Catching Latrophilin With Lasso: A Universal Mechanism for Axonal Attraction and Synapse Formation

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

C-terminal phosphorylation of latrophilin-1/ADGRL1 affects the interaction between its fragments

Latrophilin-1 is an Adhesion G protein-coupled receptor, which mediates the effect of α-latrotoxin, causing massive release of neurotransmitters from nerve terminals and endocrine cells. Autoproteolysis cleaves latrophilin-1 into two parts: the extracellular N-terminal fragment (NTF) and the heptahelical C-terminal fragment (CTF). NTF and CTF can exist as independent proteins in the plasma membrane, but α-latrotoxin binding to NTF induces their association and G protein-mediated signaling. We demonstrate here that CTF in synapses is phosphorylated on multiple sites. Phosphorylated CTF has a high affinity for NTF and co-purifies with it on affinity columns and on sucrose density gradients. Dephosphorylated CTF has a lower affinity for NTF and can behave as a separate protein. α-Latrotoxin (and possibly other ligands of latrophilin-1) binds both to the NTF-CTF complex and to receptor-like protein tyrosine phosphatase σ, bringing them together. This leads to CTF dephosphorylation and facilitates CTF release from the complex. We propose that ligand-dependent phosphorylationdephosphorylation of latrophilin-1 could affect the interaction between its fragments and its functions as a G protein-coupled receptor

Proteolytically released Lasso/teneurin-2 induces axonal attraction by interacting with latrophilin-1 on growth cones

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 signalling that induces axonal attraction

The tim-3-galectin-9 secretory pathway is involved in the immune escape of human acute myeloid leukemia cells

Acute myeloid leukemia (AML) is a severe and often fatal systemic malignancy. Malignant cells are capable of escaping host immune surveillance by inactivating cytotoxic lymphoid cells. In this work we discovered a fundamental molecular pathway, which includes ligand-dependent activation of ectopically expressed latrophilin 1 and possibly other G-protein coupled receptors leading to increased translation and exocytosis of the immune receptor Tim-3 and its ligand galectin-9. This occurs in a protein kinase C and mTOR (mammalian target of rapamycin)-dependent manner. Tim-3 participates in galectin-9 secretion and is also released in a free soluble form. Galectin-9 impairs the anti-cancer activity of cytotoxic lymphoid cells including natural killer (NK) cells. Soluble Tim-3 prevents secretion of interleukin-2 (IL-2) required for the activation of cytotoxic lymphoid cells. These results were validated in ex vivo experiments using primary samples from AML patients. This pathway provides reliable targets for both highly specific diagnosis and immune therapy of AML

The expanding functional roles and signaling mechanisms of adhesion G protein–coupled receptors

The adhesion class of G protein–coupled receptors (GPCRs) is the second largest family of GPCRs (33 members in humans). Adhesion GPCRs (aGPCRs) are defined by a large extracellular N‐terminal region that is linked to a C‐terminal seven transmembrane (7TM) domain via a GPCR‐autoproteolysis inducing (GAIN) domain containing a GPCR proteolytic site (GPS). Most aGPCRs undergo autoproteolysis at the GPS motif, but the cleaved fragments stay closely associated, with the N‐terminal fragment (NTF) bound to the 7TM of the C‐terminal fragment (CTF). The NTFs of most aGPCRs contain domains known to be involved in cell–cell adhesion, while the CTFs are involved in classical G protein signaling, as well as other intracellular signaling. In this workshop report, we review the most recent findings on the biology, signaling mechanisms, and physiological functions of aGPCRs

α-Latrotoxin Tetramers Spontaneously Form Two-Dimensional Crystals in Solution and Coordinated Multi-Pore Assemblies in Biological Membranes

α-Latrotoxin (α-LTX) was found to form two-dimensional (2D) monolayer arrays in solution at relatively low concentrations (0.1 mg/mL), with the toxin tetramer constituting a unit cell. The crystals were imaged using cryogenic electron microscopy (cryoEM), and image analysis yielded a ~12 Å projection map. At this resolution, no major conformational changes between the crystalline and solution states of α-LTX tetramers were observed. Electrophysiological studies showed that, under the conditions of crystallization, α-LTX simultaneously formed multiple channels in biological membranes that displayed coordinated gating. Two types of channels with conductance levels of 120 and 208 pS were identified. Furthermore, we observed two distinct tetramer conformations of tetramers both when observed as monodisperse single particles and within the 2D crystals, with pore diameters of 11 and 13.5 Å, suggestive of a flickering pore in the middle of the tetramer, which may correspond to the two states of toxin channels with different conductance levels. We discuss the structural changes that occur in α-LTX tetramers in solution and propose a mechanism of α-LTX insertion into the membrane. The propensity of α-LTX tetramers to form 2D crystals may explain many features of α-LTX toxicology and suggest that other pore-forming toxins may also form arrays of channels to exert maximal toxic effect

Latrophilin fragments behave as independent proteins that associate and signal on binding of LTX(N4C)

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