title: Identification of the ankyrin-binding domain of the mouse T-lymphoma cell inositol 1,4,5-trisphosphate (IP3) receptor and its role in the regulation of IP3-mediated internal Ca2+ release.
abstract: In this study we have used several complementary techniques to explore the interaction between the membrane linker molecule, ankyrin, and the inositol 1,4,5-trisphosphate (IP3) receptor in mouse T-lymphoma cells. Using double immunolabeling and laser confocal microscopy, we have found that both cytoplasmic IP3 receptor and ankyrin are preferentially accumulated within ligand-induced lymphocyte receptor-capped structures. The binding between ankyrin and IP3 receptor appears to be very specific. Further analyses indicate that the amino acid sequence GGVGDVLRKPS in the IP3 receptor shares a great deal of structural homology with the ankyrin-binding domain located in certain well characterized ankyrin-binding proteins such as the cell adhesion molecule, CD44. Biochemical studies using competition binding assays and a synthetic peptide identical to GGVGDVLRKPS (a sequence detected in rat brain IP3 receptor (amino acids 2548-2558) and mouse brain IP3 receptor (amino acids 2546-2556)) indicate that this 11-amino acid peptide binds specifically to ankyrin (but not fodrin or spectrin). Furthermore, this peptide competes effectively for ankyrin binding to IP3 receptor-containing vesicles and/or purified IP3 receptor, and it blocks ankyrin-induced inhibitory effects on IP3 binding and IP3-mediated internal Ca2+ release in mouse T-lymphoma cells. These findings suggest that this amino acid sequence, GGVGDVLRKPS, which is located close to the C terminus of the IP3 receptor, resides on the cytoplasmic side (not the luminal side) of IP3 receptor-containing vesicles. This unique region appears to be an important part of the IP3 receptor ankyrin-binding domain and may play an important role in the regulation of IP3 receptor-mediated internal Ca2+ release during lymphocyte activation.
authors.affiliation: Department of Cell Biology and Anatomy, University of Miami Medical School, Florida 33101, USA.
authors.forename: L Y
authors.initials: LY
authors.lastname: Bourguignon
authors.forename: H
authors.initials: H
authors.lastname: Jin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017487:Ankyrins / Q000378:metabolism*; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007457:Iodine Radioisotopes; D007700:Kinetics; D016399:Lymphoma, T-Cell / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011869:Radioligand Assay; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D017487:Ankyrins; D015220:Calcium Channels; D003598:Cytoskeletal Proteins; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007457:Iodine Radioisotopes; D018160:Receptors, Cytoplasmic and Nuclear; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium