Bourguignon L Y, Jin H
Department of Cell Biology and Anatomy, University of Miami Medical School, Florida 33101, USA.
J Biol Chem. 1995 Mar 31;270(13):7257-60. doi: 10.1074/jbc.270.13.7257.
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.
在本研究中，我们运用了多种互补技术来探究膜连接分子锚蛋白与小鼠T淋巴瘤细胞中肌醇1,4,5 - 三磷酸（IP3）受体之间的相互作用。通过双重免疫标记和激光共聚焦显微镜技术，我们发现细胞质中的IP3受体和锚蛋白都优先聚集在配体诱导的淋巴细胞受体帽状结构内。锚蛋白与IP3受体之间的结合似乎非常特异。进一步分析表明，IP3受体中的氨基酸序列GGVGDVLRKPS与某些特征明确的锚蛋白结合蛋白（如细胞粘附分子CD44）中位于锚蛋白结合域的结构具有高度同源性。使用竞争结合试验和与GGVGDVLRKPS相同的合成肽（在大鼠脑IP3受体（氨基酸2548 - 2558）和小鼠脑IP3受体（氨基酸2546 - 2556）中检测到的序列）进行的生化研究表明，这种11个氨基酸的肽特异性结合锚蛋白（而非血影蛋白或肌动蛋白）。此外，该肽能有效竞争锚蛋白与含IP3受体的囊泡和/或纯化的IP3受体的结合，并且能阻断锚蛋白对小鼠T淋巴瘤细胞中IP3结合和IP3介导的细胞内Ca2+释放的抑制作用。这些发现表明，位于IP3受体C末端附近的氨基酸序列GGVGDVLRKPS位于含IP3受体囊泡的细胞质侧（而非腔侧）。这一独特区域似乎是IP3受体锚蛋白结合域的重要组成部分，可能在淋巴细胞激活过程中对IP介导的细胞内Ca2+释放的调节中发挥重要作用。
