Electron microscopic structure of agrin and mapping of its binding site in laminin-1.

Agrin is a large, multidomain heparan sulfate proteoglycan that is associated with basement membranes of several tissues. Particular splice variants of agrin are essential for the formation of synaptic structures at the neuromuscular junction. The binding of agrin to laminin appears to be required for its localization to synaptic basal lamina and other basement membranes. Here, electron microscopy was used to determine the structure of agrin and to localize its binding site in laminin-1. Agrin appears as an approximately 95 nm long particle that consists of a globular, N-terminal laminin-binding domain, a central rod predominantly formed by the follistatin-like domains and three globular, C-terminal laminin G-like domains. In a few cases, heparan sulfate glycosaminoglycan chains were seen emerging from the central portion of the core protein. Moreover, we show that agrin binds to the central region of the three-stranded, coiled-coil oligomerization domain in the long arm of laminin-1, which mediates subunit assembly of the native laminin molecule. In summary, our data show for the first time a protein-protein interaction of the extracellular matrix that involves a coiled-coil domain, and they assign a novel role to this domain of laminin-1. Based on this, we propose that agrin associates with basal lamina in a polarized way.     

Mapping sites responsible for interactions of agrin with neurons

The multidomain proteoglycan agrin is a critical organizer of postsynaptic differentiation at the skeletal neuromuscular junction. Agrin is also abundant in the brain, but its roles there are unknown. As a step toward understanding these roles, we mapped sites responsible for interactions of neurons with agrin. First, we used a series of recombinant agrin fragments to show that at least four sites on agrin interact with chick ciliary neurons. Use of blocking antibodies and peptides indicated that neurons adhere to a site in the second of three G domains by means of alphaVbeta1 integrin, and to a site in the last of four epidermal growth factor (EGF) repeats via a distinct beta1 integrin. A third, integrin-independent adhesion site is near to but distinct from the site that induces postsynaptic differentiation in muscles. These domains are insufficient, however, to account for neurite outgrowth-inhibiting properties of full-length agrin, which are mediated by the N-terminal half of the molecule. We then used a second set of agrin mutants to demonstrate and map a transmembrane domain in the amino-terminus of the SN-isoform of agrin. The extracellular matrix-bound form of agrin, called LN, bears an amino-terminus required for secretion and binding to laminin. The SN form, which is selectively expressed by neurons, bears a variant amino terminus that converts agrin from a secreted, matrix-associated protein to a type-II transmembrane protein, providing a mechanism for presenting agrin in central, as opposed to neuromuscular, synaptic clefts. The SN-amino terminus can mediate externalization and membrane anchoring of heterologous proteins, but is insufficient to target them to the synapse. Together, these studies define sites that contribute to the subcellular localization of and signaling by neuronal agrin.     

Agrin is a chimeric proteoglycan with the attachment sites for heparan sulfate/chondroitin sulfate located in two multiple serine-glycine clusters

Agrin is a large extracellular matrix protein that plays a key role in the formation and maintenance of the vertebrate neuromuscular junction. The amino acid sequence of agrin encodes a protein with a molecular size of 220 kDa, whereas SDS-PAGE shows a diffuse band around 400 kDa. Further studies showed that agrin is highly glycosylated and belongs to the family of heparan sulfate proteoglycans. By expressing different protein fragments, we localized the glycosaminoglycan (GAG) attachment sites to two locations within the agrin molecule. One site that is located between the seventh and eight follistatin-like domain includes 3 closely spaced serine-glycine (SG) consensus sequences and carries exclusively heparan sulfate side chains. The second site is located further downstream in the centrally located serine-threonine-rich domain and contains a cluster of 4 closely packed SG consensus sequences. This site predominantly carries chondroitin sulfate side chains. Investigating the contribution of individual serines in GAG priming by site-directed mutagenesis showed that each serine of the two SG clusters has the potential to carry GAGs. In accordance with the mixed GAG glycosylation of agrin peptide fragments, it was found that recombinant and in vivo-derived full-length agrin are not exclusively heparan sulfate proteoglycans but also carry chondroitin sulfate side chains.     

