Yuri A. Ushkaryov

CARTOGRAPHY OF NEUREXINS : MORE THAN 1000 ISOFORMS GENERATED BY ALTERNATIVE SPLICING AND EXPRESSED IN DISTINCT SUBSETS OF NEURONS

Neurexins, a family of cell surface proteins specific to brain, are transcribed from two promoters in three genes, resulting in three alpha- and three beta-neurexins. In situ hybridization revealed differential but overlapping distributions of neurexin isoforms in different classes of neurons. PCRs demonstrated that alpha-neurexins are alternatively spliced at five canonical positions, and beta-neurexins at two. Characterization of many independent bovine neurexin I alpha cDNAs suggests that different splice sites are used independently. This creates the potential to express more than 1000 distinct neurexin proteins in brain. The splicing pattern is conserved in rat and cow. Thus, in addition to somatic gene rearrangement (immunoglobulins and T cell receptors) and large gene families (odorant receptors), alternative splicing potentially represents a third mechanism for creating a large number of cell surface receptors that are expressed by specific subsets of cells.

α-Latrotoxin Receptor, Latrophilin, Is a Novel Member of the Secretin Family of G Protein-coupled Receptors

α-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 Ca2+-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 Gαosubunit of heterotrimeric G proteins, because the two proteins co-purify in a two-step 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.

VESICLE EXOCYTOSIS STIMULATED BY ALPHA -LATROTOXIN IS MEDIATED BY LATROPHILIN AND REQUIRES BOTH EXTERNAL AND STORED CA2+

α‐Latrotoxin (LTX) stimulates massive neurotransmitter release by two mechanisms: Ca2+‐dependent and ‐independent. Our studies on norepinephrine secretion from nerve terminals now reveal the different molecular basis of these two actions. The Ca2+‐dependent LTX‐evoked vesicle exocytosis (abolished by botulinum neurotoxins) is 10‐fold more sensitive to external Ca2+ than secretion triggered by depolarization or A23187; it does not, however, depend on the cation entry into terminals but requires intracellular Ca2+ and is blocked by drugs depleting Ca2+ 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 Ca2+‐independent LTX‐stimulated release is not inhibited by botulinum neurotoxins or drugs interfering with Ca2+ 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 toxins effects and suggest that LTX‐stimulated exocytosis depends upon the co‐operative action of external and intracellular Ca2+ involving G proteins and PLC, whereas the Ca2+‐independent release is largely non‐vesicular.

Structure of α-latrotoxin oligomers reveals that divalent cation-dependent tetramers form membrane pores

We report here the first three-dimensional structure of α-latrotoxin, a black widow spider neurotoxin, which forms membrane pores and stimulates secretion in the presence of divalent cations. We discovered that α-latrotoxin exists in two oligomeric forms: it is dimeric in EDTA but forms tetramers in the presence of Ca2+ or Mg2+. The dimer and tetramer structures were determined independently at 18 Å and 14 Å resolution, respectively, using cryo-electron microscopy and angular reconstitution. The α-latrotoxin monomer consists of three domains. The N- and C-terminal domains have been identified using antibodies and atomic fitting. The C4-symmetric tetramers represent the active form of α-latrotoxin; they have an axial channel and can insert into lipid bilayers with their hydrophobic base, providing the first model of α-latrotoxin pore formation.

Latrophilin 1 and its endogenous ligand Lasso/teneurin-2 form a high-affinity transsynaptic receptor pair with signaling capabilities.

Latrophilin 1 (LPH1), a neuronal receptor of α-latrotoxin, is implicated in neurotransmitter release and control of presynaptic Ca2+. 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 Ca2+ 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 Ca2+ signals, which might affect synaptic functions.

The latrophilin family: multiply spliced G protein-coupled receptors with differential tissue distribution.

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.

Latrophilin fragments behave as independent proteins that associate and signal on binding of LTXN4C

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.

