Antony A. Boucard

A Splice Code for trans-Synaptic Cell Adhesion Mediated by Binding of Neuroligin 1 to α- and β-Neurexins

Previous studies suggested that postsynaptic neuroligins form a trans-synaptic complex with presynaptic β-neurexins, but not with presynaptic α-neurexins. Unexpectedly, we now find that neuroligins also bind α-neurexins and that α- and β-neurexin binding by neuroligin 1 is regulated by alternative splicing of neuroligin 1 (at splice site B) and of neurexins (at splice site 4). In neuroligin 1, splice site B is a master switch that determines α-neurexin binding but leaves β-neurexin binding largely unaffected, whereas alternative splicing of neurexins modulates neuroligin binding. Moreover, neuroligin 1 splice variants with distinct neurexin binding properties differentially regulate synaptogenesis: neuroligin 1 that binds only β-neurexins potently stimulates synapse formation, whereas neuroligin 1 that binds to both α- and β-neurexins more effectively promotes synapse expansion. These findings suggest that neuroligin binding to α- and β-neurexins mediates trans-synaptic cell adhesion but has distinct effects on synapse formation, indicating that expression of different neuroligin and neurexin isoforms specifies a trans-synaptic signaling code.

A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis.

The G protein‐coupled receptor (GPCR) Proteolysis Site (GPS) of cell‐adhesion GPCRs and polycystic kidney disease (PKD) proteins constitutes a highly conserved autoproteolysis sequence, but its catalytic mechanism remains unknown. Here, we show that unexpectedly the ∼40‐residue GPS motif represents an integral part of a much larger ∼320‐residue domain that we termed GPCR‐Autoproteolysis INducing (GAIN) domain. Crystal structures of GAIN domains from two distantly related cell‐adhesion GPCRs revealed a conserved novel fold in which the GPS motif forms five β‐strands that are tightly integrated into the overall GAIN domain. The GAIN domain is evolutionarily conserved from tetrahymena to mammals, is the only extracellular domain shared by all human cell‐adhesion GPCRs and PKD proteins, and is the locus of multiple human disease mutations. Functionally, the GAIN domain is both necessary and sufficient for autoproteolysis, suggesting an autoproteolytic mechanism whereby the overall GAIN domain fine‐tunes the chemical environment in the GPS to catalyse peptide bond hydrolysis. Thus, the GAIN domain embodies a unique, evolutionarily ancient and widespread autoproteolytic fold whose function is likely relevant for GPCR signalling and for multiple human diseases.

Structures of Neuroligin-1 and the Neuroligin-1/Neurexin-1β Complex Reveal Specific Protein-Protein and Protein-Ca2+ Interactions

Neurexins and neuroligins provide trans-synaptic connectivity by the Ca2+-dependent interaction of their alternatively spliced extracellular domains. Neuroligins specify synapses in an activity-dependent manner, presumably by binding to neurexins. Here, we present the crystal structures of neuroligin-1 in isolation and in complex with neurexin-1 beta. Neuroligin-1 forms a constitutive dimer, and two neurexin-1 beta monomers bind to two identical surfaces on the opposite faces of the neuroligin-1 dimer to form a heterotetramer. The neuroligin-1/neurexin-1 beta complex exhibits a nanomolar affinity and includes a large binding interface that contains bound Ca2+. Alternatively spliced sites in neurexin-1 beta and in neuroligin-1 are positioned nearby the binding interface, explaining how they regulate the interaction. Structure-based mutations of neuroligin-1 at the interface disrupt binding to neurexin-1 beta, but not the folding of neuroligin-1 and confirm the validity of the binding interface of the neuroligin-1/neurexin-1 beta complex. Our results provide molecular insights for understanding the role of cell-adhesion proteins in synapse function.

Neuroligin‐1 performs neurexin‐dependent and neurexin‐independent functions in synapse validation

Postsynaptic neuroligins are thought to perform essential functions in synapse validation and synaptic transmission by binding to, and dimerizing, presynaptic α‐ and β‐neurexins. To test this hypothesis, we examined the functional effects of neuroligin‐1 mutations that impair only α‐neurexin binding, block both α‐ and β‐neurexin binding, or abolish neuroligin‐1 dimerization. Abolishing α‐neurexin binding abrogated neuroligin‐induced generation of neuronal synapses onto transfected non‐neuronal cells in the so‐called artificial synapse‐formation assay, even though β‐neurexin binding was retained. Thus, in this assay, neuroligin‐1 induces apparent synapse formation by binding to presynaptic α‐neurexins. In transfected neurons, however, neither α‐ nor β‐neurexin binding was essential for the ability of postsynaptic neuroligin‐1 to dramatically increase synapse density, suggesting a neurexin‐independent mechanism of synapse formation. Moreover, neuroligin‐1 dimerization was not required for either the non‐neuronal or the neuronal synapse‐formation assay. Nevertheless, both α‐neurexin binding and neuroligin‐1 dimerization were essential for the increase in apparent synapse size that is induced by neuroligin‐1 in transfected neurons. Thus, neuroligin‐1 performs diverse synaptic functions by mechanisms that include as essential components of α‐neurexin binding and neuroligin dimerization, but extend beyond these activities.

