Mahata S K, Mahata M, Wakade A R, O'Connor D T
Department of Medicine and Center for Molecular Genetics, University of California, and Veterans Affairs San Diego Healthcare System, 92161, USA.
Mol Endocrinol. 2000 Oct;14(10):1525-35. doi: 10.1210/mend.14.10.0531.
The novel chromogranin A fragment catestatin (bovine chromogranin A(344-364); RSMRLSFRARGYGFRGPGLQL) is a potent inhibitor of catecholamine release (IC50, approximately 0.2-0.3 microM) by acting as a nicotinic cholinergic antagonist. To define the minimal active region within catestatin, we tested the potencies of synthetic serial three-residue deletion (amino-terminal, carboxyl-terminal, or bidirectional) fragments to inhibit nicotine-stimulated catecholamine secretion from PC12 pheochromocytoma cells. The results revealed that a completely active core sequence of catestatin was constituted by chromogranin A(344-364). Nicotinic cationic signal transduction was affected by catestatin fragments in a manner similar to that for secretion (confirming the functional importance of the amino-terminus). To identify crucial residues within the active core, we tested serial single amino acid truncations or single residue substitutions by alanine on nicotine-induced catecholamine secretion and desensitization. Nicotinic inhibition by the active catestatin core was diminished by even single amino acid deletions. Selective alanine substitution mutagenesis of the active core revealed important roles for Met346, Leu348, Phe350, Arg351, Arg353, Gly354, Tyr355, Phe357, and Arg358 on catecholamine secretion, whereas crucial roles to inhibit desensitization of catecholamine release were noted for Arg344, Met346, Leu348, Ser349, Phe350, Arg353, Gly354, Tyr355, Gly356, and Arg358. We conclude that a small, 15-amino acid core of catestatin (chromogranin A(344-364)) is sufficient to exert the peptide's typical inhibitory effects on nicotinic cholinergic-stimulated catecholamine secretion, signal transduction, and desensitization. These studies refine the biologically active domains of catestatin and suggest that the pharmacophores for inhibition of nicotinic secretion and desensitization may not be identical.
新型嗜铬粒蛋白A片段癌抑素（牛嗜铬粒蛋白A(344 - 364)；RSMRLSFRARGYGFRGPGLQL）是一种有效的儿茶酚胺释放抑制剂（IC50约为0.2 - 0.3 microM），它通过作为烟碱型胆碱能拮抗剂发挥作用。为了确定癌抑素内的最小活性区域，我们测试了合成的连续三个残基缺失（氨基末端、羧基末端或双向）片段抑制PC12嗜铬细胞瘤细胞中尼古丁刺激的儿茶酚胺分泌的效力。结果显示，癌抑素的一个完全活性核心序列由嗜铬粒蛋白A(344 - 364)构成。烟碱型阳离子信号转导受癌抑素片段的影响方式与分泌相似（证实了氨基末端的功能重要性）。为了确定活性核心内的关键残基，我们测试了连续单个氨基酸截短或用丙氨酸进行单个残基替换对尼古丁诱导的儿茶酚胺分泌和脱敏的影响。即使单个氨基酸缺失也会降低活性癌抑素核心对烟碱的抑制作用。活性核心的选择性丙氨酸替换诱变揭示了Met346、Leu348、Phe350、Arg351、Arg353、Gly354、Tyr355、Phe357和Arg358在儿茶酚胺分泌中的重要作用，而对于抑制儿茶酚胺释放脱敏的关键作用则见于Arg344、Met346、Leu348、Ser349、Phe350、Arg353、Gly354、Tyr355、Gly356和Arg358。我们得出结论，癌抑素的一个15个氨基酸的小核心（嗜铬粒蛋白A(344 - 364)）足以对烟碱型胆碱能刺激的儿茶酚胺分泌、信号转导和脱敏发挥该肽的典型抑制作用。这些研究细化了癌抑素的生物活性结构域，并表明抑制烟碱型分泌和脱敏的药效基团可能并不相同。
