neuropeptides has only been studied in locusts, beetles and ants, but their physiology continues to be poorly understood. One reason for this knowledge deficit is the lack of available research tools to complement functional genomics efforts. Consequently, ligands to probe insect inotocin receptors are essential. In this study, we sought to identify novel agonists and antagonists of the inotocin receptor from the representative model species Tribolium castaneum and Lasius niger. Drawing upon known ligands of the human receptors, we examined the pharmacology of the plant-derived cyclotide kalata B7 and the synthetic oxytocin analogue atosiban. Kalata B7 is a weak partial agonist of both inotocin receptors. This is the first reported direct interaction of cyclotides with an insect receptor, an observation that may explain their presumed role in herbivore defence. Furthermore, we discovered atosiban is an antagonist of the Tribolium receptor, which may provide a useful probe to investigate the functionality of inotocin signalling in beetles and related insect species. Our findings will enable further examination of insect inotocin receptor pharmacology and physiology, and may trigger studies to comprehend the interaction of plant cyclotides and insects.

The gut-brain peptide glucagon-like peptide-1 (GLP-1) reduces reward from palatable food and drugs of abuse. Recent rodent studies show that activation of GLP-1 receptors (GLP-1R) within the nucleus of the solitary tract (NTS) not only suppresses the motivation and intake of palatable food, but also reduces alcohol-related behaviors. As reward induced by addictive drugs and sexual behaviors involve similar neurocircuits, we hypothesized that activation of GLP-1R suppresses sexual behavior in sexually naïve male mice. We initially identified that systemic administration of the GLP-1R agonist, exendin-4 (Ex4), decreased the frequency and duration of mounting behaviors, but did not alter the preference for females or female bedding. Thereafter infusion of Ex4 into the NTS decreased various behaviors of the sexual interaction chain, namely social, mounting and self-grooming behaviors. In male mice tested in the sexual interaction test, NTS-Ex4 increased dopamine turnover and enhanced serotonin levels in the nucleus accumbens (NAc). In addition, these mice displayed higher corticosterone, but not testosterone, levels in plasma. Finally, GLP-1R antagonist, exendin-3 (9-39) amide (Ex9), infused into the NTS differentially altered the ability of systemic-Ex4 to suppress the various behaviors of the sexual interaction chain, indicating that GLP-1R within the NTS is one of many sub-regions contributing to the GLP-1 dependent sexual behavior link. In these mice NTS-Ex9 partly blocked the systemic-Ex4 enhancement of corticosterone levels. Collectively, these data highlight that activation of GLP-1R, specifically those in the NTS, reduces sexual interaction behaviors in sexually naïve male mice and further provide a link between NTS-GLP-1R activation and reward-related behaviors.

Glucagon-like peptide-1 receptor (GLP-1R) agonists, such as exendin-4 (Ex4), liraglutide and dulaglutide, regulate glucose homeostasis and are thus used to treat diabetes type II. GLP-1 also contributes towards a variety of additional physiological functions, including suppression of reward and improvement of learning. Acute activation of GLP-1R in the nucleus accumbens (NAc) shell, an area essential for motivation, reduces the motivation to consume sucrose or alcohol when assessed in a simple motor task. However, the effects of repeated administration of the different GLP-1R agonists on behaviours in a more complex motor task are unknown. The aim was therefore to investigate the effects of repeated Ex4, liraglutide or dulaglutide on the motivation and learning of a complex motor tasks such as skilled reach foraging in the Montoya staircase test. To explore the neurophysiological correlates of the different GLP-1R agonists on motivation, ex vivo electrophysiological recordings were conducted. In rats with an acquired skilled reach performance, Ex4 or liraglutide but not dulaglutide reduced the motivation of skilled reach foraging. In trained rats, Ex4 infusion into NAc shell decreased this motivated behaviour, and both Ex4 and liraglutide supressed the evoked field potentials in NAc shell. In rats without prior Montoya experience, dulaglutide but not Ex4 or liraglutide enhanced the learning of skilled reach foraging. Taken together, these findings indicate that the tested GLP-1R agonists have different behavioural outcomes depending on the context.

