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ADGRL1/latrophilin-1, a well-characterized adhesion G protein-coupled receptor, has been implicated in synaptic development, maturation and activity. However, the role of ADGRL1 in human disease has been elusive. Here, we describe 10 individuals with variable neurodevelopmental features including developmental delay, intellectual disability, attention deficit hyperactivity and autism spectrum disorders, and epilepsy, all featuring heterozygous variants in ADGRL1. In vitro, human ADGRL1 variants expressed in neuroblastoma cells showed faulty ligand-induced regulation of intracellular Ca2+ influx, consistent with haploinsufficiency. In vivo, Adgrl1 was knocked out in mice and studied on two genetic backgrounds. On a non-permissive background, mice carrying a heterozygous Adgrl1 null allele exhibited neurological and developmental abnormalities while homozygous mice were non-viable. On a permissive background, the null allele also appeared at sub-Mendelian frequency, but many Adgrl1 null mice survived the gestation and reached adulthood. The Adgrl1-/- mice demonstrated stereotypic behaviors, sexual dysfunction, bimodal extremes of locomotion, augmented startle reflex and attenuated pre-pulse inhibition, which responded to risperidone. Ex vivo synaptic preparations displayed increased spontaneous exocytosis of dopamine, acetylcholine and glutamate, but Adgrl1-/- neurons formed synapses in vitro poorly. Overall, our findings demonstrate that ADGRL1 haploinsufficiency leads to consistent developmental, neurological and behavioral abnormalities in mice and humans.

Latrophilin-1 (LPHN1) was isolated as the main high-affinity receptor for α-latrotoxin from black widow spider venom, a powerful presynaptic secretagogue. As an adhesion G-protein-coupled receptor, LPHN1 is cleaved into two fragments, which can behave independently on the cell surface, but re-associate upon binding the toxin. This triggers intracellular signaling that involves the Gαq/phospholipase C/inositol 1,4,5-trisphosphate cascade and an increase in cytosolic Ca2+, leading to vesicular exocytosis. Using affinity chromatography on LPHN1, we isolated its endogenous ligand, teneurin-2/Lasso. Both LPHN1 and Ten2/Lasso are expressed early in development and are enriched in neurons. LPHN1 primarily resides in axons, growth cones and presynaptic terminals, while Lasso largely localizes on dendrites. LPHN1 and Ten2/Lasso form a trans-synaptic receptor pair that has both structural and signaling functions. However, Lasso is proteolytically cleaved at multiple sites and its extracellular domain is partially released into the intercellular space, especially during neuronal development, suggesting that soluble Lasso has additional functions. We discovered that the soluble fragment of Lasso can diffuse away and bind to LPHN1 on axonal growth cones, triggering its redistribution on the cell surface and intracellular signaling which leads to local exocytosis. This causes axons to turn in the direction of spatio-temporal Lasso gradients, while LPHN1 knockout blocks this effect. These results suggest that the LPHN1-Ten2/Lasso pair can participate in long- and short-distance axonal guidance and synapse formation.

