Novel cytosolic binding partners of the neural cell adhesion molecule: mapping the binding domains of PLC gamma, LANP, TOAD-64, syndapin, PP1, and PP2A.
The neural cell adhesion molecule (NCAM) is implicated in important functions during development and maintenance of the nervous system. Two of the three major isoforms, NCAM 140 and NCAM 180, are transmembrane glycoproteins with large cytoplasmic domains of different length. The purpose of this study was to identify novel intracellular binding partners of NCAM 140 and NCAM 180. We expressed both cytoplasmic domains, as well as cytoplasmic fragments of NCAM, as fusion proteins in Escherichia coli and used them for ligand affinity chromatography or glutathione S-transferase (GST) pull-down assays. By peptide mass fingerprinting Western blot analysis, or both, we identified PLCgamma, LANP, syndapin, PP1, and PP2A as binding partners for both NCAM 140 and NCAM 180, whereas TOAD-64 was identified as a NCAM 180-specific interacting protein. Furthermore, we were able to show that binding of these novel binding proteins, as well as the previously described interaction partners ROK alpha (rho A binding kinase alpha) and alpha- and beta-tubulin, bind to specific cytosolic sequences of NCAM. For this purpose, we performed GST pull-down experiments using cytosolic fragments of NCAM as GST-fusion proteins and cytosolic- or cytoskeleton-enriched protein fractions of rat brain.
Büttner
Bettina
B
Institut für Biochemie und Molekularbiologie, Charité - Universitätsmedizin Berlin, Arnimallee 22, D-14195 Berlin-Dahlem, Germany.
Kannicht
Christoph
C
Reutter
Werner
W
Horstkorte
Rüdiger
R
D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003598:Cytoskeletal Proteins; D003600:Cytosol / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007424:Intracellular Fluid / Q000737:chemistry / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019006:Neural Cell Adhesion Molecules / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009479:Neuropeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051966:Phospholipase C gamma; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D010738:Type C Phospholipases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
C490338:Anp32a protein, rat; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; C449760:Dpysl3 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D019006:Neural Cell Adhesion Molecules; D009479:Neuropeptides; D009687:Nuclear Proteins; C117738:Pacsin1 protein, rat; D010750:Phosphoproteins; D020033:Protein Isoforms; D010749:Phosphoprotein Phosphatases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma