Molecular sensing of mechano- and ligand-dependent adhesion GPCR dissociation.

Adhesion G-protein-coupled receptors (aGPCRs) bear notable similarity to Notch proteins1, a class of surface receptors poised for mechano-proteolytic activation2-4, including an evolutionarily conserved mechanism of cleavage5-8. However, so far there is no unifying explanation for why aGPCRs are autoproteolytically processed. Here we introduce a genetically encoded sensor system to detect the dissociation events of aGPCR heterodimers into their constituent N-terminal and C-terminal fragments (NTFs and CTFs, respectively). An NTF release sensor (NRS) of the neural latrophilin-type aGPCR Cirl (ADGRL)9-11, from Drosophila melanogaster, is stimulated by mechanical force. Cirl-NRS activation indicates that receptor dissociation occurs in neurons and cortex glial cells. The release of NTFs from cortex glial cells requires trans-interaction between Cirl and its ligand, the Toll-like receptor Tollo (Toll-8)12, on neural progenitor cells, whereas expressing Cirl and Tollo in cis suppresses dissociation of the aGPCR. This interaction is necessary to control the size of the neuroblast pool in the central nervous system. We conclude that receptor autoproteolysis enables non-cell-autonomous activities of aGPCRs, and that the dissociation of aGPCRs is controlled by their ligand expression profile and by mechanical force. The NRS system will be helpful in elucidating the physiological roles and signal modulators of aGPCRs, which constitute a large untapped reservoir of drug targets for cardiovascular, immune, neuropsychiatric and neoplastic diseases13.

Tethered agonist exposure in intact adhesion/class B2 GPCRs through intrinsic structural flexibility of the GAIN domain.

Adhesion G protein-coupled receptors (aGPCRs)/family B2 GPCRs execute critical tasks during development and the operation of organs, and their genetic lesions are associated with human disorders, including cancers. Exceptional structural aGPCR features are the presence of a tethered agonist (TA) concealed within a GPCR autoproteolysis-inducing (GAIN) domain and their non-covalent heteromeric two-subunit layout. How the TA is poised for activation while maintaining this delicate receptor architecture is central to conflicting signaling paradigms that either involve or exclude aGPCR heterodimer separation. We investigated this matter in five mammalian aGPCR homologs (ADGRB3, ADGRE2, ADGRE5, ADGRG1, and ADGRL1) and demonstrate that intact aGPCR heterodimers exist at the cell surface, that the core TA region becomes unmasked in the cleaved GAIN domain, and that intra-GAIN domain movements regulate the level of tethered agonist exposure, thereby likely controlling aGPCR activity. Collectively, these findings delineate a unifying mechanism for TA-dependent signaling of intact aGPCRs.

7TM domain structures of adhesion GPCRs: what's new and what's missing?

Adhesion-type G protein-coupled receptors (aGPCRs) have long resisted approaches to resolve the structural details of their heptahelical transmembrane (7TM) domains. Single-particle cryogenic electron microscopy (cryo-EM) has recently produced aGPCR 7TM domain structures for ADGRD1, ADGRG1, ADGRG2, ADGRG3, ADGRG4, ADGRG5, ADGRF1, and ADGRL3. We review the unique properties, including the position and conformation of their activating tethered agonist (TA) and signaling motifs within the 7TM bundle, that the novel structures have helped to identify. We also discuss questions that the kaleidoscope of novel aGPCR 7TM domain structures have left unanswered. These concern the relative positions, orientations, and interactions of the 7TM and GPCR autoproteolysis-inducing (GAIN) domains with one another. Clarifying their interplay remains an important goal of future structural studies on aGPCRs.

Unveiling Mechanical Activation: GAIN Domain Unfolding and Dissociation in Adhesion GPCRs.

