RESUMEN
Sequence homology predicts that the extracellular domain of the sodium channel beta1 subunit forms an immunoglobulin (Ig) fold and functions as a cell adhesion molecule. We show here that beta1 subunits associate with neurofascin, a neuronal cell adhesion molecule that plays a key role in the assembly of nodes of Ranvier. The first Ig-like domain and second fibronectin type III-like domain of neurofascin mediate the interaction with the extracellular Ig-like domain of beta1, confirming the proposed function of this domain as a cell adhesion molecule. beta1 subunits localize to nodes of Ranvier with neurofascin in sciatic nerve axons, and beta1 and neurofascin are associated as early as postnatal day 5, during the period that nodes of Ranvier are forming. This association of beta1 subunit extracellular domains with neurofascin in developing axons may facilitate recruitment and concentration of sodium channel complexes at nodes of Ranvier.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Inmunoglobulinas/genética , Factores de Crecimiento Nervioso/metabolismo , Subunidades de Proteína , Canales de Sodio/metabolismo , Envejecimiento/metabolismo , Animales , Axones/metabolismo , Sitios de Unión/fisiología , Encéfalo/metabolismo , Línea Celular , Fibronectinas/genética , Humanos , Unión Proteica/fisiología , Estructura Terciaria de Proteína/fisiología , Nódulos de Ranvier/metabolismo , Ratas , Nervio Ciático/metabolismo , Canales de Sodio/genética , TransfecciónRESUMEN
We report a novel method for the analysis of protein ligands using a whole molecule mutagenesis/phage display system. The cDNA for the inflammatory polypeptide C5a was used as template in a PCR reaction doped with mutagenic nucleoside triphosphates (dP and 8-oxo-2'deoxyguanosine (8-oxodG)) to allow introduction of mutations in a highly controlled manner throughout the cDNA. The resultant library of mutants was displayed on the surface of phage and functional polypeptides were selected by several rounds of selection against the cells bearing the receptor for C5a. Following selection only a limited number of residues in C5a were found to be mutated, suggesting that mutations in key residues involved in the maintenance of structure and in receptor binding had been eliminated. The selected C5a sequences had a higher affinity for receptor than wild type phage-C5a conjugates. As this method for analysing the functional characteristics of proteins does not rely on knowledge a priori of structure, it may be useful for affinity maturation or analysis in a wide range of protein ligand/receptor systems.
Asunto(s)
Biblioteca de Péptidos , Proteínas/genética , Proteínas/metabolismo , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Células CHO , Línea Celular , Complemento C5a/genética , Complemento C5a/metabolismo , Cricetinae , Humanos , Ligandos , Datos de Secuencia Molecular , Mutagénesis , Receptor de Anafilatoxina C5a , Receptores de Superficie Celular/genética , Receptores de Complemento/genética , Receptores de Complemento/metabolismoRESUMEN
Voltage-gated sodium channels in brain neurons were found to associate with receptor protein tyrosine phosphatase beta (RPTPbeta) and its catalytically inactive, secreted isoform phosphacan, and this interaction was regulated during development. Both the extracellular domain and the intracellular catalytic domain of RPTPbeta interacted with sodium channels. Sodium channels were tyrosine phosphorylated and were modulated by the associated catalytic domains of RPTPbeta. Dephosphorylation slowed sodium channel inactivation, positively shifted its voltage dependence, and increased whole-cell sodium current. Our results define a sodium channel signaling complex containing RPTPbeta, which acts to regulate sodium channel modulation by tyrosine phosphorylation.