Formation of functional synaptic connections between cultured cortical neurons from agrin‐deficient mice

Numerous studies suggest that the extracellular matrix protein agrin directs the formation of the postsynaptic apparatus at the neuromuscular junction (NMJ). Strong support for this hypothesis comes from the observation that the high density of acetylcholine receptors (AChR) normally present at the neuromuscular junction fails to form in muscle of embryonic agrin mutant mice. Agrin is expressed by many populations of neurons in the central nervous system (CNS), suggesting that this molecule may also play a role in neuron–neuron synapse formation. To test this hypothesis, we examined synapse formation between cultured cortical neurons isolated from agrin‐deficient mouse embryos. Our data show that glutamate receptors accumulate at synaptic sites on agrin‐deficient neurons. Moreover, electrophysiological analysis demonstrates that functional glutamatergic and gamma‐aminobutyric acid (GABA)ergic synapses form between mutant neurons. The frequency and amplitude of miniature postsynaptic glutamatergic and GABAergic currents are similar in mutant and age‐matched wild‐type neurons during the first 3 weeks in culture. These results demonstrate that neuron‐specific agrin is not required for formation and early development of functional synaptic contacts between CNS neurons, and suggest that mechanisms of interneuronal synaptogenesis are distinct from those regulating synapse formation at the neuromuscular junction. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 547–557, 1999     

Structural domains of agrin required for clustering of nicotinic acetylcholine receptors.

Agrin is an extracellular matrix component which promotes the clustering of nicotinic acetylcholine receptors (nAChRs) and other proteins at the neuromuscular junction. This aggregation process is one of the earliest steps in synapse formation. Expression of highly active isoforms of agrin, generated by alternative splicing, is restricted to neurons in the central nervous system (CNS) including motoneurons. In the experiments reported here we investigate the regions of agrin necessary for nAChR clustering activity using two different methods. First, we expressed truncated soluble forms of the agrin protein in mammalian cells and assessed their clustering activity. Second, we generated a panel of monoclonal antibodies (mAbs) against agrin and mapped their epitopes. Several mAbs block agrin-induced aggregation of nAChRs. One of the mAbs, Agr86, binds exclusively to the CNS-specific splicing variants and thus identifies an epitope common only to these more active isoforms. Mapping of the Agr86 epitope suggests that alternative splicing results in a distributed conformational change in the agrin protein. Taken together our data suggest that four domains in the C-terminal 55 kDa of agrin contribute to its nAChR clustering activity.     

Prm1 Functions as a Disulfide-linked Complex in Yeast Mating

Prm1 is a pheromone-induced membrane glycoprotein that promotes plasma membrane fusion in yeast mating pairs. HA-Prm1 migrates at twice its expected molecular weight on non-reducing SDS-PAGE gels and coprecipitates with Prm1-TAP, indicating that Prm1 is a disulfide-linked homodimer. The N terminus of a plasma membrane-localized GFP-Prm1 endocytic mutant projects into the cytoplasm, where it is protected from low pH quenching in live cells and from external protease in spheroplasts. In a revised topological map, Prm1 has four transmembrane domains and two large extracellular loops. Mutation of all four cysteines in the extracellular loops blocked disulfide bond formation and destabilized the Prm1 homodimer without preventing Prm1 transport to contact sites in mating pairs. Cys¹²⁰ in loop 1 and Cys⁵⁴⁵ in loop 2 form disulfide cross-links in the Prm1 homodimer and are required for fusion activity. Cys¹²⁰ lies between a hydrophobic segment formerly thought to be a transmembrane domain and an amphipathic helix. An interaction between either of these regions and the opposing membrane could promote fusion.     