Adhesion G protein-coupled receptors (aGPCRs) have extracellular regions (ECRs) containing GPCR autoproteolysis-inducing (GAIN) domains. The GAIN domain enables the ECR to self-cleave into N- and C-terminal fragments. However, the impact of force on the GAIN domain's conformation, critical for mechanosensitive aGPCR activation, remains unclear. Our study investigated the mechanical stability of GAIN domains in three aGPCRs (B, G, and L subfamilies) at a loading rate of 1 pN/s. We discovered that forces of a few piconewtons can destabilize the GAIN domains. In autocleaved aGPCRs ADGRG1/GPR56 and ADGRL1/LPHN1, these forces cause the GAIN domain detachment from the membrane-proximal Stachel sequence, preceded by partial unfolding. In noncleavable aGPCR ADGRB3/BAI3 and cleavage-deficient mutant ADGRG1/GPR56-T383G, complex mechanical unfolding of the GAIN domain occurs. Additionally, GAIN domain detachment happens during cell migration. Our findings support the mechanical activation hypothesis of aGPCRs, emphasizing the sensitivity of the GAIN domain structure and detachment to physiological force ranges.

The human cognition-enhancing CORD7 mutation increases active zone number and synaptic release.

Humans carrying the CORD7 (cone-rod dystrophy 7) mutation possess increased verbal IQ and working memory. This autosomal dominant syndrome is caused by the single-amino acid R844H exchange (human numbering) located in the 310 helix of the C2A domain of RIMS1/RIM1 (Rab3-interacting molecule 1). RIM is an evolutionarily conserved multi-domain protein and essential component of presynaptic active zones, which is centrally involved in fast, Ca2+-triggered neurotransmitter release. How the CORD7 mutation affects synaptic function has remained unclear thus far. Here, we established Drosophila melanogaster as a disease model for clarifying the effects of the CORD7 mutation on RIM function and synaptic vesicle release. To this end, using protein expression and X-ray crystallography, we solved the molecular structure of the Drosophila C2A domain at 1.92 Šresolution and by comparison to its mammalian homologue ascertained that the location of the CORD7 mutation is structurally conserved in fly RIM. Further, CRISPR/Cas9-assisted genomic engineering was employed for the generation of rim alleles encoding the R915H CORD7 exchange or R915E, R916E substitutions (fly numbering) to effect local charge reversal at the 310 helix. Through electrophysiological characterization by two-electrode voltage clamp and focal recordings we determined that the CORD7 mutation exerts a semi-dominant rather than a dominant effect on synaptic transmission resulting in faster, more efficient synaptic release and increased size of the readily releasable pool but decreased sensitivity for the fast calcium chelator BAPTA. In addition, the rim CORD7 allele increased the number of presynaptic active zones but left their nanoscopic organization unperturbed as revealed by super-resolution microscopy of the presynaptic scaffold protein Bruchpilot/ELKS/CAST. We conclude that the CORD7 mutation leads to tighter release coupling, an increased readily releasable pool size and more release sites thereby promoting more efficient synaptic transmitter release. These results strongly suggest that similar mechanisms may underlie the CORD7 disease phenotype in patients and that enhanced synaptic transmission may contribute to their increased cognitive abilities.

Publisher Correction: G12/13 is activated by acute tethered agonist exposure in the adhesion GPCR ADGRL3.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

G12/13 is activated by acute tethered agonist exposure in the adhesion GPCR ADGRL3.

The adhesion G-protein-coupled receptor (GPCR) latrophilin 3 (ADGRL3) has been associated with increased risk of attention deficit hyperactivity disorder (ADHD) and substance use in human genetic studies. Knockdown in multiple species leads to hyperlocomotion and altered dopamine signaling. Thus, ADGRL3 is a potential target for treatment of neuropsychiatric disorders that involve dopamine dysfunction, but its basic signaling properties are poorly understood. Identification of adhesion GPCR signaling partners has been limited by a lack of tools to acutely activate these receptors in living cells. Here, we design a novel acute activation strategy to characterize ADGRL3 signaling by engineering a receptor construct in which we could trigger acute activation enzymatically. Using this assay, we found that ADGRL3 signals through G12/G13 and Gq, with G12/13 the most robustly activated. Gα12/13 is a new player in ADGRL3 biology, opening up unexplored roles for ADGRL3 in the brain. Our methodological advancements should be broadly useful in adhesion GPCR research.