A presynaptic adhesion G-protein-coupled receptor, latrophilin-1, and a postsynaptic transmembrane protein, Lasso/teneurin-2, are implicated in trans-synaptic interaction that contributes to synapse formation. Surprisingly, during neuronal development, a substantial proportion of Lasso is released into the intercellular space by regulated proteolysis, potentially precluding its function in synaptogenesis. We found that released Lasso binds to cell-surface latrophilin-1 on axonal growth cones. Using microfluidic devices to create stable gradients of soluble Lasso, we show that it induces axonal attraction, without increasing neurite outgrowth. Using latrophilin-1 knockout in mice, we demonstrate that latrophilin-1 is required for this effect. After binding latrophilin-1, Lasso causes downstream signaling, which leads to an increase in cytosolic calcium and enhanced exocytosis, processes that are known to mediate growth cone steering. These findings reveal a novel mechanism of axonal pathfinding, whereby latrophilin-1 and Lasso mediate both short-range interaction that supports synaptogenesis, and long-range signaling that induces axonal attraction., eLife digest The brain is a complex mesh of interconnected neurons, with each cell making tens, hundreds, or even thousands of connections. These links can stretch over long distances, and establishing them correctly during development is essential. Developing neurons send out long and thin structures, called axons, to reach distant cells. To guide these growing axons, neurons release molecules that work as traffic signals: some attract axons whilst others repel them, helping the burgeoning structures to twist and turn along their travel paths. When an axon reaches its target cell, the two cells join to each other by forming a structure called a synapse. To make the connection, surface proteins on the axon latch onto matching proteins on the target cell, zipping up the synapse. There are many different types of synapses in the brain, but we only know a few of the surface molecules involved in their creation – not enough to explain synaptic variety. Two of these surface proteins are latrophilin-1, which is produced by the growing axon, and Lasso, which sits on the membrane of the target cell. The two proteins interact strongly, anchoring the axon to the target cell and allowing the synapse to form. However, a previous recent discovery by Vysokov et al. has revealed that an enzyme can also cut Lasso from the membrane of the target cell. The ‘free’ protein can still interact with latrophilin-1, but as it is shed by the target cell, it can no longer serve as an anchor for the synapse. Could it be that free Lasso acts as a traffic signal instead? Here, Vysokov et al. tried to answer this by growing neurons from a part of the brain called the hippocampus in a special labyrinth dish. When free Lasso was gradually introduced in the culture through microscopic channels, it interacted with latrophilin-1 on the surface of the axons. This triggered internal changes that led the axons to add more membrane where they had sensed Lasso, making them grow towards the source of the signal. The results demonstrate that a target cell can both carry and release Lasso, using this duplicitous protein to help attract growing axons as well as anchor them. The work by Vysokov et al. contributes to our knowledge of how neurons normally connect, which could shed light on how this process can go wrong. This may be relevant to understand conditions such as schizophrenia and ADHD, where patients’ brains often show incorrect wiring.

Teneurins are large cell-surface receptors involved in axon guidance. Teneurin-2 (also known as latrophilin-1-associated synaptic surface organizer (Lasso)) interacts across the synaptic cleft with presynaptic latrophilin-1, an adhesion G-protein-coupled receptor that participates in regulating neurotransmitter release. Lasso-latrophilin-1 interaction mediates synapse formation and calcium signaling, highlighting the important role of this trans-synaptic receptor pair. However, Lasso is thought to be proteolytically cleaved within its ectodomain and released into the medium, making it unclear whether it acts as a proper cell-surface receptor or a soluble protein. We demonstrate here that during its intracellular processing Lasso is constitutively cleaved at a furin site within its ectodomain. The cleaved fragment, which encompasses almost the entire ectodomain of Lasso, is potentially soluble; however, it remains anchored on the cell surface via its non-covalent interaction with the transmembrane fragment of Lasso. Lasso is also constitutively cleaved within the intracellular domain (ICD). Finally, Lasso can be further proteolytically cleaved within the transmembrane domain. The third cleavage is regulated and releases the entire ectodomain of Lasso into the medium. The released ectodomain of Lasso retains its functional properties and binds latrophilin-1 expressed on other cells; this binding stimulates intracellular Ca(2+) signaling in the target cells. Thus, Lasso not only serves as a bona fide cell-surface receptor, but also as a partially released target-derived signaling factor.

