Dose-Dependent Effects of Lipopolysaccharide on the Endothelium-Sepsis versus Metabolic Endotoxemia-Induced Cellular Senescence.

The endothelium, the innermost cell layer of blood vessels, is not only a physical barrier between the bloodstream and the surrounding tissues but has also essential functions in vascular homeostasis. Therefore, it is not surprising that endothelial dysfunction is associated with most cardiovascular diseases. The functionality of the endothelium is compromised by endotoxemia, the presence of bacterial endotoxins in the bloodstream with the main endotoxin lipopolysaccharide (LPS). Therefore, this review will focus on the effects of LPS on the endothelium. Depending on the LPS concentration, the outcomes are either sepsis or, at lower concentrations, so-called low-dose or metabolic endotoxemia. Sepsis, a life-threatening condition evoked by hyperactivation of the immune response, includes breakdown of the endothelial barrier resulting in failure of multiple organs. A deeper understanding of the underlying mechanisms in the endothelium might help pave the way to new therapeutic options in sepsis treatment to prevent endothelial leakage and fatal septic shock. Low-dose endotoxemia or metabolic endotoxemia results in chronic inflammation leading to endothelial cell senescence, which entails endothelial dysfunction and thus plays a critical role in cardiovascular diseases. The identification of compounds counteracting senescence induction in endothelial cells might therefore help in delaying the onset or progression of age-related pathologies. Interestingly, two natural plant-derived substances, caffeine and curcumin, have shown potential in preventing endothelial cell senescence.

Dysfunction of the adhesion G protein-coupled receptor latrophilin 1 (ADGRL1/LPHN1) increases the risk of obesity.

Obesity is one of the diseases with severe health consequences and rapidly increasing worldwide prevalence. Understanding the complex network of food intake and energy balance regulation is an essential prerequisite for pharmacological intervention with obesity. G protein-coupled receptors (GPCRs) are among the main modulators of metabolism and energy balance. They, for instance, regulate appetite and satiety in certain hypothalamic neurons, as well as glucose and lipid metabolism and hormone secretion from adipocytes. Mutations in some GPCRs, such as the melanocortin receptor type 4 (MC4R), have been associated with early-onset obesity. Here, we identified the adhesion GPCR latrophilin 1 (ADGRL1/LPHN1) as a member of the regulating network governing food intake and the maintenance of energy balance. Deficiency of the highly conserved receptor in mice results in increased food consumption and severe obesity, accompanied by dysregulation of glucose homeostasis. Consistently, we identified a partially inactivating mutation in human ADGRL1/LPHN1 in a patient suffering from obesity. Therefore, we propose that LPHN1 dysfunction is a risk factor for obesity development.

The adhesion GPCR and PCP component flamingo (FMI-1) alters body size and regulates the composition of the extracellular matrix.

The extracellular matrix (ECM) is a network of macromolecules that presents a vital scaffold for cells and enables multiple ways of cellular communication. Thus, it is essential for many physiological processes such as development, tissue morphogenesis, homeostasis, the shape and partially the size of the body and its organs. To ensure these, the composition of the ECM is tissue-specific and highly dynamic. ECM homeostasis is therefore tightly controlled by several mechanisms. Here, we show that FMI-1, the homolog of the Adhesion GPCR Flamingo/CELSR/ADGRC in the nematode Caenorhabditis elegans, modulates the composition of the ECM by controlling the production both of ECM molecules such as collagens and also of ECM modifying enzymes. Thereby, FMI-1 affects the morphology and functionality of the nematode´s cuticle, which is mainly composed of ECM, and also modulates the body size. Mechanistic analyses highlight the fact that FMI-1 exerts its function from neurons non-cell autonomously (trans) solely via its extracellular N terminus. Our data support a model, by which the activity of the receptor, which has a well-described role in the planar cell polarity (PCP) pathway, involves the PCP molecule VANG-1, but seems to be independent of the DBL-1/BMP pathway.

NanoBRET in C. elegans illuminates functional receptor interactions in real time.

