Adhesion G protein-coupled receptors (aGPCR) are unique molecules. They are able to transmit classical signals via G-protein activation as well as mediate functions solely through their extracellular N termini, completely independently of the seven transmembrane helices domain (7TM) and the C terminus. This dual mode of action is highly unusual for GPCRs and allows for a plethora of possible cellular consequences. However, the physiological implications and molecular details of this N terminus-mediated signaling are poorly understood. Here, we show that several distinct 7TM-independent/trans functions of the aGPCR Latrophilin homolog LAT-1 in the nematode Caenorhabditis elegans together regulate reproduction: sperm guidance, ovulation, and germ cell apoptosis. In these contexts, the receptor elicits its functions in a non-cell autonomous manner. The functions might be realized through alternative splicing of the receptor specifically generating N terminus-only variants. Thus, our findings shed light on the versatility of 7TM-independent/N terminus-only/trans functions of aGPCR and discuss possible molecular details.

Latrophilins (LPHNs), a group of the G-protein-coupled receptor to which a spider venom latrotoxin (LTX) is known to bind, remain largely uncharacterized in neoplastic diseases. In the present study, we aimed to determine the role of LPHNs in the progression of prostate cancer. We assessed the actions of LPHNs, including LPHN1, LPHN2, and LPHN3, in human prostate cancer lines via their ligand (e.g., α-LTX, FLRT3) treatment or shRNA infection, as well as in surgical specimens. In androgen receptor (AR)-positive LNCaP/C4-2/22Rv1 cells, dihydrotestosterone considerably increased the expression levels of LPHNs, while chromatin immunoprecipitation assay revealed the binding of endogenous ARs, including AR-V7, to the promoter region of each LPHN. Treatment with α-LTX or FLRT3 resulted in induction in the cell viability and migration of both AR-positive and AR-negative lines. α-LTX and FLRT3 also enhanced the expression of Bcl-2 and phosphorylated forms of JAK2 and STAT3. Meanwhile, the knockdown of each LPHN showed opposite effects on all of those mediated by ligand treatment. Immunohistochemistry in radical prostatectomy specimens further showed the significantly elevated expression of each LPHN in prostate cancer, compared with adjacent normal-appearing prostate, which was associated with a significantly higher risk of postoperative biochemical recurrence in both univariate and multivariable settings. These findings indicate that LPHNs function as downstream effectors of ARs and promote the growth of androgen-sensitive, castration-resistant, or even AR-negative prostate cancer.

Endothelial cell responses to fluid shear stress from blood flow are crucial for vascular development, function, and disease. A complex of PECAM-1, VE-cadherin, VEGF receptors (VEGFRs), and Plexin D1 located at cell-cell junctions mediates many of these events. However, available evidence suggests that another mechanosensor upstream of PECAM-1 initiates signaling. Hypothesizing that GPCR and Gα proteins may serve this role, we performed siRNA screening of Gα subunits and found that Gαi2 and Gαq/11 are required for activation of the junctional complex. We then developed a new activation assay, which showed that these G proteins are activated by flow. We next mapped the Gα residues required for activation and developed an affinity purification method that used this information to identify latrophilin-2 (Lphn2/ADGRL2) as the upstream GPCR. Latrophilin-2 is required for all PECAM-1 downstream events tested. In both mice and zebrafish, latrophilin-2 is required for flow-dependent angiogenesis and artery remodeling. Furthermore, endothelial-specific knockout demonstrates that latrophilin plays a role in flow-dependent artery remodeling. Human genetic data reveal a correlation between the latrophilin-2-encoding Adgrl2 gene and cardiovascular disease. Together, these results define a pathway that connects latrophilin-dependent G protein activation to subsequent endothelial signaling, vascular physiology, and disease.

Latrophilin-1 (Lphn1, aka CIRL1 and CL1; gene symbol Adgrl1) is an adhesion GPCR that has been implicated in excitatory synaptic transmission as a candidate receptor for α-latrotoxin. Here we analyzed conditional knock-in/knock-out mice for Lphn1 that contain an extracellular myc epitope tag. Mice of both sexes were used in all experiments. Surprisingly, we found that Lphn1 is localized in cultured neurons to synaptic nanoclusters that are present in both excitatory and inhibitory synapses. Conditional deletion of Lphn1 in cultured neurons failed to elicit a detectable impairment in excitatory synapses but produced a decrease in inhibitory synapse numbers and synaptic transmission that was most pronounced for synapses close to the neuronal soma. No changes in axonal or dendritic outgrowth or branching were observed. Our data indicate that Lphn1 is among the few postsynaptic adhesion molecules that are present in both excitatory and inhibitory synapses and that Lphn1 by itself is not essential for excitatory synaptic transmission but is required for some inhibitory synaptic connections.

