AFFINITY LABELS FOR INOSITOL POLYPHOSPHATE RECEPTORS

脑细胞通过神经递质和精神活性药物作出反应 大量的细胞特异性受体蛋白。为了理解如何 大脑在健康和病理条件下整合不同的信号， 人们的注意力集中在两个第二信使系统上，环状AMP和 磷脂酰肌醇。第二信使D-肌醇-1，4，5- 三磷酸(INS(1，4，5)P3，以下简称IP3)以立体特异性相互作用 通过膜受体促进钙离子的释放 细胞内存储。识别IP3并介导IP3的受体蛋白 它在钙释放中的作用最近得到了鉴定。这个 高级肌醇多磷酸盐，主要是Ins(1，3，4，5)P4(IP4)和 INS(1，2，3，4，5，6)P6(IP6)，也有特异性的胞内受体 它们的特征还有待充分研究。 化学亲和标记、光亲和标记和固定化亲和标记 识别IP3、IP4和IP6的蛋白质矩阵为 化学合成的。三种肌醇多磷酸盐中的每一种都将 用C-1或C-2磷酸选择性官能化制备 作为系留伯胺的磷酸二酯衍生物。四个1-O- 将检查不同链长和不同链长的氨基烷基系链剂 僵硬。伯胺基团将在化学上转化为两个- 反应性亲和标记和三种不同的光活化 衍生品也将准备就绪。一种新的费里尔重排路线 P-1系留D-myo-IP4和[~3H]D-myo-IP4将被开发出来。 类似物的药理和生物学特性也将 由合作实验室进行检查。例如，IP3类比将是 与[~3H]Ins(1，4，5)P3竞争结合粗品或纯化的IP3 大鼠脑、大鼠纤毛或鲶鱼纤毛的受体(IP3Rs)。 重组受体脂质体将用于显示钙释放 当受到类似物刺激时。竞争性约束性分析将是 与IP4和IP6衍生物一起进行。亲和层析 对于固定化的IP3、IP4和IP6衍生物将用于受体 净化。正确的D-MYO表格预计将显示改进 已经成功的外消旋IP3和IP4配体的特异性。 亲和探针在受体纯化和测序中的应用 大鼠配体结合部位共价修饰多肽的研究 脑IP3R、IP4R和IP6R以及两个新的嗅觉IP3R将被 与石溪研究人员联合进行的。免疫原性IPN 将合成偶联物，并将使用抗血清开发IPN ELISAS。将准备用于组织化学的荧光IPN探针 学习。

Brain cells respond to neurotransmitters and psychoactive drugs through a multitude of cell-specific receptor proteins. To understand how the brain integrates diverse signals in healthy and pathological conditions, attention has focused on two second messenger systems, cyclic AMP and the phosphoinositides. The second messenger D-myo-inositol 1,4,5- trisphosphate (Ins(1,4,5)P3, hereafter IP3) interacts stereospecifically with membrane receptors to promote the release of Ca2+ from intracellular stores. Receptor proteins which recognize IP3 and mediate its role in calcium release have been characterized very recently. The higher inositol polyphosphates, principally Ins(1,3,4,5)P4 (IP4) and Ins(1,2,3,4,5,6)P6 (IP6), also have specific intracellular receptors which remain to be fully characterized. Chemical affinity labels, photoaffinity labels, and immobilized affinity matrices for proteins which recognize IP3, IP4, and IP6 will be chemically synthesized. Each of the three inositol polyphosphates will be prepared with selective functionalization of the C-1 or C-2 phosphate as a phosphodiester derivative of a tethered primary amine. Four 1-O- aminoalkyl tethers will be examined which vary in chain length and rigidity. The primary amine groups will be converted to two chemically- reactive affinity labels, and three different photoactivatable derivatives will also be prepared. A novel Ferrier rearrangement route to P-1 tethered D-myo-IP4 and [3H]D-myo-IP4 will be developed. The pharmacological and biological properties of the analogs will also be examined by collaborating labs. For example, IP3 analogs will be competed with [3H]Ins(1,4,5)P3 for binding to crude or purified IP3 receptors (IP3Rs) from rat brain, rat cilia, or catfish cilia. Reconstituted receptor liposomes will be used to show calcium release when stimulated by the analogs. Competitive binding assays will be carried out with the IP4 and IP6 derivatives. Affinity chromatography with immobilized IP3, IP4, and IP6 derivatives will be used for receptor purification. The correct D-myo forms are expected to show improved specificity over the already successful racemic IP3 and IP4 ligands. The use of the affinity probes for receptor purification and sequencing of covalently-modified polypeptides in the ligand binding site of rat brain IP3R, IP4R, and IP6R and of two new olfactory IP3Rs will be conducted jointly with Stony Brook researchers. Immunogenic IPn conjugates will be synthesized, and antisera will be used to develop IPn ELISAs. Fluorescent IPn probes will be prepared for histochemical studies.