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Mol Cell Neurosci. 2002 Jan;19(1):18-31.

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

Author information

1
Department of Pharmacology and Program in Cell and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York 13210, USA.

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.

PMID:
11817895
DOI:
10.1006/mcne.2001.1060
[Indexed for MEDLINE]

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