RESUMO
Cell-surface protein-protein interactions (PPIs) mediate cell-cell communication, recognition, and responses. We executed an interactome screen of 564 human cell-surface and secreted proteins, most of which are immunoglobulin superfamily (IgSF) proteins, using a high-throughput, automated ELISA-based screening platform employing a pooled-protein strategy to test all 318,096 PPI combinations. Screen results, augmented by phylogenetic homology analysis, revealed â¼380 previously unreported PPIs. We validated a subset using surface plasmon resonance and cell binding assays. Observed PPIs reveal a large and complex network of interactions both within and across biological systems. We identified new PPIs for receptors with well-characterized ligands and binding partners for "orphan" receptors. New PPIs include proteins expressed on multiple cell types and involved in diverse processes including immune and nervous system development and function, differentiation/proliferation, metabolism, vascularization, and reproduction. These PPIs provide a resource for further biological investigation into their functional relevance and may offer new therapeutic drug targets.
Assuntos
Ligantes , Mapas de Interação de Proteínas/fisiologia , Receptores de Superfície Celular/metabolismo , Receptor DCC/química , Receptor DCC/metabolismo , Humanos , Filogenia , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/química , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/classificação , Receptores de Interleucina-1/química , Receptores de Interleucina-1/metabolismo , Família de Moléculas de Sinalização da Ativação Linfocitária/química , Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
In the inner plexiform layer (IPL) of the mouse retina, ~70 neuronal subtypes organize their neurites into an intricate laminar structure that underlies visual processing. To find recognition proteins involved in lamination, we utilized microarray data from 13 subtypes to identify differentially-expressed extracellular proteins and performed a high-throughput biochemical screen. We identified ~50 previously-unknown receptor-ligand pairs, including new interactions among members of the FLRT and Unc5 families. These proteins show laminar-restricted IPL localization and induce attraction and/or repulsion of retinal neurites in culture, placing them in an ideal position to mediate laminar targeting. Consistent with a repulsive role in arbor lamination, we observed complementary expression patterns for one interaction pair, FLRT2-Unc5C, in vivo. Starburst amacrine cells and their synaptic partners, ON-OFF direction-selective ganglion cells, express FLRT2 and are repelled by Unc5C. These data suggest a single molecular mechanism may have been co-opted by synaptic partners to ensure joint laminar restriction.
Assuntos
Comunicação Celular , Glicoproteínas de Membrana/metabolismo , Neurônios/fisiologia , Receptores de Superfície Celular/metabolismo , Retina/anatomia & histologia , Retina/fisiologia , Animais , Bioquímica/métodos , Perfilação da Expressão Gênica , Camundongos , Análise em Microsséries , Receptores de Netrina , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteoma/análiseRESUMO
The Dscam family of immunoglobulin cell surface proteins mediates recognition events between neurons that play an essential role in the establishment of neural circuits. The Drosophila Dscam1 locus encodes tens of thousands of cell surface proteins via alternative splicing. These isoforms exhibit exquisite isoform-specific binding in vitro that mediates homophilic repulsion in vivo. These properties provide the molecular basis for self-avoidance, an essential developmental mechanism that allows axonal and dendritic processes to uniformly cover their synaptic fields. In a mechanistically similar fashion, homophilic repulsion mediated by Drosophila Dscam2 prevents processes from the same class of cells from occupying overlapping synaptic fields through a process called tiling. Genetic studies in the mouse visual system support the view that vertebrate DSCAM also promotes both self-avoidance and tiling. By contrast, DSCAM and DSCAM-L promote layer-specific targeting in the chick visual system, presumably through promoting homophilic adhesion. The fly and mouse studies underscore the importance of homophilic repulsion in regulating neural circuit assembly, whereas the chick studies suggest that DSCAM proteins may mediate a variety of different recognition events during wiring in a context-dependent fashion.
Assuntos
Moléculas de Adesão Celular/metabolismo , Proteínas de Drosophila/metabolismo , Rede Nervosa , Neurônios/metabolismo , Isoformas de Proteínas/metabolismo , Animais , Moléculas de Adesão Celular/genética , Drosophila/anatomia & histologia , Drosophila/fisiologia , Proteínas de Drosophila/genética , Humanos , Rede Nervosa/fisiologia , Neurônios/citologia , Células Fotorreceptoras de Invertebrados/citologia , Células Fotorreceptoras de Invertebrados/fisiologia , Ligação Proteica , Isoformas de Proteínas/genéticaRESUMO
Drosophila Dscam encodes a vast family of immunoglobulin (Ig)-containing proteins that exhibit isoform-specific homophilic binding. This diversity is essential for cell recognition events required for wiring the brain. Each isoform binds to itself but rarely to other isoforms. Specificity is determined by "matching" of three variable Ig domains within an approximately 220 kD ectodomain. Here, we present the structure of the homophilic binding region of Dscam, comprising the eight N-terminal Ig domains (Dscam(1-8)). Dscam(1-8) forms a symmetric homodimer of S-shaped molecules. This conformation, comprising two reverse turns, allows each pair of the three variable domains to "match" in an antiparallel fashion. Structural, genetic, and biochemical studies demonstrate that, in addition to variable domain "matching," intramolecular interactions between constant domains promote homophilic binding. These studies provide insight into how "matching" at all three pairs of variable domains in Dscam mediates isoform-specific recognition.