Atrial- and brain-type natriuretic peptides (ANP and BNP, respectively) have been shown to exert potent lipolytic action in adipocytes. A family of natriuretic peptide receptors (NPRs), NPR-1, NPR-2, and NPR-3, mediates their physiologic effects. NPR-1 and NPR-2 are receptor guanylyl cyclases, while NPR-3 lacks enzymatic activity and functions primarily as a clearance receptor for natriuretic peptides. ANP has a high affinity for NPR-1 and NPR-3 than other natriuretic peptides. There is a possibility that ANP may exhibit its lipolytic effect through the balance of NPR-1 and NPR-3 expressions in adipocytes. However, the regulation of adipose NPRs has not been fully elucidated. We here examined the regulation of mouse adipose NPRs by insulin, an anti-lipolytic hormone. Among the insulin target organs, NPR-1 mRNA levels were higher in white adipose tissue (WAT) than in liver and skeletal muscle. NPR-3 mRNA was expressed most abundantly in WAT. Fasting condition induced NPR-1 mRNA level while suppressed NPR-3 mRNA level in WAT. Administration of streptozotocin resulted in the increase of NPR-1 mRNA level while the decrease of NPR-3 mRNA level in WAT. In ob/ob mice, hyperinsulinemic model, NPR-1 mRNA level was lower whereas NPR-3 mRNA level was higher compared to lean control mice. In 3T3-L1 adipocytes, insulin significantly reduced NPR-1 mRNA level while increased NPR-3 mRNA levels both through phosphatidylinositol 3-kinase (PI3-kinase) pathway. In summary, NPR-1 and NPR-3 were highly expressed in WAT and adipose NPR-1 and NPR-3 were reciprocally regulated by insulin. This study suggests that insulin may efficiently promote lipogenesis partly by reducing the lipolytic action of ANP through the opposite regulation of NPR-1 and NPR-3.

The formyl peptide receptor (Fpr) family is well known for its contribution to immune defense against pathogens in human and rodent leukocytes. Recently, several structurally related members of these receptors were discovered in sensory neurons of the mouse vomeronasal organ (VNO), key detectors of pheromones and related semiochemicals. Although the biological role of vomeronasal Fprs is not yet clear, the known contribution of other Fprs to host immune defense suggested that they could contribute to vomeronasal pathogen sensing. Precise knowledge about the agonist properties of mouse Fprs is required to determine their function. We expressed all seven mouse and three human Fprs using an in vitro system and tested their activation with 32 selected compounds by conducting high throughput calcium measurements. We found an intriguing functional conservation between human and mouse immune Fprs that is most likely a consequence of closely similar biological constraints. By contrast, our data suggest a neofunctionalization of the vomeronasal Fprs. We show that the vomeronasal receptor mFpr-rs1 can be activated robustly by W-peptide and structural derivatives but not by other typical ligands of immune Fprs. mFpr-rs1 exhibits a stereo-selective preference for peptides containing d-amino acids. The same peptide motifs are contained in pathogenic microorganisms. Thus, the ligand profile of mFpr-rs1 is consistent with a role in vomeronasal pathogen sensing.

CART (cocaine- and amphetamine-regulated transcript) peptide is a neuropeptide with a powerful central anorexigenic effect. Specific CART peptide binding sites, most likely CART peptide receptors, have been found in PC12 cells. This study further characterizes the CART peptide binding sites in PC12 cells. After differentiation to a neuronal phenotype with nerve growth factor, the number of CART peptide binding sites in PC12 cells tripled. Following dexamethasone treatment, which transforms PC12 cells into chromaffin-like cells, the number of CART peptide binding sites substantially decreased. CART peptide did not affect the differentiation or acetylcholinesterase activity of PC12 cells, indicating that CART peptide does not participate in differentiation or neuronal activity. CART peptide increased the phosphorylation of SAPK/JNK (stress-activated protein kinase/c-Jun-amino-terminal kinase) and subsequent c-Jun protein expression. These effects were reversed by SP600125, a specific JNK-kinase inhibitor. CART peptide did not significantly affect ERK (extracellular signal-regulated kinase), CREB (cAMP responsive element binding protein), or p38 phosphorylation and c-Fos protein expression. Central administration of CART peptide into mice also resulted in increased c-Jun positive cells in dorsomedial hypothalamic nucleus and nucleus of the solitary tract, areas involved in food intake regulation. Activation of c-Jun by CART peptide might indicate a possible role of CART peptide in managing stress conditions rather than a role in cell proliferation or differentiation as well as the more complex and/or specific regulation ways by transcription factors in some nuclei involved in food intake regulation. The characteristics of stress that CART peptide potentially mediates should be further studied.