Adhesion G protein-coupled receptors in nervous system development and disease.

Members of the adhesion G protein-coupled receptor (aGPCR) class have emerged as crucial regulators of nervous system development, with important implications for human health and disease. In this Review, we discuss the current understanding of aGPCR functions during key steps in neural development, including cortical patterning, dendrite and synapse formation, and myelination. We focus on aGPCR modulation of cell-cell and cell-matrix interactions and signalling to control these varied aspects of neural development, and we discuss how impaired aGPCR function leads to neurological disease. We further highlight the emerging hypothesis that aGPCRs can be mechanically activated and the implications of this property in the nervous system.

Implications of the Sap47 null mutation for synapsin phosphorylation, longevity, climbing proficiency and behavioural plasticity in adult Drosophila.

The Sap47 gene of Drosophila melanogaster encodes a highly abundant 47 kDa synaptic vesicle-associated protein. Sap47 null mutants show defects in synaptic plasticity and larval olfactory associative learning but the molecular function of Sap47 at the synapse is unknown. We demonstrate that Sap47 modulates the phosphorylation of another highly abundant conserved presynaptic protein, synapsin. Site-specific phosphorylation of Drosophila synapsin has repeatedly been shown to be important for behavioural plasticity but it was not known where these phospho-synapsin isoforms are localized in the brain. Here, we report the distribution of serine-6-phosphorylated synapsin in the adult brain and show that it is highly enriched in rings of synapses in the ellipsoid body and in large synapses near the lateral triangle. The effects of knockout of Sap47 or synapsin on olfactory associative learning/memory support the hypothesis that both proteins operate in the same molecular pathway. We therefore asked if this might also be true for other aspects of their function. We show that knockout of Sap47 but not synapsin reduces lifespan, whereas knockout of Sap47 and synapsin, either individually or together, affects climbing proficiency, as well as plasticity in circadian rhythms and sleep. Furthermore, electrophysiological assessment of synaptic properties at the larval neuromuscular junction (NMJ) reveals increased spontaneous synaptic vesicle fusion and reduced paired pulse facilitation in Sap47 and synapsin single and double mutants. Our results imply that Sap47 and synapsin cooperate non-uniformly in the control of synaptic properties in different behaviourally relevant neuronal networks of the fruitfly.

International Union of Basic and Clinical Pharmacology. XCIV. Adhesion G protein-coupled receptors.

The Adhesion family forms a large branch of the pharmacologically important superfamily of G protein-coupled receptors (GPCRs). As Adhesion GPCRs increasingly receive attention from a wide spectrum of biomedical fields, the Adhesion GPCR Consortium, together with the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification, proposes a unified nomenclature for Adhesion GPCRs. The new names have ADGR as common dominator followed by a letter and a number to denote each subfamily and subtype, respectively. The new names, with old and alternative names within parentheses, are: ADGRA1 (GPR123), ADGRA2 (GPR124), ADGRA3 (GPR125), ADGRB1 (BAI1), ADGRB2 (BAI2), ADGRB3 (BAI3), ADGRC1 (CELSR1), ADGRC2 (CELSR2), ADGRC3 (CELSR3), ADGRD1 (GPR133), ADGRD2 (GPR144), ADGRE1 (EMR1, F4/80), ADGRE2 (EMR2), ADGRE3 (EMR3), ADGRE4 (EMR4), ADGRE5 (CD97), ADGRF1 (GPR110), ADGRF2 (GPR111), ADGRF3 (GPR113), ADGRF4 (GPR115), ADGRF5 (GPR116, Ig-Hepta), ADGRG1 (GPR56), ADGRG2 (GPR64, HE6), ADGRG3 (GPR97), ADGRG4 (GPR112), ADGRG5 (GPR114), ADGRG6 (GPR126), ADGRG7 (GPR128), ADGRL1 (latrophilin-1, CIRL-1, CL1), ADGRL2 (latrophilin-2, CIRL-2, CL2), ADGRL3 (latrophilin-3, CIRL-3, CL3), ADGRL4 (ELTD1, ETL), and ADGRV1 (VLGR1, GPR98). This review covers all major biologic aspects of Adhesion GPCRs, including evolutionary origins, interaction partners, signaling, expression, physiologic functions, and therapeutic potential.

