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

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.