Latrophilin 1 and Its Endogenous Ligand Lasso/ Teneurin-2 Form a High-Affinity Transsynaptic Receptor Pair with Signaling Capabilities
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Latrophilin 1 and its endogenous ligand Lasso/ teneurin-2 form a high-affinity transsynaptic receptor pair with signaling capabilities John-Paul Silvaa, Vera G. Lelianovaa, Yaroslav S. Ermolyukb, Nickolai Vysokova, Paul G. Hitchenc, Otto Berninghausena,1, M. Atiqur Rahmana,2, Alice Zangrandia,3, Sara Fidalgoa,4, Alexander G. Tonevitskyd, Anne Dellc, Kirill E. Volynskib, and Yuri A. Ushkaryova,5 aDivision of Cell and Molecular Biology, Imperial College London, London SW7 2AZ, United Kingdom; bUniversity College London Institute of Neurology, London, WC1N 3BG, United Kingdom; cDivision of Molecular Biosciences, Imperial College London, London SW7 2AZ, United Kingdom; and dMoscow State University, Moscow, 119991, Russia Edited by Peter Scheiffele, Biozentrum, University of Basel, Basel, Switzerland, and accepted by the Editorial Board June 5, 2011 (received for review December 24, 2010) Latrophilin 1 (LPH1), a neuronal receptor of α-latrotoxin, is impli- LPH1 gene (lphn1) is up-regulated by antipsychotic drugs (19). cated in neurotransmitter release and control of presynaptic Ca2+. In humans, deletions of a chromosome 19 fragment containing As an “adhesion G-protein-coupled receptor,” LPH1 can convert lphn1 lead to brain malformations, mental retardation, and hy- cell surface interactions into intracellular signaling. To examine peractivity (20). Mutations in LPH3 are linked to attention the physiological functions of LPH1, we used LPH1’s extracellular deficit/hyperactivity disorder (21). However, the endogenous li- domain to purify its endogenous ligand. A single protein of ∼275 gand of LPH1 has not yet been identified, hampering the elu- kDa was isolated from rat brain and termed Lasso. Peptide se- cidation of its physiological functions. quencing and molecular cloning have shown that Lasso is a splice Teneurins (also called odd Oz, tenascin-m, neurestin, or variant of teneurin-2, a brain-specific orphan cell surface receptor DOC4) are large cell surface glycoproteins with a single TMR with a function in neuronal pathfinding and synaptogenesis. We found in all animal species (22–24). Their ectodomains contain show that LPH1 and Lasso interact strongly and specifically. They eight EGF-like repeats, two of which have unpaired cysteine NEUROSCIENCE are always copurified from rat brain extracts. Coculturing cells residues that mediate covalent homodimerization of the mole- expressing LPH1 with cells expressing Lasso leads to their mutual cule (25). The distal part of the C terminus, containing 6 NHL attraction and formation of multiple junctions to which both pro- and 26 YD repeats, forms a 13-nm globule (25). teins are recruited. Cells expressing LPH1 form chimerical synapses All four teneurins found in vertebrates are expressed specifi- with hippocampal neurons in cocultures; LPH1 and postsynaptic cally in the brain (25), although teneurin-3 and -4 also show low neuronal protein PSD-95 accumulate on opposite sides of these expression in other tissues (25–27). Teneurins play a role in structures. Immunoblotting and immunoelectron microscopy of development, neurite outgrowth, axon guidance, neuronal con- purified synapses and immunostaining of cultured hippocampal nectivity, and synaptogenesis (reviewed in refs. 28 and 29). In C. neurons show that LPH1 and Lasso are enriched in synapses; in elegans, mutations in ten-1 lead to aberrations in axon guidance both systems, LPH1 is presynaptic, whereas Lasso is postsynaptic. and morphogenesis (30, 31). Knockout of Ten-m3 in mice A C-terminal fragment of Lasso interacts with LPH1 and induces impairs vision, consistent with abnormal patterning of visual 2+ Ca signals in presynaptic boutons of hippocampal neurons and pathways (32). In humans, ablation of the teneurin-1 gene and in neuroblastoma cells expressing LPH1. Thus, LPH1 and Lasso can rearrangements of the teneurin-2 gene lead to mental re- 2+ form transsynaptic complexes capable of inducing presynaptic Ca tardation (33, 34). Teneurins are hypothesized to engage in signals, which might affect synaptic functions. homophilic interactions (25, 35), but attempts to isolate an en- dogenous ligand of teneurins have not been described. atrophilin 1 (LPH1) is a neuronal G protein–coupled re- In the present study, we used the NTF of LPH1 to isolate its Lceptor (GPCR) that binds α-latrotoxin (α-LTX) (1–3), or its natural ligand. Only one protein, Lasso (a splice variant of ten- recombinant mutant LTXN4C (4), and sends intracellular signals eurin-2), has been found to bind LPH1. We demonstrate that the (5) leading to a massive increase in spontaneous and evoked interaction of Lasso and LPH1 is specific, strong, and functional, exocytosis of neurotransmitters (6–8). The dramatic inhibition of the presynaptic action of α-LTX in the LPH1 knockout mouse (9) indicates that LPH1 is localized in presynaptic terminals and Author contributions: K.E.V. and Y.A.U. designed research; J.