Agrin is required for posterior development and motor axon outgrowth and branching in embryonic zebrafish

Although recent studies have extended our understanding of agrin's function during development, its function in the central nervous system (CNS) is not clearly understood. To address this question, zebrafish agrin was identified and characterized. Zebrafish agrin is expressed in the developing CNS and in nonneural structures such as somites and notochord. In agrin morphant embryos, acetylcholine receptor (AChR) cluster number and size on muscle fibers at the choice point were unaffected, whereas AChR clusters on muscle fibers in the dorsal and ventral regions of the myotome were reduced or absent. Defects in the axon outgrowth by primary motor neurons, subpopulations of branchiomotor neurons, and Rohon-Beard sensory neurons were also observed, which included truncation of axons and increased branching of motor axons. Moreover, agrin morphants exhibit significantly inhibited tail development in a dose-dependent manner, as well as defects in the formation of the midbrain-hindbrain boundary and reduced size of eyes and otic vesicles. Together these results show that agrin plays an important role in both peripheral and CNS development and also modulates posterior development in zebrafish.     

Identification and characterization of TMEFF2, a novel survival factor for hippocampal and mesencephalic neurons

We have identified a novel mammalian gene, TMEFF2, that encodes a putative transmembrane protein containing two follistatin-like domains and one epidermal growth factor (EGF)-like domain. The TMEFF2 gene is predominantly expressed in the brain. In situ hybridization analysis revealed that TMEFF2 is widely expressed in the brain, including hippocampal cornu ammonis, dentate gyrus, and substantia nigra pars compacta. We evaluated the survival effect of TMEFF2 using primary cultured neurons from several regions of fetal rat brain following treatment with a recombinant TMEFF2 protein fragment consisting of the putative extracellular domain. TMEFF2 increased survival of neurons from the hippocampus and midbrain, but not from the cerebral cortex, indicating that the survival effects of TMEFF2 are specific to certain cell types. Recombinant TMEFF2 also promoted survival of mesencephalic dopaminergic neurons. Together, these findings suggest that TMEFF2 may be a novel survival factor for hippocampal and mesencephalic, but not for cortical, neurons.     

The COOH-terminal domain of agrin signals via a synaptic receptor in central nervous system neurons

Agrin is a motor neuron-derived factor that directs formation of the postsynaptic apparatus of the neuromuscular junction. Agrin is also expressed in the brain, raising the possibility that it might serve a related function at neuron-neuron synapses. Previously, we identified an agrin signaling pathway in central nervous system (CNS) neurons, establishing the existence of a neural receptor that mediates responses to agrin. As a step toward identifying this agrin receptor, we have characterized the minimal domains in agrin that bind and activate it. Structures required for agrin signaling in CNS neurons are contained within a 20-kD COOH-terminal fragment of the protein. Agrin signaling is independent of alternative splicing at the z site, but requires sequences that flank it because their deletion results in a 15-kD fragment that acts as an agrin antagonist. Thus, distinct regions within agrin are responsible for receptor binding and activation. Using the minimal agrin fragments as affinity probes, we also studied the expression of the agrin receptor on CNS neurons. Our results show that both agrin and its receptor are concentrated at neuron-neuron synapses. These data support the hypothesis that agrin plays a role in formation and/or function of CNS synapses.     

Formation of functional synaptic connections between cultured cortical neurons from agrin-deficient mice.