BACKGROUND: Protein-protein interactions form the basis of every organism and thus, investigating their dynamics, intracellular protein localization, trafficking and interactions of distinct proteins such as receptors and their ligand-binding are of general interest. Bioluminescence resonance energy transfer (BRET) is a powerful tool to investigate these aspects in vitro. Since in vitro approaches mostly neglect the more complex in vivo situation, we established BRET as an in vivo tool for studying protein interactions in the nematode C. elegans. RESULTS: We generated worms expressing NanoBRET sensors and elucidated the interaction of two ligand-G protein-coupled receptor (GPCR) pairs, the neuropeptide receptor NPR-11 and the Adhesion GPCR LAT-1. Furthermore, we adapted the enhanced bystander BRET technology to measure subcellular protein localization. Using this approach, we traced ligand-induced internalization of NPR-11 in vivo. CONCLUSIONS: Our results indicate that in vivo NanoBRET is a tool to investigate specific protein interactions and localization in a physiological setting in real time in the living organism C. elegans.

A guide to adhesion GPCR research.

Adhesion G protein-coupled receptors (aGPCRs) are a class of structurally and functionally highly intriguing cell surface receptors with essential functions in health and disease. Thus, they display a vastly unexploited pharmacological potential. Our current understanding of the physiological functions and signaling mechanisms of aGPCRs form the basis for elucidating further molecular aspects. Combining these with novel tools and methodologies from different fields tailored for studying these unusual receptors yields a powerful potential for pushing aGPCR research from singular approaches toward building up an in-depth knowledge that will facilitate its translation to applied science. In this review, we summarize the state-of-the-art knowledge on aGPCRs in respect to structure-function relations, physiology, and clinical aspects, as well as the latest advances in the field. We highlight the upcoming most pressing topics in aGPCR research and identify strategies to tackle them. Furthermore, we discuss approaches how to promote, stimulate, and translate research on aGPCRs 'from bench to bedside' in the future.

The relevance of adhesion G protein-coupled receptors in metabolic functions.

G protein-coupled receptors (GPCRs) modulate a variety of physiological functions and have been proven to be outstanding drug targets. However, approximately one-third of all non-olfactory GPCRs are still orphans in respect to their signal transduction and physiological functions. Receptors of the class of Adhesion GPCRs (aGPCRs) are among these orphan receptors. They are characterized by unique features in their structure and tissue-specific expression, which yields them interesting candidates for deorphanization and testing as potential therapeutic targets. Capable of G-protein coupling and non-G protein-mediated function, aGPCRs may extend our repertoire of influencing physiological function. Besides their described significance in the immune and central nervous systems, growing evidence indicates a high importance of these receptors in metabolic tissue. RNAseq analyses revealed high expression of several aGPCRs in pancreatic islets, adipose tissue, liver, and intestine but also in neurons governing food intake. In this review, we focus on aGPCRs and their function in regulating metabolic pathways. Based on current knowledge, this receptor class represents high potential for future pharmacological approaches addressing obesity and other metabolic diseases.

Latrophilin-1 drives neuron morphogenesis and shapes chemo- and mechanosensation-dependent behavior in C. elegans via a trans function.

Latrophilins are highly conserved Adhesion GPCRs playing essential roles in the mammalian nervous system and are associated with severe neurological disorders. Recently, it has been shown that murine Latrophilins mediate classical G-protein signals to drive synaptogenesis. However, there is evidence that Latrophilins in the nematode Caenorhabditis elegans can also function independently of their seven-transmembrane domain and C terminus (trans function). Here, we show that Latrophilin-1 acts in trans to mediate morphogenesis of sensory structures in the C. elegans nervous system. This trans function is physiologically relevant in copulation behavior. Detailed expression and RNA-Seq analyses revealed specific LAT-1-positive neurons and first insights into the genetic network that is modulated by the receptor function. We conclude that 7TM-independent functions of Latrophilins are essential for neuronal physiology, possibly complementing canonical functions via G protein-mediated signaling.