Channelrhodopsin-2-XXL, a powerful optogenetic tool for low-light applications.

Channelrhodopsin-2 (ChR2) has provided a breakthrough for the optogenetic control of neuronal activity. In adult Drosophila melanogaster, however, its applications are severely constrained. This limitation in a powerful model system has curtailed unfolding the full potential of ChR2 for behavioral neuroscience. Here, we describe the D156C mutant, termed ChR2-XXL (extra high expression and long open state), which displays increased expression, improved subcellular localization, elevated retinal affinity, an extended open-state lifetime, and photocurrent amplitudes greatly exceeding those of all heretofore published ChR variants. As a result, neuronal activity could be efficiently evoked with ambient light and even without retinal supplementation. We validated the benefits of the variant in intact flies by eliciting simple and complex behaviors. We demonstrate efficient and prolonged photostimulation of monosynaptic transmission at the neuromuscular junction and reliable activation of a gustatory reflex pathway. Innate male courtship was triggered in male and female flies, and olfactory memories were written through light-induced associative training.

Effects of transgenic expression of botulinum toxins in Drosophila.

Clostridial neurotoxins (botulinum toxins and tetanus toxin) disrupt neurotransmitter release by cleaving neuronal SNARE proteins. We generated transgenic flies allowing for conditional expression of different botulinum toxins and evaluated their potential as tools for the analysis of synaptic and neuronal network function in Drosophila melanogaster by applying biochemical assays and behavioral analysis. On the biochemical level, cleavage assays in cultured Drosophila S2 cells were performed and the cleavage efficiency was assessed via western blot analysis. We found that each botulinum toxin cleaves its Drosophila SNARE substrate but with variable efficiency. To investigate the cleavage efficiency in vivo, we examined lethality, larval peristaltic movements and vision dependent motion behavior of adult Drosophila after tissue-specific conditional botulinum toxin expression. Our results show that botulinum toxin type B and botulinum toxin type C represent effective alternatives to established transgenic effectors, i.e. tetanus toxin, interfering with neuronal and non-neuronal cell function in Drosophila and constitute valuable tools for the analysis of synaptic and network function.

Adhesion GPCRs as a Putative Class of Metabotropic Mechanosensors.

Adhesion GPCRs as mechanosensors. Different aGPCR homologs and their cognate ligands have been described in settings, which suggest that they function in a mechanosensory capacity. For details, see text G protein-coupled receptors (GPCRs) constitute the most versatile superfamily of biosensors. This group of receptors is formed by hundreds of GPCRs, each of which is tuned to the perception of a specific set of stimuli a cell may encounter emanating from the outside world or from internal sources. Most GPCRs are receptive for chemical compounds such as peptides, proteins, lipids, nucleotides, sugars, and other organic compounds, and this capacity is utilized in several sensory organs to initiate visual, olfactory, gustatory, or endocrine signals. In contrast, GPCRs have only anecdotally been implicated in the perception of mechanical stimuli. Recent studies, however, show that the family of adhesion GPCRs (aGPCRs), which represents a large panel of over 30 homologs within the GPCR superfamily, displays molecular design and expression patterns that are compatible with receptivity toward mechanical cues (Fig. 1). Here, we review physiological and molecular principles of established mechanosensors, discuss their relevance for current research of the mechanosensory function of aGPCRs, and survey the current state of knowledge on aGPCRs as mechanosensing molecules.