The unusual adhesion G-protein-coupled receptors (aGPCRs) contain large extracellular N-terminal domains, which resemble cell-adhesion receptors, and C-terminal heptahelical domains, which may couple to G-proteins. These receptors are cleaved post-translationally between these domains into two fragments (NTF and CTF). Using the aGPCR latrophilin 1, we previously demonstrated that the fragments behave as independent cell-surface proteins. Upon binding the agonist, α-latrotoxin (LTX), latrophilin fragments reassemble and induce intracellular signaling. Our observations raised important questions: is the aGPCR signaling mediated by reassembled fragments or by any non-cleaved receptors? Also, can the fragments originating from distinct aGPCRs form hybrid complexes? To answer these questions, we created two types of chimerical constructs. One contained the CTF of latrophilin joined to the NTF of another aGPCR, EMR2; the resulting protein did not bind LTX but, similar to latrophilin, could couple to G-proteins. In another construct, the NTF of latrophilin was fused with the C terminus of neurexin; this chimera bound LTX but could not signal via G-proteins. Both constructs were efficiently cleaved in cells. When the two constructs were co-expressed, their fragments could cross-interact, as shown by immunoprecipitation. Furthermore, LTXN4C induced intracellular Ca2+ signaling only in cells expressing both constructs but not each individual construct. Finally, we demonstrated that fragments of unrelated aGPCRs can be cross-immunoprecipitated from live tissues. Thus, (i) aGPCR fragments behave as independent proteins, (ii) the complementary fragments from distinct aGPCRs can cross-interact, and (iii) these cross-complexes are functionally active. This unusual cross-assembly of aGPCR fragments could couple cell-surface interactions to multiple signaling pathways.

Heptahelical, or G-protein-coupled, receptors control many cellular functions and normally consist of one polypeptide chain. In contrast, heptahelical receptors that belong to the long N-terminus, group B (LNB) family are cleaved constitutively into two fragments. The N-terminal fragments (NTFs) resemble cell-adhesion proteins and the C-terminal fragments (CTFs) are typical G-protein-coupled receptors (GPCRs) with seven transmembrane regions. However, the functional roles of this cleavage and of any subsequent NTF-CTF interactions remain to be identified. Using latrophilin, a well-studied member of the LNB family, we now demonstrate that cleavage is critical for delivery of this receptor to the cell surface. On the plasma membrane, NTF and CTF behave as separate membrane proteins involved, respectively, in cell-surface reception and signalling. The two fragments can also internalise independently. However, separated NTF and CTF can re-associate on solubilisation. Agonist binding to NTF on the cell surface also induces re-association of fragments and provokes signal transduction via CTF. These findings define a novel principle of structural and functional organisation of the cleaved, two-subunit GPCRs.

Pure alpha-latrotoxin is very inefficient at forming channels/pores in artificial lipid bilayers or in the plasma membrane of non-secretory cells. However, the toxin induces pores efficiently in COS-7 cells transfected with the heptahelical receptor latrophilin or the monotopic receptor neurexin. Signaling-deficient (truncated) mutants of latrophilin and latrophilin-neurexin hybrids also facilitate pore induction, which correlates with toxin binding irrespective of receptor structure. This rules out the involvement of signaling in pore formation. With any receptor, the alpha-latrotoxin pores are permeable to Ca(2+) and small molecules including fluorescein isothiocyanate and norepinephrine. Bound alpha-latrotoxin remains on the cell surface without penetrating completely into the cytosol. Higher temperatures facilitate insertion of the toxin into the plasma membrane, where it co-localizes with latrophilin (under all conditions) and with neurexin (in the presence of Ca(2+)). Interestingly, on subsequent removal of Ca(2+), alpha-latrotoxin dissociates from neurexin but remains in the membrane and continues to form pores. These receptor-independent pores are inhibited by anti-alpha-latrotoxin antibodies. Our results indicate that (i) alpha-latrotoxin is a pore-forming toxin, (ii) receptors that bind alpha-latrotoxin facilitate its insertion into the membrane, (iii) the receptors are not physically involved in the pore structure, (iv) alpha-latrotoxin pores may be independent of the receptors, and (v) pore formation does not require alpha-latrotoxin interaction with other neuronal proteins.