The Evolutionary History of Vertebrate Adhesion GPCRs and Its Implication on Their Classification.

Adhesion G protein-coupled receptors (aGPCRs) form a structurally separate class of GPCRs with an unresolved evolutionary history and classification. Based on phylogenetic relations of human aGPCRs, nine families (A-G, L, V) were distinguished. Taking advantage of available genome data, we determined the aGPCR repertoires in all vertebrate classes. Although most aGPCR families show a high numerical stability in vertebrate genomes, the full repertoire of family E, F, and G members appeared only after the fish-tetrapod split. We did not find any evidence for new aGPCR families in vertebrates which are not present in the human genome. Based on ortholog sequence alignments, selection analysis clearly indicated two types of tetrapod aGPCRs: (i) aGPCR under strong purifying selection in tetrapod evolution (families A, B, D, L, V); and (ii) aGPCR with signatures of positive selection in some tetrapod linages (families C, E, G, F). The alignments of aGPCRs also allowed for a revised definition of reference positions within the seven-transmembrane-helix domain (relative position numbering scheme). Based on our phylogenetic cluster analysis, we suggest a revised nomenclature of aGPCRs including their transcript variants. Herein, the former families E and L are combined to one family (L) and GPR128/ADGRG7 forms a separate family (E). Furthermore, our analyses provide valuable information about the (patho)physiological relevance of individual aGPCR members.

Structural Perspective on Ancient Neuropeptide Y-like System reveals Hallmark Features for Peptide Recognition and Receptor Activation.

The neuropeptide Y (NPY) family is a peptide-activated G protein-coupled receptor system conserved across all bilaterians, and is involved in food intake, learning, and behavior. We hypothesized that comparing the NPY system in evolutionarily ancient organisms can reveal structural determinants of peptide recognition and receptor activation conserved in evolution. To test this hypothesis, we investigated the homologous FLP/NPR system of the protostome C.elegans. For three prototypic peptide-receptor complexes representing different ligand types, we integrate extensive functional data into structural models of the receptors. Common features include acidic patches in the extracellular loops (ECLs) of the receptors that cooperatively 'draw' the peptide into the binding pocket, which was functionally validated in vivo. A structurally conserved glutamate in the ECL2 anchors the peptides by a conserved salt bridge to the arginine of the RFamide motif. Beyond this conserved interaction, peptide binding show variability enabled by receptor-specific interactions. The family-conserved residue Q3.32 is a key player for peptide binding and receptor activation. Altered interaction patterns at Q3.32 may drastically increase the efficacy to activate the receptor.

Pharmacological and functional similarities of the human neuropeptide Y system in C. elegans challenges phylogenetic views on the FLP/NPR system.

BACKGROUND: The neuropeptide Y system affects various processes, among others food intake, and is frequently discussed in the context of targeting obesity. Studies in model organisms are indispensable to enable molecular studies in a physiological context. Although the NPY system is evolutionarily conserved in all bilaterians, in the widely used model Caenorhabditis elegans there is controversy on the existence of NPY orthologous molecules. While the FMRFamide-like peptide (FLP)/Neuropeptide receptor-Resemblance (NPR) system in the nematode was initially suggested to be orthologous to the mammalian NPY system, later global phylogenetic studies indicate that FLP/NPR is protostome-specific. METHODS: We performed a comprehensive pharmacological study of the FLP/NPR system in transfected cells in vitro, and tested for functional substitution in C. elegans knockout strains. Further, we phenotypically compared different flp loss-of-function strains. Differences between groups were compared by ANOVA and post-hoc testing (Dunnett, Bonferroni). RESULTS: Our pharmacological analysis of the FLP/NPR system including formerly functionally uncharacterized NPY-like peptides from C. elegans demonstrates that G protein-coupling and ligand requirements for receptor activation are similar to the human NPY system. In vitro and in vivo analyses show cross-reactivity of NPY with the FLP/NPR system manifesting in the ability of the human GPCRs to functionally substitute FLP/NPR signaling in vivo. The high pharmacological/functional similarities enabled us to identify C. elegans FLP-14 as a key molecule in avoidance behavior. CONCLUSIONS: Our data demonstrate the pharmacological and functional similarities of human NPY and C. elegans NPR systems. This adds a novel perspective to current phylogenetic reconstructions of the neuropeptide Y system. NPY and NPR receptors are pharmacologically so similar that the human receptors can functionally compensate for the C. elegans ones, suggesting orthologous relationships. This is also underlined by the presence of NPY-like peptides and parallels in peptide requirements for receptor activation. Further, the results presented here highlight the potential of this knowledge for physiological as well as molecular studies on neuropeptide GPCRs such as the NPY system in the future.