Annexin A1 (ANXA1, lipocortin-1) is a glucocorticoid-regulated 37-kDa protein, so called since its main property is to bind (i.e. to annex) to cellular membranes in a Ca(2+)-dependent manner. Although ANXA1 has predominantly been studied in the context of immune responses and cancer, the protein can affect a larger variety of biological phenomena, including cell proliferation and migration. Our previous results show that endogenous ANXA1 positively modulates myoblast cell differentiation by promoting migration of satellite cells and, consequently, skeletal muscle differentiation. In this work, we have evaluated the hypothesis that ANXA1 is able to exert effects on myoblast cell migration acting through formyl peptide receptors (FPRs) following changes in its subcellular localization as in other cell types and tissues. The analysis of the subcellular localization of ANXA1 in C2C12 myoblasts during myogenic differentiation showed an interesting increase of extracellular ANXA1 starting from the initial phases of skeletal muscle cell differentiation. The investigation of intracellular Ca(2+) perturbation following exogenous administration of the ANXA1 N-terminal derived peptide Ac2-26 established the engagement of the FPRs which expression in C2C12 cells was assessed by qualitative PCR. Wound healing assay experiments showed that Ac2-26 peptide is able to increase migration of C2C12 skeletal muscle cells and to induce cell surface translocation and secretion of ANXA1. Our results suggest a role for ANXA1 as a highly versatile component in the signaling chains triggered by the proper calcium perturbation that takes place during active migration and differentiation or membrane repair since the protein is strongly redistributed onto the plasma membranes after an rapid increase of intracellular levels of Ca(2+). These properties indicate that ANXA1 may be involved in a novel repair mechanism for skeletal muscle and may have therapeutic implications with respect to the development of ANXA1 mimetics.

To summarize the pharmacology of the calcitonin peptide family of receptors and explore their relationship to migraine and current migraine therapies.

Disruption of the circadian rhythm can lead to obesity and cardiovascular disease. In white adipose tissue, activation of the natriuretic peptide receptors (NPRs) stimulates lipolysis. We have previously shown that natriuretic peptides are expressed in a circadian manner in the heart, but the temporal expression profile of their cognate receptors has not been examined in white adipose tissue. We therefore collected peri-renal white adipose tissue and serum from WT mice. Tissue mRNA contents of NPRs - NPR-A and NPR-C, the clock genes Per1 and Bmal1, and transcripts involved in lipid metabolism were quantified at 4-h intervals: in the diurnal study, mice were exposed to a period of 12 h light followed by 12 h darkness (n=52). In the circadian study, mice were kept in darkness for 24 h (n=47). Concomitant serum concentrations of free fatty acids, glycerol, triglycerides (TGs), and insulin were measured. Per1 and Bmal1 mRNA contents showed reciprocal circadian profiles (P<0.0001). NPR-A mRNA contents followed a temporal pattern (P=0.01), peaking in the dark (active) period. In contrast, NPR-C mRNA was expressed in an antiphase manner with nadir in the active period (P=0.007). TG concentrations in serum peaked in the active dark period (P=0.003). In conclusion, NPR-A and NPR-C gene expression is associated with the expression of clock genes in white adipose tissue. The reciprocal expression may thus contribute to regulate lipolysis and energy homeostasis in a diurnal manner.