RESUMO
BACKGROUND: DNA aptamers generated by cell-SELEX offer an attractive alternative to antibodies, but generating aptamers to specific, known membrane protein targets has proven challenging, and has severely limited the use of aptamers as affinity reagents for cell identification and purification. METHODOLOGY: We modified the BJAB lymphoblastoma cell line to over-express the murine c-kit cell surface receptor. After six rounds of cell-SELEX, high-throughput sequencing and bioinformatics analysis, we identified aptamers that bound BJAB cells expressing c-kit but not wild-type BJAB controls. One of these aptamers also recognizes c-kit endogenously expressed by a mast cell line or hematopoietic progenitor cells, and specifically blocks binding of the c-kit ligand stem cell factor (SCF). This aptamer enables better separation by fluorescence-activated cell sorting (FACS) of c-kit(+) hematopoietic progenitor cells from mixed bone marrow populations than a commercially available antibody, suggesting that this approach may be broadly useful for rapid isolation of affinity reagents suitable for purification of other specific cell types. CONCLUSIONS/SIGNIFICANCE: Here we describe a novel procedure for the efficient generation of DNA aptamers that bind to specific cell membrane proteins and can be used as high affinity reagents. We have named the procedure STACS (Specific TArget Cell-SELEX).
Assuntos
Aptâmeros de Nucleotídeos , Técnica de Seleção de Aptâmeros , Animais , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Células da Medula Óssea/metabolismo , Linhagem Celular Tumoral , Biologia Computacional/métodos , Citometria de Fluxo , Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Masculino , Camundongos , Ligação Proteica , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Fator de Células-Tronco/metabolismoRESUMO
Precise trafficking, localization, and activity of inward rectifier potassium Kir2 channels are important for shaping the electrical response of skeletal muscle. However, how coordinated trafficking occurs to target sites remains unclear. Kir2 channels are tetrameric assemblies of Kir2.x subunits. By immunocytochemistry we show that endogenous Kir2.1 and Kir2.2 are localized at the plasma membrane and T-tubules in rodent skeletal muscle. Recently, a new subunit, Kir2.6, present in human skeletal muscle, was identified as a gene in which mutations confer susceptibility to thyrotoxic hypokalemic periodic paralysis. Here we characterize the trafficking and interaction of wild type Kir2.6 with other Kir2.x in COS-1 cells and skeletal muscle in vivo. Immunocytochemical and electrophysiological data demonstrate that Kir2.6 is largely retained in the endoplasmic reticulum, despite high sequence identity with Kir2.2 and conserved endoplasmic reticulum and Golgi trafficking motifs shared with Kir2.1 and Kir2.2. We identify amino acids responsible for the trafficking differences of Kir2.6. Significantly, we show that Kir2.6 subunits can coassemble with Kir2.1 and Kir2.2 in vitro and in vivo. Notably, this interaction limits the surface expression of both Kir2.1 and Kir2.2. We provide evidence that Kir2.6 functions as a dominant negative, in which incorporation of Kir2.6 as a subunit in a Kir2 channel heterotetramer reduces the abundance of Kir2 channels on the plasma membrane.
Assuntos
Membrana Celular/metabolismo , Regulação da Expressão Gênica/fisiologia , Músculo Esquelético/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/biossíntese , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Animais , Células COS , Membrana Celular/genética , Chlorocebus aethiops , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Humanos , Paralisia Periódica Hipopotassêmica/genética , Paralisia Periódica Hipopotassêmica/metabolismo , Camundongos , Músculo Esquelético/citologia , Mutação , Canais de Potássio Corretores do Fluxo de Internalização/genética , Crise Tireóidea/genética , Crise Tireóidea/metabolismoRESUMO
Inward rectifier potassium (Kir) channels play important roles in the maintenance and control of cell excitability. Both intracellular trafficking and modulation of Kir channel activity are regulated by protein-protein interactions. We adopted a proteomics approach to identify proteins associated with Kir2 channels via the channel C-terminal PDZ binding motif. Detergent-solubilized rat brain and heart extracts were subjected to affinity chromatography using a Kir2.2 C-terminal matrix to purify channel-interacting proteins. Proteins were identified with multidimensional high pressure liquid chromatography coupled with electrospray ionization tandem mass spectrometry, N-terminal microsequencing, and immunoblotting with specific antibodies. We identified eight members of the MAGUK family of proteins (SAP97, PSD-95, Chapsyn-110, SAP102, CASK, Dlg2, Dlg3, and Pals2), two isoforms of Veli (Veli-1 and Veli-3), Mint1, and actin-binding LIM protein (abLIM) as Kir2.2-associated brain proteins. From heart extract purifications, SAP97, CASK, Veli-3, and Mint1 also were found to associate with Kir2 channels. Furthermore, we demonstrate for the first time that components of the dystrophin-associated protein complex, including alpha1-, beta1-, and beta2-syntrophin, dystrophin, and dystrobrevin, interact with Kir2 channels, as demonstrated by immunoaffinity purification and affinity chromatography from skeletal and cardiac muscle and brain. Affinity pull-down experiments revealed that Kir2.1, Kir2.2, Kir2.3, and Kir4.1 all bind to scaffolding proteins but with different affinities for the dystrophin-associated protein complex and SAP97, CASK, and Veli. Immunofluorescent localization studies demonstrated that Kir2.2 co-localizes with syntrophin, dystrophin, and dystrobrevin at skeletal muscle neuromuscular junctions. These results suggest that Kir2 channels associate with protein complexes that may be important to target and traffic channels to specific subcellular locations, as well as anchor and stabilize channels in the plasma membrane.