Latrophilins are adhesion G-protein coupled receptors (aGPCRs) that control excitatory synapse formation. Most aGPCRs, including latrophilins, are autoproteolytically cleaved at their GPCR-autoproteolysis inducing (GAIN) domain, but the two resulting fragments remain noncovalently associated on the cell surface. Force-mediated dissociation of the fragments is thought to activate G-protein signaling, but how this mechanosensitivity arises is poorly understood. Here, we use magnetic tweezer assays to show that physiologically relevant forces in the 1-10 pN range lead to dissociation of the latrophilin-3 GAIN domain on the seconds-to-minutes time scale, compared to days in the absence of force. In addition, we find that the GAIN domain undergoes large changes in length in response to increasing mechanical load. These data are consistent with a model in which a force-sensitive equilibrium between compact and extended GAIN domain states precedes dissociation, suggesting a mechanism by which latrophilins and other aGPCRs may mediate mechanically induced signal transduction.

Latrophilin is a member of adhesion GPCRs involved in various physiological pro1cesses. The extracellular fragment of Tribolium castaneum Latrophilin (TcLph) contains a galactose-binding lectin (GBL) domain. However, the biological function of GBL domain remains mysterious. Here, we initially studied the role of TcLph in recognizing pathogens through its GBL domain and then triggering immune defense in invertebrates. Results showed that GBL domain was highly conserved, and its predicted 3D structure was similar to rhamnose-binding lectin domain of mouse Latrophilin-1 with a unique α/β fold and two long loops. Molecular docking and ELISA results revealed the GBL domain can bind to D-galactose, L-rhamnose, lipopolysaccharide and peptidoglycan. The recombinant extracellular segment of TcLph and the recombinant GBL exhibited strong agglutinating and binding activities to all tested bacteria in a Ca2+-dependent manner. Moreover, TcLph was markedly induced after infection by Escherichia coli or Staphylococcus aureus, while its silencing exacerbated bacterial loads and larvae mortality. TcLph-deficient larvae significantly decreased the transcription levels of antimicrobial peptides and prophenoloxidase activating system-related genes, leading to a significant reduction in phenoloxidase activity. It indicated that TcLph functioned as a pattern recognition receptor in pathogen recognition and activated immune responses to eliminate invasive microbes, suggesting a potential target for insecticides.

During nervous system development, neurons choose synaptic partners with remarkable specificity; however, the cell-cell recognition mechanisms governing rejection of inappropriate partners remain enigmatic. Here, we show that mouse retinal neurons avoid inappropriate partners by using the FLRT2-uncoordinated-5 (UNC5) receptor-ligand system. Within the inner plexiform layer (IPL), FLRT2 is expressed by direction-selective (DS) circuit neurons, whereas UNC5C/D are expressed by non-DS neurons projecting to adjacent IPL sublayers. In vivo gain- and loss-of-function experiments demonstrate that FLRT2-UNC5 binding eliminates growing DS dendrites that have strayed from the DS circuit IPL sublayers. Abrogation of FLRT2-UNC5 binding allows mistargeted arbors to persist, elaborate, and acquire synapses from inappropriate partners. Conversely, UNC5C misexpression within DS circuit sublayers inhibits dendrite growth and drives arbors into adjacent sublayers. Mechanistically, UNC5s promote dendrite elimination by interfering with FLRT2-mediated adhesion. Based on their broad expression, FLRT-UNC5 recognition is poised to exert widespread effects upon synaptic partner choices across the nervous system.

Neuronally orchestrated muscular movement and locomotion are defining faculties of multicellular animals. Due to its simple brain and genetic accessibility, the larva of the fruit fly Drosophila melanogaster allows one to study these processes at tractable levels of complexity. However, although the faculty of locomotion clearly pertains to the individual, most studies of locomotion in larvae use measurements aggregated across animals, or animals tested one by one, an extravagance for larger-scale analyses. This prevents grasping the inter- and intra-individual variability in locomotion and its neurogenetic determinants. Here, we present the IMBA (individual maggot behaviour analyser) for analysing the behaviour of individual larvae within groups, reliably resolving individual identity across collisions. We use the IMBA to systematically describe the inter- and intra-individual variability in locomotion of wild-type animals, and how the variability is reduced by associative learning. We then report a novel locomotion phenotype of an adhesion GPCR mutant. We further investigated the modulation of locomotion across repeated activations of dopamine neurons in individual animals, and the transient backward locomotion induced by brief optogenetic activation of the brain-descending \'mooncrawler\' neurons. In summary, the IMBA is an easy-to-use toolbox allowing an unprecedentedly rich view of the behaviour and its variability of individual larvae, with utility in multiple biomedical research contexts.

