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.