Numerous studies suggest that the extracellular matrix protein agrin directs the formation of the postsynaptic apparatus at the neuromuscular junction (NMJ). Strong support for this hypothesis comes from the observation that the high density of acetylcholine receptors (AChR) normally present at the neuromuscular junction fails to form in muscle of embryonic agrin mutant mice. Agrin is expressed by many populations of neurons in the central nervous system (CNS), suggesting that this molecule may also play a role in neuron-neuron synapse formation. To test this hypothesis, we examined synapse formation between cultured cortical neurons isolated from agrin-deficient mouse embryos. Our data show that glutamate receptors accumulate at synaptic sites on agrin-deficient neurons. Moreover, electrophysiological analysis demonstrates that functional glutamatergic and gamma-aminobutyric acid (GABA)ergic synapses form between mutant neurons. The frequency and amplitude of miniature postsynaptic glutamatergic and GABAergic currents are similar in mutant and age-matched wild-type neurons during the first 3 weeks in culture. These results demonstrate that neuron-specific agrin is not required for formation and early development of functional synaptic contacts between CNS neurons, and suggest that mechanisms of interneuronal synaptogenesis are distinct from those regulating synapse formation at the neuromuscular junction.     

Protein Kinase B (PknB) of Mycobacterium tuberculosis Is Essential for Growth of the Pathogen in Vitro as well as for Survival within the Host

The Mycobacterium tuberculosis protein kinase B (PknB) comprises an intracellular kinase domain, connected through a transmembrane domain to an extracellular region that contains four PASTA domains. The present study describes the comprehensive analysis of different domains of PknB in the context of viability in avirulent and virulent mycobacteria. We find stringent regulation of PknB expression necessary for cell survival, with depletion or overexpression of PknB leading to cell death. Although PknB-mediated kinase activity is essential for cell survival, active kinase lacking the transmembrane or extracellular domain fails to complement conditional mutants not expressing PknB. By creating chimeric kinases, we find that the intracellular kinase domain has unique functions in the virulent strain, which cannot be substituted by other kinases. Interestingly, we find that although the presence of the C-terminal PASTA domain is dispensable in the avirulent M. smegmatis, all four PASTA domains are essential in M. tuberculosis. The differential behavior of PknB vis-à-vis the number of essential PASTA domains and the specificity of kinase domain functions suggest that PknB-mediated growth and signaling events differ in virulent compared with avirulent mycobacteria. Mouse infection studies performed to determine the role of PknB in mediating pathogen survival in the host demonstrate that PknB is not only critical for growth of the pathogen in vitro but is also essential for the survival of the pathogen in the host.     

Base of the thumb domain modulates epithelial sodium channel gating

The activity of the epithelial sodium channel (ENaC) is modulated by multiple external factors, including proteases, cations, anions and shear stress. The resolved crystal structure of acid-sensing ion channel 1 (ASIC1), a structurally related ion channel, and mutagenesis studies suggest that the large extracellular region is involved in recognizing external signals that regulate channel gating. The thumb domain in the extracellular region of ASIC1 has a cylinder-like structure with a loop at its base that is in proximity to the tract connecting the extracellular region to the transmembrane domains. This loop has been proposed to have a role in transmitting proton-induced conformational changes within the extracellular region to the gate. We examined whether loops at the base of the thumb domains within ENaC subunits have a similar role in transmitting conformational changes induced by external Na(+) and shear stress. Mutations at selected sites within this loop in each of the subunits altered channel responses to both external Na(+) and shear stress. The most robust changes were observed at the site adjacent to a conserved Tyr residue. In the context of channels that have a low open probability due to retention of an inhibitory tract, mutations in the loop activated channels in a subunit-specific manner. Our data suggest that this loop has a role in modulating channel gating in response to external stimuli, and are consistent with the hypothesis that external signals trigger movements within the extracellular regions of ENaC subunits that are transmitted to the channel gate.     