The formation of neural circuits requires extensive interactions of cell-surface proteins to guide axons to their correct target neurons. Trans-cellular interactions of the adhesion G protein-coupled receptor latrophilin-2 (Lphn2) with its partner teneurin-3 instruct the precise assembly of hippocampal networks by reciprocal repulsion. Lphn2 acts as a repulsive receptor in distal CA1 neurons to direct their axons to the proximal subiculum, and as a repulsive ligand in the proximal subiculum to direct proximal CA1 axons to the distal subiculum. It remains unclear if Lphn2-mediated intracellular signaling is required for its role in either context. Here, we show that Lphn2 couples to Gα12/13 in heterologous cells; this coupling is increased by constitutive exposure of the tethered agonist. Specific mutations of Lphn2\'s tethered agonist region disrupt its G protein coupling and autoproteolytic cleavage, whereas mutating the autoproteolytic cleavage site alone prevents cleavage but preserves a functional tethered agonist. Using an in vivo misexpression assay, we demonstrate that wild-type Lphn2 misdirects proximal CA1 axons to the proximal subiculum and that Lphn2 tethered agonist activity is required for its role as a repulsive receptor in axons. By contrast, neither tethered agonist activity nor autoproteolysis were necessary for Lphn2\'s role as a repulsive ligand in the subiculum target neurons. Thus, tethered agonist activity is required for Lphn2-mediated neural circuit assembly in a context-dependent manner.
The complex brain circuits that allow animals to sense and interact with their environment start to form early during development. Throughout this period, neurons extend fiber-like projections to establish precise wiring patterns. Various types of proteins at the surface of both incoming fibers and target cells ensure that only the right partners will connect together. Latrophilin-2, for example, is a neuronal surface protein essential for the formation of accurate connections in the hippocampus, a brain region important for memory. Studded through the membrane of certain neurons, it acts as a signal-sending ligand to direct incoming fibers, with neurons that carry Latrophilin-2 repelling projections from cells that display certain protein partners. At the same time, Latrophilin-2 also allows neurons to receive chemical signals by working with intracellular signaling proteins known as G proteins, which help to relay information between cells. It remained unclear how this role as a signalling receptor participates in the wiring of the hippocampus during development. To explore this question, Pederick, Perry-Hauser et al. examined the impact of Latrophilin-2 on the connection patterns of mouse hippocampal neurons that do not normally carry this protein. Introducing Latrophilin-2 into these ‘proximal CA1 cells’ misdirected them away from their usual partners – unless Latrophilin-2 was altered so that it could not interact with G proteins. In contrast, forcing the connecting partners of CA1 cells to display normal or altered versions of Latrophilin-2 did not interfere with the protein acting as a repulsive ligand. Taken together, these results suggest that the ability of Latrophilin-2 to signal through G proteins is important for neurons that are attempting to project their fibers onto other cells, but not important when Latrophilin-2 acts in targets to direct incoming fibers from other neurons. These results show that a single protein can shape neural circuits by acting both as a signal-receiving receptor and a signal-sending ligand depending on the context. In the future, Pederick, Perry-Hauser et al. hope that their findings will shed new light on how the wiring of the brain is disrupted in neurodevelopmental disorders.

Neural activity-dependent synaptic plasticity is an important physiological phenomenon underlying environmental adaptation, memory and learning. However, its molecular basis, especially in presynaptic neurons, is not well understood. Previous studies have shown that the number of presynaptic active zones in the Drosophila melanogaster photoreceptor R8 is reversibly changed in an activity-dependent manner. During reversible synaptic changes, both synaptic disassembly and assembly processes were observed. Although we have established a paradigm for screening molecules involved in synaptic stability and several genes have been identified, genes involved in stimulus-dependent synaptic assembly are still elusive. Therefore, the aim of this study was to identify genes regulating stimulus-dependent synaptic assembly in Drosophila using an automated synapse quantification system. To this end, we performed RNAi screening against 300 memory-defective, synapse-related or transmembrane molecules in photoreceptor R8 neurons. Candidate genes were narrowed down to 27 genes in the first screen using presynaptic protein aggregation as a sign of synaptic disassembly. In the second screen, we directly quantified the decreasing synapse number using a GFP-tagged presynaptic protein marker. We utilized custom-made image analysis software, which automatically locates synapses and counts their number along individual R8 axons, and identified cirl as a candidate gene responsible for synaptic assembly. Finally, we present a new model of stimulus-dependent synaptic assembly through the interaction of cirl and its possible ligand, ten-a. This study demonstrates the feasibility of using the automated synapse quantification system to explore activity-dependent synaptic plasticity in Drosophila R8 photoreceptors in order to identify molecules involved in stimulus-dependent synaptic assembly.