The involvement of ankyrin in the regulation of inositol 1,4,5-trisphosphate receptor-mediated internal Ca2+ release from Ca2+ storage vesicles in mouse T-lymphoma cells.
Mouse T-lymphoma cells contain a unique type of internal vesicle which bands at the relatively light density of 1.07 g/cc. These vesicles do not contain any detectable Golgi, endoplasmic reticulum, plasma membrane, or lysosomal marker protein activities. Binding of [3H]inositol 1,4,5-trisphosphate (IP3) to these internal vesicles reveals the presence of a single, high affinity class of IP3 receptor with a dissociation constant (Kd) of 1.6 +/- 0.3 nM. Using a panel of monoclonal and polyclonal antibodies against IP3 receptor, we have established that the IP3 receptor (approximately 260 kDa) displays immunological cross-reactivity with the rat brain IP3 receptor. Polymerase chain reaction analysis of first-strand cDNAs from both mouse T-lymphoma cells and rat brain tissues reveals that the IP3 receptor transcript in mouse T-lymphoma cells belongs to the short form (non-neuronal form) and not the long form (neuronal form) detected in rat brain tissue. Scatchard plot analysis shows that high affinity binding occurs between ankyrin and the IP3 receptor with a Kd of 0.2 nM. Most importantly, the binding of ankyrin to the light density vesicles significantly inhibits IP3 binding and IP3-induced internal Ca2+ release. These findings suggest that the cytoskeleton plays a pivotal role in the regulation of IP3 receptor-mediated internal Ca2+ release during lymphocyte activation.
Department of Cell Biology and Anatomy, School of Medicine, University of Miami, Florida 33101.
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Bourguignon
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Jin
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Iida
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Brandt
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Zhang
D000818:Animals; D017487:Ankyrins / Q000378:metabolism*; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels; D003599:Cytoskeleton / Q000378:metabolism; D004277:DNA, Single-Stranded; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates / Q000378:metabolism*; D050356:Lipid Metabolism; D016399:Lymphoma, T-Cell / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D017487:Ankyrins; D015220:Calcium Channels; D004277:DNA, Single-Stranded; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D002118:Calcium