Large-scale cultivation of Caenorhabditis elegans in a bioreactor using a labor-friendly fed-batch approach.

Caenorhabditis elegans is an invertebrate model organism used in many areas of biology including developmental biology and the identification of molecular mechanisms and pathways. However, several experimental approaches require large quantities of worms, which is limiting and time-consuming. We present a protocol that uses modern fermentation methodology to effectively produce large numbers of C. elegans using a 7-l bioreactor in a fed-batch cultivation procedure. The production is modular and flexible as well as being a self-controlled system, thus not much labor is required until harvesting C. elegans. The high-yield worm cultivation is flexible and simple to amend, and now allows for the extended application of C. elegans as a model organism and expression system, including large-scale protein production.

The expanding functional roles and signaling mechanisms of adhesion G protein-coupled receptors.

The adhesion class of G protein-coupled receptors (GPCRs) is the second largest family of GPCRs (33 members in humans). Adhesion GPCRs (aGPCRs) are defined by a large extracellular N-terminal region that is linked to a C-terminal seven transmembrane (7TM) domain via a GPCR-autoproteolysis inducing (GAIN) domain containing a GPCR proteolytic site (GPS). Most aGPCRs undergo autoproteolysis at the GPS motif, but the cleaved fragments stay closely associated, with the N-terminal fragment (NTF) bound to the 7TM of the C-terminal fragment (CTF). The NTFs of most aGPCRs contain domains known to be involved in cell-cell adhesion, while the CTFs are involved in classical G protein signaling, as well as other intracellular signaling. In this workshop report, we review the most recent findings on the biology, signaling mechanisms, and physiological functions of aGPCRs.

Latrophilins and Teneurins in Invertebrates: No Love for Each Other?

Transsynaptic connections enabling cell-cell adhesion and cellular communication are a vital part of synapse formation, maintenance and function. A recently discovered interaction between the Adhesion GPCRs Latrophilins and the type II single transmembrane proteins Teneurins at mammalian synapses is vital for synapse formation and dendrite branching. While the understanding of the effects and the molecular interplay of this Latrophilin-Teneurin partnership is not entirely understood, its significance is highlighted by behavioral and neurological phenotypes in various animal models. As both groups of molecules, Latrophilins and Teneurins, are generally highly conserved, have overlapping expression and often similar functions across phyla, it can be speculated that this interaction, which has been proven essential in mammalian systems, also occurs in invertebrates to control shaping of synapses. Knowledge of the generality of this interaction is especially of interest due to its possible involvement in neuropathologies. Further, several invertebrates serve as model organisms for addressing various neurobiological research questions. So far, an interaction of Latrophilins and Teneurins has not been observed in invertebrates, but our knowledge on both groups of molecules is by far not complete. In this review, we give an overview on existing experimental evidence arguing for as well as against a potential Latrophilin-Teneurin interaction beyond mammals. By combining these insights with evolutionary aspects on each of the interaction partners we provide and discuss a comprehensive picture on the functions of both molecules in invertebrates and the likeliness of an evolutionary conservation of their interaction.

Involvement of the Adhesion GPCRs Latrophilins in the Regulation of Insulin Release.

