Characterization of a mouse amelogenin [A-4]/M59 cell surface receptor.
Amelogenin proteins comprise up to 90% of the organic matrix of developing enamel in the vertebrate tooth. Alternative splicing of mouse amelogenin pre-mRNA leads to the production of more than 14 protein isoforms, the functions of which are not totally understood. The smaller splice products, [A + 4] or M73 and [A - 4] or M59, have been shown to act differently as signaling molecules affecting odontogenic and other cell types. The mechanisms of these signaling processes, beginning with receptor identification, are not well understood. Utilizing radiolabeled [A - 4], we show here that 3H[A - 4] binds in a saturable fashion to the cell surface of C2C12 mouse fetal myoblasts at 4 degrees C, and not only binds at the surface but is internalized at 37 degrees C. "Far Western" immunohistochemistry performed on sections of E18 mouse incisors and molars with biotin-labeled [A - 4] as the primary ligand demonstrates [A - 4]-biotin binding to polarizing ameloblasts and odontoblasts, cells of the dental follicle, and along the stratum intermedium. Using [A - 4] affinity column chromatography and [A - 4]-biotin label transfer reaction, we have identified a 95 kDa C2C12 cell surface protein which bound [A - 4]. Utilizing Tandem MS (MS/MS) sequencing, we report the novel finding of the 95 kDa murine transmembrane protein, LAMP-1, originally identified as a lysosomal membrane protein that is also found at the cell surface, as an [A - 4] cell binding protein.
Tompkins
Kevin
K
Department of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, 303 E. Chicago Ave Ward-13-100 Chicago, IL 60611, USA.
George
Anne
A
Veis
Arthur
A
D017398:Alternative Splicing; D000565:Ameloblasts / Q000378:metabolism*; D053523:Amelogenin; D000595:Amino Acid Sequence; D000818:Animals; D001710:Biotin / Q000378:metabolism; D026481:Blotting, Far-Western; D002460:Cell Line; D003746:Dental Enamel Proteins / Q000235:genetics* / Q000378:metabolism*; D007180:Incisor; D051907:Lysosomal Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D051379:Mice; D008963:Molar; D008969:Molecular Sequence Data; D009804:Odontoblasts / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D052118:Lysosomal-Associated Membrane Protein 1
D053523:Amelogenin; D001710:Biotin; D003746:Dental Enamel Proteins; D051907:Lysosomal Membrane Proteins; D011956:Receptors, Cell Surface; C507200:Amelx protein, mouse; C052255:Lamp1 protein, mouse; D052118:Lysosomal-Associated Membrane Protein 1