Assuntos
Proteínas de Drosophila/química , Drosophila melanogaster/metabolismo , Animais , Sítios de Ligação , Moléculas de Adesão Celular , Cristalografia por Raios X , Proteínas de Drosophila/metabolismo , Imunoglobulinas/química , Imunoglobulinas/metabolismo , Modelos Moleculares , Dobramento de Proteína , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de ProteínaRESUMO
Dscam encodes a family of cell surface proteins required for establishing neural circuits in Drosophila. Alternative splicing of Drosophila Dscam can generate 19,008 distinct extracellular domains containing different combinations of three variable immunoglobulin domains. To test the binding properties of many Dscam isoforms, we developed a high-throughput ELISA-based binding assay. We provide evidence that 95% (>18,000) of Dscam isoforms exhibit striking isoform-specific homophilic binding. We demonstrate that each of the three variable domains binds to the same variable domain in an opposing isoform and identify the structural elements that mediate this self-binding of each domain. These studies demonstrate that self-binding domains can assemble in different combinations to generate an enormous family of homophilic binding proteins. We propose that this vast repertoire of Dscam recognition molecules is sufficient to provide each neuron with a unique identity and homotypic binding specificity, thereby allowing neuronal processes to distinguish between self and nonself.
Assuntos
Proteínas de Drosophila/metabolismo , Região Variável de Imunoglobulina/metabolismo , Neurônios/metabolismo , Processamento Alternativo , Animais , Sítios de Ligação , Moléculas de Adesão Celular , Clonagem Molecular , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Ensaio de Imunoadsorção Enzimática , Humanos , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Modelos Moleculares , Modelos Neurológicos , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , Reprodutibilidade dos TestesRESUMO
The Drosophila gene Dscam, encoding Down syndrome cell-adhesion molecule, is required for the development of neural circuits. Alternative splicing of Dscam mRNA potentially generates 38016 isoforms of a cell-surface recognition protein of the immunoglobulin superfamily. These isoforms include 19008 different ectodomains joined to one of two alternative transmembrane segments. Each ectodomain comprises a unique combination of three variable immunoglobulin domains. Biochemical studies support a model in which each isoform preferentially binds to the same isoform on opposing cell surfaces. This homophilic binding requires matching at all three variable immunoglobulin domains. These findings raise the intriguing possibility that specificity of binding by the Dscam isoforms mediates cell-surface recognition events required for wiring the fly brain.
Assuntos
Encéfalo/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/genética , Processamento Alternativo/genética , Animais , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Moléculas de Adesão Celular , Drosophila/embriologia , Drosophila/crescimento & desenvolvimento , Proteínas de Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Modelos Biológicos , Neurônios/citologia , Neurônios/metabolismo , Ligação ProteicaRESUMO
Dscam is an immunoglobulin (Ig) superfamily protein required for the formation of neuronal connections in Drosophila. Through alternative splicing, Dscam potentially gives rise to 19,008 different extracellular domains linked to one of two alternative transmembrane segments, resulting in 38,016 isoforms. All isoforms share the same domain structure but contain variable amino acid sequences within three Ig domains in the extracellular region. We demonstrate that different isoforms exhibit different binding specificity. Each isoform binds to itself but does not bind or binds poorly to other isoforms. The amino acid sequences of all three variable Ig domains determine binding specificity. Even closely related isoforms sharing nearly identical amino acid sequences exhibit isoform-specific binding. We propose that this preferential homophilic binding specificity regulates interactions between cells and contributes to the formation of complex patterns of neuronal connections.
Assuntos
Processamento Alternativo/genética , Proteínas de Drosophila , Embrião não Mamífero/embriologia , Cones de Crescimento/metabolismo , Sistema Nervoso/embriologia , Proteínas/genética , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos/genética , Animais , Sítios de Ligação/genética , Células COS , Moléculas de Adesão Celular , Comunicação Celular/genética , Diferenciação Celular/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Drosophila melanogaster , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Cones de Crescimento/ultraestrutura , Sistema Nervoso/citologia , Sistema Nervoso/metabolismo , Ligação Proteica/genética , Biossíntese de Proteínas , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína/genética , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/genéticaRESUMO
Alzheimer's disease is characterized by deposition of beta-amyloid peptide (Abeta) into plaques in the brain, leading to neuronal toxicity and dementia. Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system can also cause a dementia, and amyloid deposition in the central nervous system is significantly higher in HIV-1-infected individuals compared with uninfected controls. Here we report that Abeta fibrils stimulated, by 5-20-fold, infection of target cells expressing CD4 and an appropriate coreceptor by multiple HIV-1 isolates but did not permit infection of cells lacking these receptors. Abeta enhanced infection at the stage of virus attachment or entry into the cell. Abeta fibrils also stimulated infection by amphotrophic Moloney leukemia virus, herpes simplex virus, and viruses pseudotyped with the envelope glycoprotein of vesicular stomatitis virus. Other synthetic fibril-forming peptides similarly enhanced viral infection and may be useful in gene delivery applications utilizing retroviral vectors. These data suggest that Abeta deposition may increase the vulnerability of the central nervous system to enveloped viral infection and that amyloidogenic peptides could be useful in enhancing gene transfer by enveloped viral vectors.