inositol-1-4-5-trisphosphate and Lymphoma--T-Cell

There are at least three types of inositol 1,4,5-trisphosphate receptor (IP(3)R) [IP(3)-gated Ca(2+)channels], which are expressed in different cell types and mammalian tissues. In this study, we have identified three IP(3)R subtypes in human Jurkat T-lymphoma cells. All three subtypes have a molecular mass of about 260 kDa, and display Ca(2+)channel properties in an IP(3)-dependent manner. We have also demonstrated that TNFalpha promotes the activity of different proteases (e.g. caspase-8, caspase-3 and calpain), alters the TCR-mediated Ca(2+)response and subsequently induces apoptosis in Jurkat cells. During the first 6 h of incubation with TNFalpha, several IP(3)R subtype-related changes occur (e.g. proteolysis of IP(3)R subtypes, inhibition of IP(3)binding and impairment of IP(3)-mediated Ca(2+)flux) concomitantly with an elevation of protease (caspase-8, caspase-3 and calpain) activity. Furthermore, the caspase inhibitor, Z-VAD-fmk, significantly reduces TNFalpha-mediated perturbation of IP(3)R1 and IP(3)R2 (but not IP(3)R3) function; whereas the calpain inhibitor I, ALLN, is capable of blocking the inhibitory effect of TNFalpha on IP(3)R3 function. These findings suggest that IP(3)R1 and IP(3)R2 serve as cellular substrates for caspases, and IP(3)R3 is a substrate for calpain. We propose that the selective down-regulation of IP(3)R subtype-mediated Ca(2+)function by caspase-dependent and calpain-sensitive mechanisms may be responsible for the early onset of the apoptotic signal by TNFalpha in human T-cells.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Binding Sites; Calcium; Calcium Channels; Calpain; Caspase Inhibitors; Caspases; Down-Regulation; Glycoproteins; Humans; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Jurkat Cells; Lymphoma, T-Cell; Microscopy, Confocal; Receptors, Cytoplasmic and Nuclear; Tumor Necrosis Factor-alpha

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.

Topics: Amino Acid Sequence; Animals; Ankyrins; Binding Sites; Brain; Calcium; Calcium Channels; Cell Line; Cytoskeletal Proteins; Fluorescent Antibody Technique; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Iodine Radioisotopes; Kinetics; Lymphoma, T-Cell; Mice; Molecular Sequence Data; Radioligand Assay; Rats; Receptors, Cytoplasmic and Nuclear

Studies were carried out to characterize the interaction between inositol 1,4,5-trisphosphate (IP3) receptors and the plasma membrane fraction. Extraction of the membranes with the nonionic detergents Nonidet P-40 and Triton X-100, followed by centrifugation at 100,000 g, resulted in the doubling of the IP3 receptor in the pellets, whereas no detectable binding was found in the supernatants. These data indicate that the detergents did not solubilize the receptor, that it remained associated with membrane particles, and that it is likely to be associated with the cytoskeleton. The cytoskeleton proteins actin, ankyrin, and spectrin were identified in the plasma membrane fraction. However, comparison of the amount of these proteins in different fractions of the detergent, or otherwise treated plasma membrane fractions, showed no direct correlation between the presence of any of these proteins in the plasma membrane fraction and their ability to bind [3H]IP3. This is in contrast to the brain and T-lymphoma cells in which the IP3 receptor is attached to ankyrin (L. Y. W. Bourguigon, H. Jin, N. Iida, N. R. Brandt, and S. H. Zhang. J. Biol. Chem. 268: 6477-6486, 1993; and S. K. Joseph and S. Samanta. J. Biol. Chem 268: 6477-6486, 1993). Thus the hepatic IP3 receptor, which is different from the brain receptor, might attach to the cytoskeleton by anchoring to a different protein. Because cytochalasin D treatment of livers diminishes the ability of IP3 to raise cytosolic free Ca2+ levels, the attachment of the IP3 receptor to the cytoskeleton seems to involve an association with microfilaments.

Topics: Actins; Animals; Ankyrins; Blotting, Western; Brain; Calcium Channels; Cell Membrane; Cytochalasin D; Cytoskeleton; Electrophoresis, Polyacrylamide Gel; Gelsolin; Gluconeogenesis; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Kinetics; Liver; Lymphoma, T-Cell; Male; Oxygen Consumption; Phosphorylase a; Rats; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; Tetracaine; Vasopressins