Assuntos
Proteínas Associadas à Distrofina , Canais de Potássio Corretores do Fluxo de Internalização/química , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Western Blotting , Encéfalo/embriologia , Encéfalo/metabolismo , Células COS , Membrana Celular/metabolismo , Cerebelo/metabolismo , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , DNA Complementar/metabolismo , Detergentes/farmacologia , Glutationa Transferase/metabolismo , Guanilato Quinases , Immunoblotting , Espectrometria de Massas , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Peptídeos/química , Testes de Precipitina , Isoformas de Proteínas , Estrutura Terciária de Proteína , Transporte Proteico , Proteoma , Proteômica/métodos , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Coloração pela Prata , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Strong inward rectifier potassium (Kir2) channels are important in the control of cell excitability, and their functions are modulated by interactions with intracellular proteins. Here we identified a complex of scaffolding/trafficking proteins in brain that associate with Kir2.1, Kir2.2, and Kir2.3 channels. By using a combination of affinity interaction pulldown assays and co-immunoprecipitations from brain and transfected cells, we demonstrated that a complex composed of SAP97, CASK, Veli, and Mint1 associates with Kir2 channels via the C-terminal PDZ-binding motif. We further demonstrated by using in vitro protein interaction assays that SAP97, Veli-1, or Veli-3 binds directly to the Kir2.2 C terminus and recruits CASK. Co-immunoprecipitations indicated that specific Veli isoforms participate in forming distinct protein complexes in brain, where Veli-1 stably associates with CASK and SAP97, Veli-2 associates with CASK and Mint1, and Veli-3 associates with CASK, SAP97, and Mint1. Additionally, immunocytochemistry of rat cerebellum revealed overlapping expression of Kir2.2, SAP97, CASK, Mint1, with Veli-1 in the granule cell layer and Veli-3 in the molecular layer. We propose a model whereby Kir2.2 associates with distinct SAP97-CASK-Veli-Mint1 complexes. In one complex, SAP97 interacts directly with the Kir2 channels and recruits CASK, Veli, and Mint1. Alternatively, Veli-1 or Veli-3 interacts directly with the Kir2 channels and recruits CASK and SAP97; association of Mint1 with the complex requires Veli-3. Expression of Kir2.2 in polarized epithelial cells resulted in targeting of the channels to the basolateral membrane and co-localization with SAP97 and CASK, whereas a dominant interfering form of CASK caused the channels to mislocalize. Therefore, CASK appears to be a central protein of a macromolecular complex that participates in trafficking and plasma membrane localization of Kir2 channels.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/metabolismo , Polaridade Celular , Cerebelo/química , Proteína 1 Homóloga a Discs-Large , Células Epiteliais/metabolismo , Glutationa Transferase , Guanilato Quinases , Coração , Substâncias Macromoleculares , Proteínas de Membrana/metabolismo , Complexos Multiproteicos , Ligação Proteica , Mapeamento de Interação de Proteínas , Transporte Proteico , Ratos , Proteínas Recombinantes de Fusão/isolamento & purificação , TransfecçãoRESUMO
Oligosaccharides play significant roles in trafficking, folding, and sorting of membrane proteins. Sulfonylurea receptors (SURx), members of the ATP binding cassette family of proteins, associate with the inward rectifier Kir6.x to form ATP-sensitive potassium channels (K(ATP)). These channels are found on the plasma membrane in many tissues and play a pivotal role in synchronizing electrical excitability with cell metabolic state. Trafficking defects resulting from three independent SUR1 mutations involved in the disease persistent hyperinsulinemic hypoglycemia of infancy have been described. Two of these mutations displayed notable decreases in glycosylation. Here we have investigated the relationship between the two N-linked glycosylation sites (Asn(10) and Asn(1050)) and SUR1 trafficking. Using patch clamp analysis, surface biotinylation, and immunofluorescence microscopy, we demonstrate a significant decrease in surface expression of SUR1 single or double glycosylation site mutants (N10Q,N1050Q) when co-expressed with Kir6.2. Additionally, we show prominent retention within the ER of the SUR1 double glycosylation mutant under the same conditions. Further investigation revealed that mutation of the ER retention signal was able to partially restore surface expression of the SUR1 double glycosylation mutant. These studies suggest that SUR1 glycosylation is a key element for the proper trafficking and surface expression of K(ATP) channels.