Isolation and characterization of detergent-resistant microdomains responsive to NCAM-mediated signaling from growth cones.
It is still largely unclear how cell adhesion molecule (CAM)-mediated signaling evokes responses from the growth cone cytoskeleton. Here we used TX-114 extraction of growth cones followed by equilibrium gradient centrifugation to isolate subfractions of detergent-resistant microdomains (DRMs) that could be structurally and functionally distinguished on the basis of localization and activation of components of CAM-mediated signaling pathways. DRMs enriched in cholesterol, caveolin, NCAM140, GPI-linked NCAM120, fyn, and GAP-43, all conventional markers of microdomains or rafts, were located in areas 2 and 3 of the gradient. Coimmunoprecipitation of specific components of CAM signaling pathways by GAP-43 then identified distinct subpopulations of DRMs. GAP-43 from area 2 DRMs coprecipitated GPI-linked NCAM120 and was inactive, i.e., PKC phosphorylation had not been stimulated. In contrast the GAP-43 from area 3 DRMs coprecipitated both transmembrane NCAM140 and caveolin and was active, i.e., highly phosphorylated by PKC. A different subset of DRMs from both area 2 and area 3 contained fyn that could not be coprecipitated with GAP-43 antibodies. In this case area 2 DRMs contained activated fyn that was phosphorylated on Y415. In contrast area 3 DRMs contained inactive fyn. Hence fyn and GAP-43, both targets of NCAM signaling, are located in distinct populations of DRMs, and their activated forms are reciprocally distributed on the gradient. A detergent-resistant membrane fraction recovered from area 4 was enriched in NCAM140, phosphorylated GAP-43, and actin, but not cholesterol, caveolin, or fyn. Immunoelectron microscopy revealed that phosphorylated GAP-43 was localized where the membranes and F-actin interacted. Our results provide evidence for NCAM-mediated signaling in DRMs and suggest that the DRMs responsible for fyn and PKC/GAP-43-mediated NCAM signaling are structurally distinct and differentially distributed in growth cones.
He
Qin
Q
Department of Pharmacology and Program in Cell and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York 13210, USA.
Meiri
Karina F
KF
D000199:Actins / Q000302:isolation & purification; D000818:Animals; D000918:Antibody Specificity; D001923:Brain Chemistry / Q000502:physiology; D051242:Caveolin 1; D022461:Caveolins / Q000276:immunology / Q000302:isolation & purification; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000737:chemistry / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient; D003599:Cytoskeleton / Q000737:chemistry / Q000648:ultrastructure; D003902:Detergents; D019922:GAP-43 Protein / Q000276:immunology / Q000302:isolation & purification; D020439:Growth Cones / Q000737:chemistry / Q000502:physiology*; D016253:Microscopy, Immunoelectron; D019006:Neural Cell Adhesion Molecules / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*; D017830:Octoxynol; D011092:Polyethylene Glycols; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D051381:Rats; D015398:Signal Transduction / Q000502:physiology*; D013395:Sucrose
D000199:Actins; C495537:Cav1 protein, rat; D051242:Caveolin 1; D022461:Caveolins; D003902:Detergents; D019922:GAP-43 Protein; D019006:Neural Cell Adhesion Molecules; D011092:Polyethylene Glycols; D013395:Sucrose; D017830:Octoxynol; C010615:Nonidet P-40