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.

␣-Latrotoxin (LTX) stimulates massive exocytosis of synaptic vesicles and may help to elucidate the mechanism of regulation of neurosecretion. We have recently isolated latrophilin, the synaptic Ca 2؉ -independent LTX receptor. Now we demonstrate that latrophilin is a novel member of the secretin family of G protein-coupled receptors that are involved in secretion. Northern blot analysis shows that latrophilin message is present only in neuronal tissue. Upon expression in COS cells, the cloned protein is indistinguishable from brain latrophilin and binds LTX with high affinity. Latrophilin physically interacts with a G␣ o subunit of heterotrimeric G proteins, because the two proteins co-purify in a twostep affinity chromatography. Interestingly, extracellular domain of latrophilin is homologous to olfactomedin, a soluble neuronal protein thought to participate in odorant binding. Our findings suggest that latrophilin may bind unidentified endogenous ligands and transduce signals into nerve terminals, thus implicating G proteins in the control of synaptic vesicle exocytosis.

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 reassociate 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.

␣-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 Ca 2؉. Neurexins, previously isolated as ␣-latrotoxin receptors, require Ca 2؉ for their interaction with the toxin and, thus, may not participate in the Ca 2؉ -independent ␣-latrotoxin activity. We now report the isolation of a novel protein that binds ␣-latrotoxin with high affinity in the presence of various divalent cations (Ca 2؉ , Mg 2؉ , Ba 2؉ , and Sr 2؉ ) as well as in EDTA. This protein, termed here latrophilin, has been purified from detergent-solubilized bovine brain membranes by affinity chromatography on immobilized ␣-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 ␣-latrotoxin form a stable equimolar complex. In the presence of the toxin, anti-␣-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 Ca 2؉ -independent receptor and/or molecular target for ␣-latrotoxin.␣-Latrotoxin (LTX), 1 a neurotoxin from the black widow spider venom, potently stimulates neurotransmitter release from all vertebrate synapses tested (1). The toxin causes a massive discharge of synaptic vesicles (2) by acting upon nerve terminals hypothetically in two stages. Initially, it binds to a high affinity presynaptic receptor and then forms nonselective cation-permeable channels in the plasma membrane (reviewed in Ref.3). The subsequent entry of Ca 2ϩ through these channels triggers fast neurotransmitter release (4). However, accumulating evidence suggests that the mode of LTX action is more complex.First, studies in neuronal and PC12 cells demonstrate that the toxin-receptor interaction does not require Ca 2ϩ , although the removal of Ca 2ϩ appreciably decreases the binding (5, 6). This result is best explained by the existence of two classes of LTX receptors, Ca 2ϩ -dependent and independent, both presumably active when Ca 2ϩ is present. These receptor types possess similar high affinities to LTX (7) but display different toxin binding properties (e.g. the Ca 2ϩ -independent binding is more sensitive to high salt) (8). Furthermore, in at least one PC12 cell line only the Ca 2ϩ -independent binding was detectable (7), indicating that the heterogeneous LTX receptors are probably differentially regulated and, thus, may also be structurally different. Ca 2ϩ is also not essential during the second p...

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 toxin's effects and suggest that LTXstimulated 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.

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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 posttranslationally 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, ␣-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, LTX N4C induced intracellular Ca 2؉ 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.

Latrophilin is a brain-specific Ca P+ -independent receptor of K K-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 K K-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.z 1999 Federation of European Biochemical Societies.