Control of Adhesion GPCR Function Through Proteolytic Processing.

Proteolytic processing events in adhesion GPCRs. aGPCRs can undergo multiple autoproteolytic (red asterisks) and proteolytic processing events by exogenous proteases (yellow asterisks) that may be involved in signaling events of the receptors. Proteolytic processing is an unusual property of adhesion family G protein-coupled receptors (aGPCRs) that was observed upon their cloning and biochemical characterization.Ever since, much effort has been dedicated to delineate the mechanisms and requirements for cleavage events in the control of aGPCR function. Most notably, all aGPCRs possess a juxtamembrane protein fold, the GPCR autoproteolysis-inducing (GAIN) domain, which operates as an autoprotease for many aGPCR homologs investigated thus far. Analysis of its autoproteolytic reaction, the consequences for receptor fate and function, and the allocation of physiological effects to this peculiar feature of aGPCRs has occupied the experimental agenda of the aGPCR field and shaped our current understanding of the signaling properties and cell biological effects of aGPCRs. Interestingly, individual aGPCRs may undergo additional proteolytic steps, one of them resulting in shedding of the entire ectodomain that is secreted and can function independently. Here, we summarize the current state of knowledge on GAIN domain-mediated and GAIN domain-independent aGPCR cleavage events and their significance for the pharmacological and cellular actions of aGPCRs. Further, we compare and contrast the proteolytic profile of aGPCRs with known signaling routes that are governed through proteolysis of surface molecules such as the Notch and ephrin pathways.

Adhesion G Protein-Coupled Receptors: From In Vitro Pharmacology to In Vivo Mechanisms.

The adhesion family of G protein-coupled receptors (aGPCRs) comprises 33 members in humans. aGPCRs are characterized by their enormous size and complex modular structures. While the physiologic importance of many aGPCRs has been clearly demonstrated in recent years, the underlying molecular functions have only recently begun to be elucidated. In this minireview, we present an overview of our current knowledge on aGPCR activation and signal transduction with a focus on the latest findings regarding the interplay between ligand binding, mechanical force, and the tethered agonistic Stachel sequence, as well as implications on translational approaches that may derive from understanding aGPCR pharmacology.

Model Organisms in G Protein-Coupled Receptor Research.

The study of G protein-coupled receptors (GPCRs) has benefited greatly from experimental approaches that interrogate their functions in controlled, artificial environments. Working in vitro, GPCR receptorologists discovered the basic biologic mechanisms by which GPCRs operate, including their eponymous capacity to couple to G proteins; their molecular makeup, including the famed serpentine transmembrane unit; and ultimately, their three-dimensional structure. Although the insights gained from working outside the native environments of GPCRs have allowed for the collection of low-noise data, such approaches cannot directly address a receptor's native (in vivo) functions. An in vivo approach can complement the rigor of in vitro approaches: as studied in model organisms, it imposes physiologic constraints on receptor action and thus allows investigators to deduce the most salient features of receptor function. Here, we briefly discuss specific examples in which model organisms have successfully contributed to the elucidation of signals controlled through GPCRs and other surface receptor systems. We list recent examples that have served either in the initial discovery of GPCR signaling concepts or in their fuller definition. Furthermore, we selectively highlight experimental advantages, shortcomings, and tools of each model organism.

Experimental modulation of physiological force application on leg joint neurons in intact Drosophila melanogaster.

