Localization of inositol 1,4,5-trisphosphate receptor-like protein in plasmalemmal caveolae.
Activation of various receptors by extracellular ligands induces an influx of Ca2+ through the plasma membrane, but its molecular mechanism remains elusive and seems variable in different cell types. In the present study, we utilized mAbs generated against the cerebellar type I inositol 1,4,5-trisphosphate (InsP3) receptor and performed immunocytochemical and immunochemical experiments to examine its localization in several non-neuronal cells. By immunogold electron microscopy of ultrathin frozen sections as well as permeabilized tissue specimens, we found that a mAb to the type I InsP3 receptor (mAb 4C11) labels the plasma membrane of the endothelium, smooth muscle cell and keratinocyte in vivo. Interestingly, the labeling with the antibody was confined to caveolae, smooth vesicular inpocketings of the plasma membrane. The reactive protein, with an M(r) of 240,000 by SDS-PAGE, could be biotinylated with a membrane-impermeable reagent, sulfo-NHS-biotin, in intact cultured endothelial cells, and recovered by streptavidin-agarose beads, which result further confirmed its presence on the cell surface. The present findings indicate that a protein structurally homologous to the type I InsP3 receptor is localized in the caveolar structure of the plasma membrane and might be involved in the Ca2+ influx.
Fujimoto
T
T
Department of Anatomy, Faculty of Medicine, Kyoto University, Japan.
Nakade
S
S
Miyawaki
A
A
Mikoshiba
K
K
Ogawa
K
K
D000818:Animals; D000911:Antibodies, Monoclonal; D001710:Biotin; D015153:Blotting, Western; D015220:Calcium Channels; D002462:Cell Membrane / Q000737:chemistry* / Q000276:immunology / Q000648:ultrastructure; D004730:Endothelium, Vascular / Q000737:chemistry / Q000276:immunology / Q000648:ultrastructure; D004817:Epidermis / Q000737:chemistry / Q000276:immunology / Q000648:ultrastructure; D007150:Immunohistochemistry; D053496:Inositol 1,4,5-Trisphosphate Receptors; D015603:Keratinocytes / Q000737:chemistry / Q000276:immunology / Q000648:ultrastructure; D051379:Mice; D008815:Mice, Inbred Strains; D016253:Microscopy, Immunoelectron; D009130:Muscle, Smooth / Q000737:chemistry / Q000276:immunology / Q000648:ultrastructure; D011956:Receptors, Cell Surface / Q000276:immunology / Q000302:isolation & purification*; D018160:Receptors, Cytoplasmic and Nuclear
D000911:Antibodies, Monoclonal; D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D001710:Biotin