RESUMO
Neuronal development and function are dependent in part on the several roles of the secreted glycoprotein Reelin. Endogenous proteases process this 400 kDa, modular protein, yielding N-terminal, central, and C-terminal fragments that each have distinct roles in Reelin's function and regulation. The C-terminal fragment comprises Reelin repeat (RR) domains seven and eight, as well as a basic stretch of 32 amino acid residues termed the C-terminal region (CTR), influences Reelin signaling intensity, and has been reported to bind to Neuropilin-1, which serves as a co-receptor in the canonical Reelin signaling pathway. Here, we present a crystal structure of RR8 at 3.0 Å resolution. Analytical ultracentrifugation and small-angle x-ray scattering confirmed that RR8 is monomeric and enabled us to identify the CTR as a flexible, yet compact subdomain. We conducted structurally informed protein engineering to design a chimeric RR8 construct guided by the structural similarities with RR6. Experimental results support a mode of Reelin-receptor interaction reliant on the multiple interfaces coordinating the binding event. Structurally, RR8 resembles other individual RRs, but its structure does show discrete differences that may account for Reelin receptor specificity toward RR6.
Assuntos
Moléculas de Adesão Celular Neuronais , Proteínas da Matriz Extracelular , Moléculas de Adesão Celular Neuronais/química , Proteínas da Matriz Extracelular/genética , Proteínas do Tecido Nervoso/química , Neurônios/metabolismo , Proteína Reelina , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismoRESUMO
Reelin is a secreted glycoprotein that is integral in neocortex development and synaptic function. Reelin exists as a homodimer with two chains linked by a disulfide bond at cysteine 2101, a feature that is vital to the protein's function. This is highlighted by the fact that only dimeric Reelin can elicit efficient, canonical signaling, even though a mutated (C2101A) monomeric construct of Reelin retains the capacity to bind to its receptors. Receptor clustering has been shown to be important in the signaling pathway, however direct evidence regarding the stoichiometry of Reelin-receptor binding interaction is lacking. Here we describe the construction and purification of a heterodimeric Reelin construct to investigate the stoichiometry of Reelin-receptor binding and how it affects Reelin pathway signaling. We have devised different strategies and have finalized a protocol to produce a heterodimer of Reelin's central fragment using differential tagging and tandem affinity chromatography, such that chain A is wild type in amino acid sequence whereas chain B includes a receptor binding site mutation (K2467A). We also validate that the heterodimer is capable of binding to the extracellular domain of one of Reelin's known receptors, calculating the KD of the interaction. This heterodimeric construct will enable us to understand in greater detail the mechanism by which Reelin interacts with its known receptors and initiates pathway signaling.
Assuntos
Moléculas de Adesão Celular Neuronais/química , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Multimerização Proteica , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Moléculas de Adesão Celular Neuronais/isolamento & purificação , Proteínas da Matriz Extracelular/isolamento & purificação , Células HEK293 , Humanos , Proteínas do Tecido Nervoso/isolamento & purificação , Neurônios/citologia , Neurônios/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Proteína Reelina , Serina Endopeptidases/isolamento & purificação , Transdução de SinaisRESUMO
Although many neuronal membrane proteins undergo proteolytic cleavage, little is known about the biological significance of neuronal ectodomain shedding (ES). Here, we show that the neuronal sheddome is detectable in human cerebrospinal fluid (hCSF) and is enriched in neurodevelopmental disorder (NDD) risk factors. Among shed synaptic proteins is the ectodomain of CNTNAP2 (CNTNAP2-ecto), a prominent NDD risk factor. CNTNAP2 undergoes activity-dependent ES via MMP9 (matrix metalloprotease 9), and CNTNAP2-ecto levels are reduced in the hCSF of individuals with autism spectrum disorder. Using mass spectrometry, we identified the plasma membrane Ca2+ ATPase (PMCA) extrusion pumps as novel CNTNAP2-ecto binding partners. CNTNAP2-ecto enhances the activity of PMCA2 and regulates neuronal network dynamics in a PMCA2-dependent manner. Our data underscore the promise of sheddome analysis in discovering neurobiological mechanisms, provide insight into the biology of ES and its relationship with the CSF, and reveal a mechanism of regulation of Ca2+ homeostasis and neuronal network synchrony by a shed ectodomain.
Assuntos
Transtorno do Espectro Autista , Proteínas de Membrana , Proteínas do Tecido Nervoso , ATPases Transportadoras de Cálcio da Membrana Plasmática , Transtorno do Espectro Autista/líquido cefalorraquidiano , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/metabolismo , Membrana Celular/metabolismo , Homeostase , Humanos , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/líquido cefalorraquidiano , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Transdução de SinaisRESUMO
Reelin operates through canonical and non-canonical pathways that mediate several aspects of brain development and function. Reelin's dimeric central fragment (CF), generated through proteolytic cleavage, is required for the lipoprotein-receptor-dependent canonical pathway activation. Here, we analyze the signaling properties of a variety of Reelin fragments and measure the differential binding affinities of monomeric and dimeric CF fragments to lipoprotein receptors to investigate the mode of canonical signal activation. We also present the cryoelectron tomography-solved dimeric structure of Reelin CF and support it using several other biophysical techniques. Our findings suggest that Reelin CF forms a covalent parallel dimer with some degree of flexibility between the two protein chains. As a result of this conformation, Reelin binds to lipoprotein receptors in a manner inaccessible to its monomeric form and is capable of stimulating canonical pathway signaling.
Assuntos
Proteína Reelina/química , Microscopia Crioeletrônica , Células HEK293 , Humanos , Domínios Proteicos , Multimerização Proteica , Receptores de LDL/metabolismo , Proteína Reelina/metabolismo , Transdução de SinaisRESUMO
In the developing brain, cell-surface proteins play crucial roles, but their protein-protein interaction network remains largely unknown. A proteomic screen identified 200 interactions, 89 of which were not previously published. Among these interactions, we find that the IgLONs, a family of five cell-surface neuronal proteins implicated in various human disorders, interact as homo- and heterodimers. We reveal their interaction patterns and report the dimeric crystal structures of Neurotrimin (NTRI), IgLON5, and the neuronal growth regulator 1 (NEGR1)/IgLON5 complex. We show that IgLONs maintain an extended conformation and that their dimerization occurs through the first Ig domain of each monomer and is Ca2+ independent. Cell aggregation shows that NTRI and NEGR1 homo- and heterodimerize in trans. Taken together, we report 89 unpublished cell-surface ligand-receptor pairs and describe structural models of trans interactions of IgLONs, showing that their structures are compatible with a model of interaction across the synaptic cleft.