-P.S., V.G.L., Y.S.E., N.V., P.G.H., mediates most of the α-LTX effect. O.B., M.A.R., A.Z., S.F., and K.E.V. performed research; A.G.T. and A.D. contributed new “ ” reagents/analytic tools; J.-P.S., Y.S.E., P.G.H., K.E.V., and Y.A.U. analyzed data; and J.-P.S. LPH1 is a so-called adhesion GPCR (10, 11). Members of this and Y.A.U. wrote the paper. family have large and divergent N-terminal ectodomains and con- The authors declare no conflict of interest. served C-terminal domains containing seven transmembrane This article is a PNAS Direct Submission. P.S. is a guest editor invited by the Editorial regions (TMRs) (12). After synthesis, adhesion GPCRs undergo Board. autoproteolysis upstream of the first TMR (13, 14). The N-terminal Data deposition: The sequences of the full-length Lasso and recombinant constructs used fragment (NTF) is not normally released into the medium, but in this study are available at GenBank (accession nos. JF784339–JF784359). remains bound to the cell surface via the C-terminal fragment 1Present address: Gene Center Munich, Ludwig-Maximilians-Universität Munich, 81377 (CTF) (13) and/or an unknown hydrophobic anchor (5). On bind- Munich, Germany. N4C ing an agonist (e.g., LTX ), the fragments of LPH1 reassemble, 2Present address: AbD Serotec, Oxford OX5 1JE, United Kingdom. 2+ eliciting an intracellular Ca signal via the CTF (5, 15). 3Present address: Helsinn Healthcare SA, 6915 Lugano/Pambio-Noranco, Switzerland. The functions of LPH1 and its homologs (LPH2 and LPH3) 4Present address: School of Pharmacy and Biomolecular Sciences, University of Brighton, (3) are unclear. In Caenorhabditis elegans, the LPH1 ortholog Brighton BN2 4GJ, United Kingdom. LAT-1 plays a role in embryo morphogenesis (16) and modula- 5To whom correspondence should be addressed. E-mail: [email protected]. tion of neurotransmitter release (17, 18). Null mutations in LAT- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1 arrest worm development at the embryo stage (16). In rats, the 1073/pnas.1019434108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1019434108 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 leading to intracellular signaling. LPH1 and Lasso interact across the nature of this protein, we used two additional V5 Ab/LPH-51 intercellular and synaptic junctions, suggesting that this trans- columns, one incubated with a detergent-free extract from brain synaptic pair might be involved in synaptic functions. and the other incubated with a detergent extract from rat liver. The 275-kDa protein did not bind to control columns (Fig. 1D). Results Initial MS analysis failed to identify the LPH1-binding protein, Isolation of the LPH1 Ligand. To create an affinity adsorbent, three but the affinity chromatography results (Fig. 1D) indicated that soluble constructs based on the NTF of LPH1 were expressed in the isolated protein was brain-specific and membrane-bound. neuroblastoma (NB2a) cells (Fig. 1A): LPH-51, containing a V5 Thus, it was operationally termed Lasso (LPH1-associated syn- tag at the N terminus and the natural cleavage sequence at the C aptic surface organizer). For further analysis, we isolated a large terminus; LPH-74, terminating exactly at the cleavage site; and amount of Lasso and elicited anti-Lasso Abs in mice. LPH-62, with a V5 tag attached at the cleavage site but not These Abs (dmAbs) were used to test whether Lasso inter- fi cleaved in cells. α-LTX binding to these proteins demonstrated acts with endogenous LPH1. We puri ed LPH1 from rat brain fi α biological activity in only LPH-51 (Fig. 1B). lysate by af nity chromatography on -LTX or anti-LPH1 Ab Purified LPH-51 was attached to an immobilized anti-V5 Ab and found that Lasso was isolated together with LPH1 in both and used for affinity chromatography of a detergent extract from cases, but not on a control column (Fig. 1E). Thus, in the brain, rat brain. Bound proteins were desorbed using high pH, which LPH1 and Lasso form strong complexes that survive stringent purification conditions. also eluted LPH-51 but not the Ab. We used two control con- We also used high-resolution tandem MS for de novo se- ditions: a V5 Ab/LPH-51 column not incubated with brain extract quencing of Lasso and determined the sequences of nine tryptic and a V5 Ab column (without LPH-51) incubated with the ex- ∼ peptides (Table S1). All of the peptides corresponded precisely tract. A single brain protein of 275 kDa was eluted from the test to neurestin (26), also known as rat teneurin-2. column, but not from the control columns (Fig. 1C). To explore Based on the peptide sequences obtained, cDNA encoding full-size (FS) human Lasso was cloned (Fig. 2A). Lasso cDNA is 12,450 bp long and encodes a protein which has a calculated molecular mass of 292 kDa, no signal peptide, one predicted TMR, and is a splice variant of teneurin-2 (Fig.