High Affinity Neurexin Binding to Cell Adhesion G-protein-coupled Receptor CIRL1/Latrophilin-1 Produces an Intercellular Adhesion Complex

Background: Neurexins and CIRL/latrophilin-1 (CL1) are independent synaptic receptors for α-latrotoxin. Results: Neurexins and CL1 form a high affinity complex that mediates intercellular adhesion and is regulated by neurexin alternative splicing. Conclusion: Thus, two independent α-latrotoxin receptors interact trans-cellularly to form a connection between neurons. Significance: The neurexin-CL1 complex may be involved in trans-synaptic cell adhesion and mediate α-latrotoxin toxicity. The G-protein-coupled receptor CIRL1/latrophilin-1 (CL1) and the type-1 membrane proteins neurexins represent distinct neuronal cell adhesion molecules that exhibit no similarities except for one common function: both proteins are receptors for α-latrotoxin, a component of black widow spider venom that induces massive neurotransmitter release at synapses. Unexpectedly, we have now identified a direct binding interaction between the extracellular domains of CL1 and neurexins that is regulated by alternative splicing of neurexins at splice site 4 (SS4). Using saturation binding assays, we showed that neurexins lacking an insert at SS4 bind to CL1 with nanomolar affinity, whereas neurexins containing an insert at SS4 are unable to bind. CL1 competed for neurexin binding with neuroligin-1, a well characterized neurexin ligand. The extracellular sequences of CL1 contain five domains (lectin, olfactomedin-like, serine/threonine-rich, hormone-binding, and G-protein-coupled receptor autoproteolysis-inducing (GAIN) domains). Of these domains, the olfactomedin-like domain mediates neurexin binding as shown by deletion mapping. Cell adhesion assays using cells expressing neurexins and CL1 revealed that their interaction produces a stable intercellular adhesion complex, indicating that their interaction can be trans-cellular. Thus, our data suggest that CL1 constitutes a novel ligand for neurexins that may be localized postsynaptically based on its well characterized interaction with intracellular SH3 and multiple ankyrin repeats adaptor proteins (SHANK) and could form a trans-synaptic complex with presynaptic neurexins.

Unusually rapid evolution of Neuroligin-4 in mice

Neuroligins (NLs) are postsynaptic cell-adhesion molecules that are implicated in humans in autism spectrum disorders because the genes encoding NL3 and NL4 are mutated in rare cases of familial autism. NLs are highly conserved evolutionarily, except that no NL4 was detected in the currently available mouse genome sequence assemblies. We now demonstrate that mice express a distant NL4 variant that rapidly evolved from other mammalian NL4 genes and that exhibits sequence variations even between different mouse strains. Despite its divergence, mouse NL4 binds neurexins and is transported into dendritic spines, suggesting that the core properties of NLs are retained in this divergent NL isoform. The selectively rapid evolution of NL4 in mice suggests that its function in the brain is under less stringent control than that of other NLs, shedding light on why its mutation in autism spectrum disorder patients is not lethal, but instead leads to a discrete developmental brain disorder.

Latrophilins function as heterophilic cell-adhesion molecules by binding to teneurins: regulation by alternative splicing.

Background: Latrophilins are large adhesion-type GPCRs that may mediate cell adhesion via heterophilic interactions. Results: Latrophilin-1 binding to teneurins exhibits nanomolar affinity, is regulated by alternative splicing, and mediates intercellular adhesion. Conclusion: Latrophilins are cell-adhesion molecules with multiple trans-synaptic ligands. Significance: Our data support a role for latrophilin in trans-neuronal interactions by binding to multiple heterophilic ligands. Latrophilin-1, -2, and -3 are adhesion-type G protein-coupled receptors that are auxiliary α-latrotoxin receptors, suggesting that they may have a synaptic function. Using pulldowns, we here identify teneurins, type II transmembrane proteins that are also candidate synaptic cell-adhesion molecules, as interactors for the lectin-like domain of latrophilins. We show that teneurin binds to latrophilins with nanomolar affinity and that this binding mediates cell adhesion, consistent with a role of teneurin binding to latrophilins in trans-synaptic interactions. All latrophilins are subject to alternative splicing at an N-terminal site; in latrophilin-1, this alternative splicing modulates teneurin binding but has no effect on binding of latrophilin-1 to another ligand, FLRT3. Addition to cultured neurons of soluble teneurin-binding fragments of latrophilin-1 decreased synapse density, suggesting that latrophilin binding to teneurin may directly or indirectly influence synapse formation and/or maintenance. These observations are potentially intriguing in view of the proposed role for Drosophila teneurins in determining synapse specificity. However, teneurins in Drosophila were suggested to act as homophilic cell-adhesion molecules, whereas our findings suggest a heterophilic interaction mechanism. Thus, we tested whether mammalian teneurins also are homophilic cell-adhesion molecules, in addition to binding to latrophilins as heterophilic cell-adhesion molecules. Strikingly, we find that although teneurins bind to each other in solution, homophilic teneurin-teneurin binding is unable to support stable cell adhesion, different from heterophilic teneurin-latrophilin binding. Thus, mammalian teneurins act as heterophilic cell-adhesion molecules that may be involved in trans-neuronal interaction processes such as synapse formation or maintenance.