Insulin secretion from pancreatic ß cells is a highly complex and tightly regulated process. Its dysregulation is one characteristic of type 2 diabetes, and thus, an in-depth understanding of the mechanisms controlling insulin secretion is essential for rational therapeutic intervention. G-protein-coupled receptors (GPCRs) have been established as major regulators of insulin exocytosis. Recent studies also suggest the involvement of adhesion GPCRs, a non-prototypical class of GPCRs. Here, we identify latrophilins, which belong to the class of adhesion GPCRs, to be highly expressed in different cell types of pancreatic islets. In vitro and ex vivo analyses show that distinct splice variants of the latrophilin LPHN3/ADGRL3 decrease insulin secretion from pancreatic ß cells by reducing intracellular cyclic AMP levels via the Gi-mediated pathway. Our data highlight the key role of LPHN3 in modulating insulin secretion and its potential as therapeutic target for type 2 diabetes.

G Proteins and GPCRs in C. elegans Development: A Story of Mutual Infidelity.

Many vital processes during C. elegans development, especially the establishment and maintenance of cell polarity in embryogenesis, are controlled by complex signaling pathways. G protein-coupled receptors (GPCRs), such as the four Frizzled family Wnt receptors, are linchpins in regulating and orchestrating several of these mechanisms. However, despite being GPCRs, which usually couple to G proteins, these receptors do not seem to activate classical heterotrimeric G protein-mediated signaling cascades. The view on signaling during embryogenesis is further complicated by the fact that heterotrimeric G proteins do play essential roles in cell polarity during embryogenesis, but their activity is modulated in a predominantly GPCR-independent manner via G protein regulators such as GEFs GAPs and GDIs. Further, the triggered downstream effectors are not typical. Only very few GPCR-dependent and G protein-mediated signaling pathways have been unambiguously defined in this context. This unusual and highly intriguing concept of separating GPCR function and G-protein activity, which is not restricted to embryogenesis in C. elegans but can also be found in other organisms, allows for essential and multi-faceted ways of regulating cellular communication and response. Although its relevance cannot be debated, its impact is still poorly discussed, and C. elegans is an ideal model to understand the underlying principles.

A Comprehensive Mutagenesis Screen of the Adhesion GPCR Latrophilin-1/ADGRL1.

Adhesion G-protein-coupled receptors (aGPCRs) play critical roles in diverse cellular processes in neurobiology, development, immunity, and numerous diseases. The lack of molecular understanding of their activation mechanisms, especially with regard to the transmembrane domains, hampers further studies to facilitate aGPCR-targeted drug development. Latrophilin-1/ADGRL1 is a model aGPCR that regulates synapse formation and embryogenesis, and its mutations are associated with cancer and attention-deficit/hyperactivity disorder. Here, we established functional assays to monitor latrophilin-1 function and showed the activation of latrophilin-1 by its endogenous agonist peptide. Via a comprehensive mutagenesis screen, we identified transmembrane domain residues essential for latrophilin-1 basal activity and for agonist peptide response. Strikingly, a cancer-associated mutation exhibited increased basal activity and failed to rescue the embryonic developmental phenotype in transgenic worms. These results provide a mechanistic foundation for future aGPCR-targeted drug design.

Mechano-dependent signaling by Latrophilin/CIRL quenches cAMP in proprioceptive neurons.

Adhesion-type G protein-coupled receptors (aGPCRs), a large molecule family with over 30 members in humans, operate in organ development, brain function and govern immunological responses. Correspondingly, this receptor family is linked to a multitude of diverse human diseases. aGPCRs have been suggested to possess mechanosensory properties, though their mechanism of action is fully unknown. Here we show that the Drosophila aGPCR Latrophilin/dCIRL acts in mechanosensory neurons by modulating ionotropic receptor currents, the initiating step of cellular mechanosensation. This process depends on the length of the extended ectodomain and the tethered agonist of the receptor, but not on its autoproteolysis, a characteristic biochemical feature of the aGPCR family. Intracellularly, dCIRL quenches cAMP levels upon mechanical activation thereby specifically increasing the mechanosensitivity of neurons. These results provide direct evidence that the aGPCR dCIRL acts as a molecular sensor and signal transducer that detects and converts mechanical stimuli into a metabotropic response.