In order to explore the mechanisms by which alpha-latrotoxin activates neurotransmitter release, we have characterized its effects by patch-clamp methods on cells heterologously expressing its receptors, latrophilin-1 or neurexin-Ialpha. Application of alpha-latrotoxin (1 nM) to cells expressing rat latrophilin or neurexin, but not mock-transfected cells, induced a cationic conductance. In cells expressing latrophilin, current development was slow in the absence of divalent cations, but was accelerated by Ca2+ or Mg2+. In cells expressing neurexin, alpha-latrotoxin did not elicit currents in the absence of Ca2+. The toxin-induced conductance was rectifying, persistent, permeable to monovalent and divalent cations, but blocked by La3+. Single-channel recording revealed a permanently open state, with the same unitary conductance irrespective of whether cells expressed latrophilin or neurexin. Therefore, while pore formation displayed differences consistent with the reported properties of alpha-latrotoxin binding to latrophilin and neurexin, the pores induced by alpha-latrotoxin had identical properties. These results suggest that after anchoring to either of its nerve terminal receptors, alpha-latrotoxin inserts into the membrane and constitutes a single type of transmembrane ion pore.

alpha-Latrotoxin, a black widow spider neurotoxin, can bind to high affinity receptors on the presynaptic plasma membrane and stimulate massive neurotransmitter release in the absence of Ca2+. Neurexins, previously isolated as alpha-latrotoxin receptors, require Ca2+ for their interaction with the toxin and, thus, may not participate in the Ca2+-independent alpha-latrotoxin activity. We now report the isolation of a novel protein that binds alpha-latrotoxin with high affinity in the presence of various divalent cations (Ca2+, Mg2+, Ba2+, and Sr2+) as well as in EDTA. This protein, termed here latrophilin, has been purified from detergent-solubilized bovine brain membranes by affinity chromatography on immobilized alpha-latrotoxin and concentrated on a wheat germ agglutinin affinity column. The single polypeptide chain of latrophilin is N-glycosylated and has an apparent molecular weight of 120,000. Sucrose gradient centrifugations demonstrated that latrophilin and alpha-latrotoxin form a stable equimolar complex. In the presence of the toxin, anti-alpha-latrotoxin antibodies precipitated iodinated latrophilin, whose binding to immobilized toxin was characterized by a dissociation constant of 0.5-0.7 nM. This presumably membrane-bound protein is localized to and differentially distributed among neuronal tissues, with about four times more latrophilin expressed in the cerebral cortex than in the cerebellum; subcellular fractionation showed that the protein is highly enriched in synaptosomal plasma membranes. Our data suggest that latrophilin may represent the Ca2+-independent receptor and/or molecular target for alpha-latrotoxin.

Latrophilin 1 (LPH1), a neuronal receptor of α-latrotoxin, is implicated in neurotransmitter release and control of presynaptic Ca 2+ . As an “adhesion G-protein-coupled receptor,” LPH1 can convert cell surface interactions into intracellular signaling. To examine the physiological functions of LPH1, we used LPH1’s extracellular domain to purify its endogenous ligand. A single protein of ∼275 kDa was isolated from rat brain and termed Lasso. Peptide sequencing and molecular cloning have shown that Lasso is a splice variant of teneurin-2, a brain-specific orphan cell surface receptor with a function in neuronal pathfinding and synaptogenesis. We show that LPH1 and Lasso interact strongly and specifically. They are always copurified from rat brain extracts. Coculturing cells expressing LPH1 with cells expressing Lasso leads to their mutual attraction and formation of multiple junctions to which both proteins are recruited. Cells expressing LPH1 form chimerical synapses with hippocampal neurons in cocultures; LPH1 and postsynaptic neuronal protein PSD-95 accumulate on opposite sides of these structures. Immunoblotting and immunoelectron microscopy of purified synapses and immunostaining of cultured hippocampal neurons show that LPH1 and Lasso are enriched in synapses; in both systems, LPH1 is presynaptic, whereas Lasso is postsynaptic. A C-terminal fragment of Lasso interacts with LPH1 and induces Ca 2+ signals in presynaptic boutons of hippocampal neurons and in neuroblastoma cells expressing LPH1. Thus, LPH1 and Lasso can form transsynaptic complexes capable of inducing presynaptic Ca 2+ signals, which might affect synaptic functions.