Analysis of the third transmembrane domain of the human type 1 angiotensin II receptor by cysteine-scanning mutagenesis

Activation of G protein-coupled receptors by agonists involves significant movement of transmembrane domains (TMD) following agonist binding. The underlying structural mechanism by which receptor activation takes place is largely unknown but can be inferred by detecting variability within the environment of the ligand-binding pocket, which is a water-accessible crevice surrounded by the seven TMD helices. Using the substituted-cysteine accessibility method, we identified the residues within the third TMD of the wild-type angiotensin II (AT1) receptor that contribute to the formation of the binding site pocket. Each residue within the Ile103–Tyr127 region was mutated one at a time to a cysteine. Treating the A104C, N111C, and L112C mutant receptors with the charged sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA) strongly inhibited ligand binding, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT1 receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD3 reporter cysteines engineered in a constitutively active AT1 receptor. Indeed, two additional mutants (S109C and V116C) were found to be sensitive to MTSEA treatment. Our results suggest that constitutive activation of the AT1 receptor causes a minor counterclockwise rotation of TMD3, thereby exposing residues, which are not present in the inactive state, to the binding pocket. This pattern of accessibility of residues in the TMD3 of the AT1 receptor parallels that of homologous residues in rhodopsin. This study identified key elements of TMD3 that contribute to the activation of class A G protein-coupled receptors through structural rearrangements.

Photolabelling the rat urotensin II/GPR14 receptor identifies a ligand-binding site in the fourth transmembrane domain.

A urotensin II (U-II) peptide analogue containing the photoreactive p -benzoyl-L-phenylalanine (Bz-Phe) in the sixth position was used to identify ligand-binding sites of the rat U-II receptor, also known as GPR14. [Bz-Phe(6)]U-II bound the receptor expressed in COS-7 cells with high affinity (IC(50) 0.7 nM) and was as potent as U-II in the agonist-induced production of inositol phosphate. Photolabelling of the U-II receptor with (125)I-[Bz-Phe(6)]U-II resulted in the specific formation of a glycosylated (125)I-[Bz-Phe(6)]U-II-U-II receptor complex of 60 kDa. Digestion of the 60 kDa complex with endoproteinase Glu-C generated a fragment of 17 kDa circumscribing the labelled fragment to residues 148-286. Digestion of the ligand-receptor complex with endoproteinase Arg-C produced a short peptide of 4 kDa corresponding to fragments 125-148, 167-192 or 210-233. CNBr treatment of the endoproteinase-Glu-C and -Arg-C fragments yielded 2 kDa fragments, defining the labelling site to methionine residues 184/185 of the fourth transmembrane domain. Photolabelling of two mutant receptors, M184L/M185L and M184A/M185A, led to a significant decrease in the overall yield of covalent labelling. Taken together, our results indicate that position 6 of U-II normally occupied by phenylalanine would interact with Met(184) and/or Met(185) of the fourth transmembrane domain of the U-II receptor. This information should be of significant value in the study of the interactions between U-II and its cognate receptor.

METHIONINE PROXIMITY ASSAY, A NOVEL METHOD FOR EXPLORING PEPTIDE LIGAND–RECEPTOR INTERACTION

ABSTRACT Probing G-protein coupled receptor (GPCR) structures is a priority in the functional and structural understanding of GPCRs. In the past, we have used several approaches around photoaffinity labeling in order to establish contact points between peptide ligands and their cognate receptors. Such contact points are helpful to build reality based molecular models of GPCRs and to elucidate their activation mechanisms. Most studies of peptidergic GPCRs have been done with photolabeling peptides containing the benzophenone moiety as a reputedly non-selective probe. However our recent results are now showing that p-benzoylphenylalanine (Bpa) has some selectivity for Met residues in the receptor protein, reducing the accuracy of this method. Turning a problem into an asset, modified analogues of Bpa, e.g. p, p″-nitrobenzoylphenylalanine (NO2Bpa), display increased selectivity for such Met residues. It means a photoprobe containing such modified benzophenone-moieties does not label a receptor protein unless a Met residue is in the immediate vicinity. This unique property allows us to propose and show the feasibility and utility of a new method for scanning the contact areas of peptidergic GPCRs, the Methionine Proximity Assay (MPA). Putative contact residues of the receptor are exchanged to Met residues by site-directed mutagenesis and are subjected to photoaffinity labeling with such modified benzophenone-containing peptides. Successful incorporation indicates physical proximity of those residues. This principle is established and explored with benzophenone-containing analogues of angiotensin II and the two known human angiotensin II receptors AT1 and AT2, determining contact points in both receptors. This approach has several important advantages over other scanning approaches, e.g., the SCAM procedure, since the MPA-method can be used in the hydrophobic core of receptors.