The study of how mechanical forces affect biological events in living tissue is important for the understanding of a multitude of physiogical and pathophysiological phenomena. However, these investigations are often impeded by insufficient knowledge about force parameters, inadequate experimental administration of force stimuli and lack of noninvasive means to record their molecular and cellular effects. We therefore introduced a procedure to study the impact of force stimulation on adhesion G-protein-coupled receptor dissociation in mechanosensory neurons. Here, we detail a procedure to harness the mechanical force spectrum that emerges during the natural flexion-extension cycle of the femorotibial joint of adult fruit flies (Drosophila melanogaster). Mechanical load generated during the joint's motion is transmitted to specialized mechanosensory neurons residing close to the joint axis, which serve as proprioceptive sensors in the peripheral nervous system of the animal. Temporary immobilization of the joint by a restraint made of a human hair allows for the observation of transgenic mechanosensitive reporters by using fluorescent readout in the neurons before, during and after cessation of mechanical stimulation. The assay harnesses physiologically adequate stimuli for joint flexion and extension, can be conducted noninvasively in live specimens and is compatible with various transgenic reporter systems beyond the initially conceived strategy and mechanobiological hypotheses tested. The application of the protocol requires knowledge in Drosophila genetics, husbandry and fluorescence imaging and micromanipulation skills. The experimental procedure can be completed in 10 h and requires an additional 30 min in advance for fly fixation and leg immobilization. The apple agar cooking and heptane glue preparation requires a maximum of 30 min on the day before the experiment is conducted.

The adhesion G-protein-coupled receptor mayo/CG11318 controls midgut development in Drosophila.

Adhesion G-protein-coupled receptors (aGPCRs) form a large family of cell surface molecules with versatile tasks in organ development. Many aGPCRs still await their functional and pharmacological deorphanization. Here, we characterized the orphan aGPCR CG11318/mayo of Drosophila melanogaster and found it expressed in specific regions of the gastrointestinal canal and anal plates, epithelial specializations that control ion homeostasis. Genetic removal of mayo results in tachycardia, which is caused by hyperkalemia of the larval hemolymph. The hyperkalemic effect can be mimicked by a raise in ambient potassium concentration, while normal potassium levels in mayoKO mutants can be restored by pharmacological inhibition of potassium channels. Intriguingly, hyperkalemia and tachycardia are caused non-cell autonomously through mayo-dependent control of enterocyte proliferation in the larval midgut, which is the primary function of this aGPCR. These findings characterize the ancestral aGPCR Mayo as a homeostatic regulator of gut development.

Characterization and functional study of a cluster of four highly conserved orphan adhesion-GPCR in mouse.

BACKGROUND: Adhesion G protein-coupled receptors (aGPCR) constitute a structurally and functionally diverse class of seven-transmembrane receptor proteins. Although for some of the members important roles in immunology, neurology, as well as developmental biology have been suggested, most receptors have been poorly characterized. RESULTS: We have studied evolution, expression, and function of an entire receptor group containing four uncharacterized aGPCR: Gpr110, Gpr111, Gpr115, and Gpr116. We show that the genomic loci of these four receptors are clustered tightly together in mouse and human genomes and that this cluster likely derives from a single common ancestor gene. Using transcriptional profiling on wild-type and knockout/LacZ reporter knockin mice strains, we have obtained detailed expression maps that show ubiquitous expression of Gpr116, co-expression of Gpr111 and Gpr115 in developing skin, and expression of Gpr110 in adult kidney. Loss of Gpr110, Gpr111, or Gpr115 function did not result in detectable defects, indicating that genes of this aGPCR group might function redundantly. CONCLUSIONS: The aGPCR cluster Gpr110, Gpr111, Gpr115, and Gpr116 developed from one common ancestor in vertebrates. Expression suggests a role in epithelia, and one can speculate about a possible redundant function of GPR111 and GPR115.

Adhesion GPCRs in glioblastoma revisited.

Glioblastoma is a devastating brain malignancy that has remained intractable to modern cancer treatments. Ravn-Boess et al.1 have discovered that the adhesion G protein-coupled receptor CD97/ADGRE5 contributes to glioblastogenesis and makes for an excellent molecular surface marker flagging the tumor cells.