CA2+-INDEPENDENT INSULIN EXOCYTOSIS INDUCED BY ALPHA -LATROTOXIN REQUIRES LATROPHILIN, A G PROTEIN-COUPLED RECEPTOR

α‐Latrotoxin (α‐LTX) induces exocytosis of small synaptic vesicles (SSVs) in neuronal cells both by a calcium‐independent mechanism and by opening cation‐permeable pores. Since the basic molecular events regulating exocytosis in neurons and endocrine cells may be similar, we have used the exocytosis of insulin‐containing large dense core vesicles (LDCVs) as a model system. In primary pancreatic β‐cells and in the derived cell lines INS‐1 and MIN6, α‐LTX increased insulin release in the absence of extracellular calcium, but the insulin‐secreting cell lines HIT‐T15 and RINm5F were unresponsive. α‐LTX did not alter membrane potential or cytosolic calcium, and its stimulatory effect on exocytosis was still observed in pre‐permeabilized INS‐1 cells kept at 0.1 μM Ca2+. Consequently, pore formation or ion fluxes induced by α‐LTX could be excluded. The Ca2+‐independent α‐LTX‐binding protein, latrophilin, is a novel member of the secretin family of G protein‐coupled receptors (GPCR). Sensitivity to α‐LTX correlated with expression of latrophilin, but not with synaptotagmin I or neurexin Iα expression. Moreover, transient expression of latrophilin in HIT‐T15 cells conferred α‐LTX‐induced exocytosis. Our results indicate that direct stimulation of exocytosis by a GPCR mediates the Ca2+‐independent effects of α‐LTX in the absence of altered ion fluxes. Therefore, direct regulation by receptor‐activated heterotrimeric G proteins constitutes an important feature of the endocrine exocytosis of insulin‐containing LDCVs and may also apply to SSV exocytosis in neurons.

α-Latrotoxin and Its Receptors

alpha-Latrotoxin (alpha-LTX) from black widow spider venom induces exhaustive release of neurotransmitters from vertebrate nerve terminals and endocrine cells. This 130-kDa protein has been employed for many years as a molecular tool to study exocytosis. However, its action is complex: in neurons, alpha-LTX induces massive secretion both in the presence of extracellular Ca(2+) (Ca(2+) (e)) and in its absence; in endocrine cells, it usually requires Ca(2+) (e). To use this toxin for further dissection of secretory mechanisms, one needs an in-depth understanding of its functions. One such function that explains some alpha-LTX effects is its ability to form cation-permeable channels in artificial lipid bilayers. The mechanism of alpha-LTX pore formation, revealed by cryo-electron microscopy, involves toxin assembly into homotetrameric complexes which harbor a central channel and can insert into lipid membranes. However, in biological membranes, alpha-LTX cannot exert its actions without binding to specific receptors of the plasma membrane. Three proteins with distinct structures have been found to bind alpha-LTX: neurexin Ialpha, latrophilin 1, and receptor-like protein tyrosine phosphatase sigma. Upon binding a receptor, alpha-LTX forms channels permeable to cations and small molecules; the toxin may also activate the receptor. To distinguish between the pore- and receptor-mediated effects, and to study structure-function relationships in the toxin, alpha-LTX mutants have been used.

Enhancement of spontaneous transmitter release at neonatal mouse neuromuscular junctions by the glial cell line-derived neurotrophic factor (GDNF)

1 The acute effects of neurotrophic factors on the frequency of spontaneous transmitter release (miniature endplate potentials (MEPPs)) from motor nerve terminals has been examined in skeletal muscles of neonatal mice aged between 9 and 20 days. The following factors were tested at a concentration of 50 ng ml−1: brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3), neurotrophin‐4 (NT‐4), ciliary neuronotrophic factor (CNTF), leukaemia inhibitory factor (LIF), insulin‐like growth factors 1 and 2 (IGF‐1 and IGF‐2), and glial cell line‐derived neurotrophic factor (GDNF). In some experiments, the responses to 2 μm LaCl3 and 10 mm K+, or to 2–5 nm purified α‐latrotoxin (α‐LTX) were also measured. 2 Neither BDNF, NT‐3, NT‐4, LIF, IGF‐1 or IGF‐2 ‐ singly or in combination ‐ caused any significant change in MEPP frequency. GDNF, however, produced a highly significant, 2‐fold increase in neurotransmitter release that was reproduced in fourteen muscles. 3 Potentiation of MEPP frequency in GDNF was of the same order as that induced by tetanic stimulation or substitution of the bathing medium with hypertonic saline; but substantially less than that induced either by lanthanum ions or α‐latrotoxin. 4 The data suggest that concentrations of GDNF that produce maximal enhancement of motoneurone survival in vitro and in vivo also produce acute, non‐saturating enhancement in transmitter release at immature mammalian neuromuscular synapses. Taken together with other reports, these findings suggest that GDNF may mediate both functional and structural plasticity of neonatal neuromuscular junctions.