Isolation and characterization of detergent-resistant microdomains responsive to NCAM-mediated signaling from growth cones

Mol Cell Neurosci. 2002 Jan;19(1):18-31. doi: 10.1006/mcne.2001.1060.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Actins / isolation & purification
  • Animals
  • Antibody Specificity
  • Brain Chemistry / physiology
  • Caveolin 1
  • Caveolins / immunology
  • Caveolins / isolation & purification
  • Cell Fractionation / methods
  • Cell Membrane / chemistry
  • Cell Membrane / ultrastructure
  • Centrifugation, Density Gradient
  • Cytoskeleton / chemistry
  • Cytoskeleton / ultrastructure
  • Detergents
  • GAP-43 Protein / immunology
  • GAP-43 Protein / isolation & purification
  • Growth Cones / chemistry
  • Growth Cones / physiology*
  • Microscopy, Immunoelectron
  • Neural Cell Adhesion Molecules / chemistry*
  • Neural Cell Adhesion Molecules / immunology
  • Neural Cell Adhesion Molecules / isolation & purification*
  • Octoxynol
  • Polyethylene Glycols
  • Precipitin Tests
  • Protein Structure, Tertiary
  • Rats
  • Signal Transduction / physiology*
  • Sucrose

Substances

  • Actins
  • Cav1 protein, rat
  • Caveolin 1
  • Caveolins
  • Detergents
  • GAP-43 Protein
  • Neural Cell Adhesion Molecules
  • Polyethylene Glycols
  • Sucrose
  • Octoxynol
  • Nonidet P-40