Agrin fragments differentially induce ectopic aggregation of acetylcholine receptors in myotomal muscles of Xenopus embryos

Agrin is an extracellular synaptic protein that organizes the postsynaptic apparatus, including acetylcholine receptors (AChRs), of the neuromuscular junction. The COOH-terminal portion of agrin has full AChR-aggregating activity in culture, and includes three globular domains, G1, G2, and G3. Portions of the agrin protein containing these domains bind to different cell surface proteins of muscle cells, including alpha-dystroglycan (G1-G2) and heparan sulfate proteoglycans (G2), whereas the G3 domain is sufficient to aggregate AChRs. We sought to determine whether the G1 and G2 domains of agrin potentiate agrin activity in vivo, as they do in culture. Fragments from the COOH-terminal of a neuronal agrin isoform (4,8) containing G3, both G2 and G3, or all three G domains were overexpressed in Xenopus embryos during neuromuscular synapse formation in myotomal muscles. RNA encoding these fragments of rat agrin was injected into one-cell embryos. All three fragments increased the ectopic aggregation of AChRs in noninnervated regions near the center of myotomes. Surprisingly, ectopic aggregation was more pronounced after overexpression of the smallest fragment, which lacks the heparin- and alpha-dystroglycan-binding domains. Synaptic AChR aggregation was decreased in embryos overexpressing the fragments, suggesting a competition between endogenous agrin secreted by nerve terminals and exogenous agrin fragments secreted by muscle cells. These results suggest that binding of the larger agrin fragments to alpha-dystroglycan and/or heparan sulfate proteoglycans may sequester the fragments and inhibit their activity in embryonic muscle. These intermolecular interactions may regulate agrin activity and differentiation of the neuromuscular junction in vivo.     

The Ig1/2 domain of MuSK binds to muscle surface and is involved in acetylcholine receptor clustering

The neuromuscular junction, the synapse between motor neurons and muscle cells, serves as an excellent model for studying synapse formation. Agrin is believed to be released by motor neurons to induce postsynaptic differentiation at the neuromuscular junction. MuSK, a receptor tyrosine kinase, appears to be a key component of the agrin receptor complex. However, how agrin activates MuSK remains unclear. To address this question, we characterized the binding of the MuSK extracellular region to the muscle cell surface. The MuSK ectodomain was found to bind to muscle cells in a manner dependent on stimulation with neural agrin. Moreover, the binding was myotube specific and appeared to be mediated by two regions in the MuSK: one region containing the first and second immunoglobin domains and the other containing the cysteine-rich domain. Importantly, recombinant proteins containing the binding activity can block full-length MuSK binding to muscle cells and agrin-induced AChR clustering. These results suggest that the Ig1/2 domain of MuSK is involved in AChR clustering by binding to the muscle surface.     

2,3-benzodiazepine-type AMPA receptor antagonists and their neuroprotective effects

AMPA receptors are fast ligand-gated members of glutamate receptors in neuronal and many types of non-neuronal cells. The heterotetramer complexes are assembled from four subunits (GluR1-4) in region-, development- and function-selective patterns. Each subunit contains three extracellular domains (a large amino terminal domain, an agonist-binding domain and a transducer domain), and three transmembrane segments with a loop (pore forming domain), as well as the intracellular carboxy terminal tail (traffic and conductance regulatory domain). The binding of the agonist (excitatory amino acids and their derivatives) initiates conformational realignments, which transmit to the transducer domain and membrane spanning segments to gate the channel permeable to Na+, K+ and more or less to Ca2+. Several 2,3-benzodiazepines act as non-competitive antagonists of the AMPA receptor (termed also negative allosteric modulators), which are thought to bind to the transducer domains and inhibit channel gating. Analysing their effects in vitro, it has been possible to recognize a structure-activity relationship, and to describe the critical parts of the molecules involved in their action at AMPA receptors. Blockade of AMPA receptors can protect the brain from apoptotic and necrotic cell death by preventing neuronal excitotoxicity during pathophysiological activation of glutamatergic neurons. Animal experiments provided evidence for the potential usefulness of non-competitive AMPA antagonists in the treatment of human ischemic and neurodegenerative disorders including stroke, multiple sclerosis, Parkinson's disease, periventricular leukomalacia and motoneuron disease. 2,3-benzodiazepine AMPA antagonists can protect against seizures, decrease levodopa-induced dyskinesia in animal models of Parkinson's disease demonstrating their utility for the treatment of a variety of CNS disorders.     