Activation of Adhesion G Protein-coupled Receptors: AGONIST SPECIFICITY OF STACHEL SEQUENCE-DERIVED PEPTIDES.

Members of the adhesion G protein-coupled receptor (aGPCR) family carry an agonistic sequence within their large ectodomains. Peptides derived from this region, called the Stachel sequence, can activate the respective receptor. As the conserved core region of the Stachel sequence is highly similar between aGPCRs, the agonist specificity of Stachel sequence-derived peptides was tested between family members using cell culture-based second messenger assays. Stachel peptides derived from aGPCRs of subfamily VI (GPR110/ADGRF1, GPR116/ADGRF5) and subfamily VIII (GPR64/ADGRG2, GPR126/ADGRG6) are able to activate more than one member of the respective subfamily supporting their evolutionary relationship and defining them as pharmacological receptor subtypes. Extended functional analyses of the Stachel sequences and derived peptides revealed agonist promiscuity, not only within, but also between aGPCR subfamilies. For example, the Stachel-derived peptide of GPR110 (subfamily VI) can activate GPR64 and GPR126 (both subfamily VIII). Our results indicate that key residues in the Stachel sequence are very similar between aGPCRs allowing for agonist promiscuity of several Stachel-derived peptides. Therefore, aGPCRs appear to be pharmacologically more closely related than previously thought. Our findings have direct implications for many aGPCR studies, as potential functional overlap has to be considered for in vitro and in vivo studies. However, it also offers the possibility of a broader use of more potent peptides when the original Stachel sequence is less effective.

Advantages and Limitations of Salmon-Gal/Tetrazolium Salt Histochemistry for the Detection of LacZ Reporter Gene Activity in Murine Epithelial Tissue.

The Escherichia coli LacZ gene is a widely used reporter for gene regulation studies in transgenic mice. It encodes bacterial ß-galactosidase (Bact ß-Gal), which causes insoluble precipitates when exposed to chromogenic homologues of galactose. We and others have recently reported that Bact ß-Gal detection with Salmon-Gal (S-Gal) in combination with nitro blue tetrazolium chloride (NBT) is very sensitive and not prone to interference by acidic endogenous ß-galactosidases. Unfortunately, as we show here, the method appears to be inadequate for evaluation of Bact ß-Gal expression in keratinized epithelial appendages but not in other keratinized epithelia. NBT in the reaction mixture, just as other tetrazolium salts, inevitably causes unwanted staining artifacts in lingual filiform papillae, penile spines, and hair fibers by interacting with keratin sulfhydryl-rich regions. The methodological limitation can be overcome in part by pretreating the tissues before the S-Gal/NBT staining with an iodine-potassium iodide solution. Alternatively, the use of iodonitrotetrazolium chloride instead of NBT in the S-Gal reaction mixture provides enough color resolution to distinguish the specific Bact ß-Gal staining in orange from the artifact staining in dark red. In summary, we provide evidence that S-Gal/NBT histochemistry has limitations, when staining keratinized epithelial appendages.

Adhesion GPCRs Govern Polarity of Epithelia and Cell Migration.

In multicellular organisms cells spatially arrange in a highly coordinated manner to form tissues and organs, which is essential for the function of an organism. The component cells and resulting structures are often polarised in one or more axes, and how such polarity is established and maintained correctly has been one of the major biological questions for many decades. Research progress has shown that many adhesion GPCRs (aGPCRs) are involved in several types of polarity. Members of the two evolutionarily oldest groups, Flamingo/Celsr and Latrophilins, are key molecules in planar cell polarity of epithelia or the propagation of cellular polarity in the early embryo, respectively. Other adhesion GPCRs play essential roles in cell migration, indicating that this receptor class includes essential molecules for the control of various levels of cellular organisation.