alpha-Latrotoxin stimulates three types of [(3)H]gamma-aminobutyric acid and [(14)C]glutamate release from synaptosomes. The Ca(2+)-independent component (i) is insensitive to SNAP-25 cleavage or depletion of vesicle contents by bafilomycin A1 and represents transmitter efflux mediated by alpha-latrotoxin pores. Two other components of release are Ca(2+)-dependent and vesicular but rely on distinct mechanisms. The fast receptor-mediated pathway (ii) involves intracellular Ca(2+) stores and acts upon sucrose-sensitive readily releasable vesicles; this mechanism is insensitive to inhibition of phosphatidylinositol 4-kinase (PI 4-kinase). The delayed pore-dependent exocytotic component (iii) is stimulated by Ca(2+) entering through alpha-latrotoxin pores; it requires PI 4-kinase and occurs mainly from depot vesicles. Lanthanum perturbs alpha-latrotoxin pores and blocks the two pore-mediated components (i, iii) but not the receptor-mediated release (ii). alpha-Latrotoxin mutant (LTX(N4C)) cannot form pores and stimulates only the Ca(2+)-dependent receptor-mediated amino acid exocytosis (ii) (detectable biochemically and electrophysiologically). These findings explain experimental data obtained by different laboratories and implicate the toxin receptors in the regulation of the readily releasable pool of synaptic vesicles. Our results also suggest that, similar to noradrenergic vesicles, amino acid-containing vesicles at some point in their cycle require PI 4-kinase.

Latrophilin is a brain-specific Ca2+-independent receptor of α-latrotoxin, a potent presynaptic neurotoxin. We now report the finding of two novel latrophilin homologues. All three latrophilins are unusual G protein-coupled receptors. They exhibit strong similarities within their lectin, olfactomedin and transmembrane domains but possess variable C-termini. Latrophilins have up to seven sites of alternative splicing; some splice variants contain an altered third cytoplasmic loop or a truncated cytoplasmic tail. Only latrophilin-1 binds α-latrotoxin; it is abundant in brain and is present in endocrine cells. Latrophilin-3 is also brain-specific, whereas latrophilin-2 is ubiquitous. Together, latrophilins form a novel family of heterogeneous G protein-coupled receptors with distinct tissue distribution and functions.

α‐Latrotoxin (LTX) stimulates massive neurotransmitter release by two mechanisms: Ca 2+ ‐dependent and ‐independent. Our studies on norepinephrine secretion from nerve terminals now reveal the different molecular basis of these two actions. The Ca 2+ ‐dependent LTX‐evoked vesicle exocytosis (abolished by botulinum neurotoxins) is 10‐fold more sensitive to external Ca 2+ than secretion triggered by depolarization or A23187; it does not, however, depend on the cation entry into terminals but requires intracellular Ca 2+ and is blocked by drugs depleting Ca 2+ stores and by inhibitors of phospholipase C (PLC). These data, together with binding studies, prove that latrophilin, which is linked to G proteins and inositol polyphosphate production, is the major functional LTX receptor. The Ca 2+ ‐independent LTX‐stimulated release is not inhibited by botulinum neurotoxins or drugs interfering with Ca 2+ metabolism and occurs via pores in the presynaptic membrane, large enough to allow efflux of neurotransmitters and other small molecules from the cytoplasm. Our results unite previously contradictory data about the toxin9s effects and suggest that LTX‐stimulated exocytosis depends upon the co‐operative action of external and intracellular Ca 2+ involving G proteins and PLC, whereas the Ca 2+ ‐independent release is largely non‐vesicular.