Opalin, a transmembrane sialylglycoprotein located in the central nervous system myelin paranodal loop membrane

In contrast to compact myelin, the series of paranodal loops located in the outermost lateral region of myelin is non-compact; the intracellular space is filled by a continuous channel of cytoplasm, the extracellular surfaces between neighboring loops keep a definite distance, but the loop membranes have junctional specializations. Although the proteins that form compact myelin have been well studied, the protein components of paranodal loop membranes are not fully understood. This report describes the biochemical characterization and expression of Opalin as a novel membrane protein in paranodal loops. Mouse Opalin is composed of a short N-terminal extracellular domain (amino acid residues 1-30), a transmembrane domain (residues 31-53), and a long C-terminal intracellular domain (residues 54-143). Opalin is enriched in myelin of the central nervous system, but not that of the peripheral nervous system of mice. Enzymatic deglycosylation showed that myelin Opalin contained N- and O-glycans, and that the O-glycans, at least, had negatively charged sialic acids. We identified two N-glycan sites at Asn-6 and Asn-12 and an O-glycan site at Thr-14 in the extracellular domain. Site-directed mutations at the glycan sites impaired the cell surface localization of Opalin. In addition to the somata and processes of oligodendrocytes, Opalin immunoreactivity was observed in myelinated axons in a spiral fashion, and was concentrated in the paranodal loop region. Immunogold electron microscopy demonstrated that Opalin was localized at particular sites in the paranodal loop membrane. These results suggest a role for highly sialylglycosylated Opalin in an intermembranous function of the myelin paranodal loops in the central nervous system.     

The fourth extracellular loop of the alpha subunit of Na,K-ATPase. Functional evidence for close proximity with the second extracellular loop

Na,K-ATPase is the main active transport system that maintains the large gradients of Na(+) and K(+) across the plasma membrane of animal cells. The crystal structure of a K(+)-occluding conformation of this protein has been recently published, but the movements of its different domains allowing for the cation pumping mechanism are not yet known. The structure of many more conformations is known for the related calcium ATPase SERCA, but the reliability of homology modeling is poor for several domains with low sequence identity, in particular the extracellular loops. To better define the structure of the large fourth extracellular loop between the seventh and eighth transmembrane segments of the alpha subunit, we have studied the formation of a disulfide bond between pairs of cysteine residues introduced by site-directed mutagenesis in the second and the fourth extracellular loop. We found a specific pair of cysteine positions (Y308C and D884C) for which extracellular treatment with an oxidizing agent inhibited the Na,K pump function, which could be rapidly restored by a reducing agent. The formation of the disulfide bond occurred preferentially under the E2-P conformation of Na,K-ATPase, in the absence of extracellular cations. Using recently published crystal structure and a distance constraint reproducing the existence of disulfide bond, we performed an extensive conformational space search using simulated annealing and showed that the Tyr(308) and Asp(884) residues can be in close proximity, and simultaneously, the SYGQ motif of the fourth extracellular loop, known to interact with the extracellular domain of the beta subunit, can be exposed to the exterior of the protein and can easily interact with the beta subunit.     

Expression and localization of agrin during sympathetic synapse formation in vitro

Agrin is a motoneuron-derived signaling factor that plays a key organizing role in the initial stages of neuromuscular synapse formation. Agrin is expressed in other regions of the developing central and peripheral nervous systems, however, raising the possibility that it also directs the formation of some interneuronal synapses. To address this question, we have examined the expression and localization of agrin during formation of cholinergic, interneuronal synapses in the sympathetic system. In the superior cervical ganglia (SCG) in vivo, we found that agrin is highly expressed, and that it is present at, but is not limited to, synapses. In SCG neuronal cultures that were treated with ciliary neurotrophic factor to induce a uniform cholinergic phenotype, we found that agrin immunostaining colocalized precisely with cholinergic terminals and aggregates of neuronal acetylcholine receptor on the neuronal cell bodies and dendrites. Moreover, we found that alpha-dystroglycan, which is a potential receptor for agrin, is also concentrated at these cholinergic synaptic contacts. Finally, the SCG neurons expressed the C-terminal isoform of agrin that is neural-specific and highly active in synaptogenesis, and also the N-terminal splice isoform that occurs as a type II transmembrane protein. These findings show that agrin is specifically localized at sympathetic synapses in vitro, and are consistent with it playing a role in interneuronal synapse formation.     

