title: Proteomic analysis of native cerebellar iFGF14 complexes.
abstract: Intracellular Fibroblast Growth Factor 14 (iFGF14) and the other intracellular FGFs (iFGF11-13) regulate the properties and densities of voltage-gated neuronal and cardiac Na(+) (Nav) channels. Recent studies have demonstrated that the iFGFs can also regulate native voltage-gated Ca(2+) (Cav) channels. In the present study, a mass spectrometry (MS)-based proteomic approach was used to identify the components of native cerebellar iFGF14 complexes. Using an anti-iFGF14 antibody, native iFGF14 complexes were immunoprecipitated from wild type adult mouse cerebellum. Parallel control experiments were performed on cerebellar proteins isolated from mice (Fgf14(-/-)) harboring a targeted disruption of the Fgf14 locus. MS analyses of immunoprecipitated proteins demonstrated that the vast majority of proteins identified in native cerebellar iFGF14 complexes are Nav channel pore-forming (α) subunits or proteins previously reported to interact with Nav α subunits. In contrast, no Cav channel α or accessory subunits were revealed in cerebellar iFGF14 immunoprecipitates. Additional experiments were completed using an anti-PanNav antibody to immunoprecipitate Nav channel complexes from wild type and Fgf14(-/-) mouse cerebellum. Western blot and MS analyses revealed that the loss of iFGF14 does not measurably affect the protein composition or the relative abundance of Nav channel interacting proteins in native adult mouse cerebellar Nav channel complexes.
authors.affiliation: a Department of Developmental Biology , Washington University School of Medicine , St. Louis , MO , USA.
authors.forename: Marie K
authors.initials: MK
authors.lastname: Bosch
authors.affiliation: a Department of Developmental Biology , Washington University School of Medicine , St. Louis , MO , USA.|b Internal Medicine, Washington University School of Medicine , St. Louis , MO , USA.
authors.forename: Jeanne M
authors.initials: JM
authors.lastname: Nerbonne
authors.affiliation: b Internal Medicine, Washington University School of Medicine , St. Louis , MO , USA.|c Cell Biology & Physiology, Washington University School of Medicine , St. Louis , MO , USA.
authors.forename: R Reid
authors.initials: RR
authors.lastname: Townsend
authors.affiliation: d Laboratory of Structural Pathology, Doshisha University, Kyotanabe-shi, Kyoto , Japan.
authors.forename: Haruko
authors.initials: H
authors.lastname: Miyazaki
authors.affiliation: d Laboratory of Structural Pathology, Doshisha University, Kyotanabe-shi, Kyoto , Japan.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Nukina
authors.affiliation: a Department of Developmental Biology , Washington University School of Medicine , St. Louis , MO , USA.
authors.forename: David M
authors.initials: DM
authors.lastname: Ornitz
authors.affiliation: e L'Institut du Thorax, INSERM UMR1087, CNRS UMR6291, Université de Nantes , Nantes , France.
authors.forename: Céline
authors.initials: C
authors.lastname: Marionneau
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002531:Cerebellum / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D005346:Fibroblast Growth Factors / Q000032:analysis* / Q000276:immunology; D047468:Immunoprecipitation; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D040901:Proteomics / Q000379:methods*; D015222:Sodium Channels / Q000032:analysis* / Q000276:immunology / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D015222:Sodium Channels; C112971:fibroblast growth factor 14; D005346:Fibroblast Growth Factors
keywords: cerebellum; intracellular fibroblast growth factors; native interactomes; proteomics; voltage-gated Na+ channels