Analysis of Transmembrane Domains 1 and 4 of the Human Angiotensin II AT1 Receptor by Cysteine-scanning Mutagenesis

The octapeptide hormone angiotensin II (AngII) exerts a wide variety of cardiovascular effects through the activation of the AT₁ receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein-coupled receptors, the AT₁ receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. Here, we investigated the role of the first and fourth transmembrane domains (TMDs) in the formation of the binding pocket of the human AT₁ receptor using the substituted-cysteine accessibility method. Each residue within the Phe-28^(1.32)–Ile-53^(1.57) fragment of TMD1 and Leu-143^(4.40)–Phe-170^(4.67) fragment of TMD4 was mutated, one at a time, to a cysteine. The resulting mutant receptors were expressed in COS-7 cells, which were subsequently treated with the charged sulfhydryl-specific alkylating agent methanethiosulfonate ethylammonium (MTSEA). This treatment led to a significant reduction in the binding affinity of TMD1 mutants M30C^(1.34)-AT₁ and T33C^(1.37)-AT₁ and TMD4 mutant V169C^(4.66)-AT₁. Although this reduction in binding of the TMD1 mutants was maintained when examined in a constitutively active receptor (N111G-AT₁) background, we found that V169C^(4.66)-AT₁ remained unaffected when treated with MTSEA compared with untreated in this context. Moreover, the complete loss of binding observed for R167C^(4.64)-AT₁ was restored upon treatment with MTSEA. Our results suggest that the extracellular portion of TMD1, particularly residues Met-30^(1.34) and Thr-33^(1.37), as well as residues Arg-167^(4.64) and Val-169^(4.66) at the junction of TMD4 and the second extracellular loop, are important binding determinants within the AT₁ receptor binding pocket but that these TMDs undergo very little movement, if at all, during the activation process.     

Reelin induces EphB activation

The integration of newborn neurons into functional neuronal networks requires migration of cells to their final position in the developing brain, the growth and arborization of neuronal processes and the formation of synaptic contacts with other neurons. A central player among the signals that coordinate this complex sequence of differentiation events is the secreted glycoprotein Reelin, which also modulates synaptic plasticity, learning and memory formation in the adult brain. Binding of Reelin to ApoER2 and VLDL receptor, two members of the LDL receptor family, initiates a signaling cascade involving tyrosine phosphorylation of the intracellular cytoplasmic adaptor protein Disabled-1, which targets the neuronal cytoskeleton and ultimately controls the positioning of neurons throughout the developing brain. However, it is possible that Reelin signals interact with other receptor-mediated signaling cascades to regulate different aspects of brain development and plasticity. EphB tyrosine kinases regulate cell adhesion and repulsion-dependent processes via bidirectional signaling through ephrin B transmembrane proteins. Here, we demonstrate that Reelin binds to the extracellular domains of EphB transmembrane proteins, inducing receptor clustering and activation of EphB forward signaling in neurons, independently of the ''classical'' Reelin receptors, ApoER2 and VLDLR. Accordingly, mice lacking EphB1 and EphB2 display a positioning defect of CA3 hippocampal pyramidal neurons, similar to that in Reelin-deficient mice, and this cell migration defect depends on the kinase activity of EphB proteins. Together, our data provide biochemical and functional evidence for signal integration between Reelin and EphB forward signaling.