Regazzi R, Sasaki T, Takahashi K, Jonas J C, Volker C, Stock J B, Takai Y, Wollheim C B
Department of Medicine, University of Geneva, Switzerland.
Biochim Biophys Acta. 1995 Sep 21;1268(3):269-78. doi: 10.1016/0167-4889(95)00085-7.
Most guanine nucleotide binding proteins (G-proteins) possess an S-prenylated C-terminal cysteine whose carboxyl group can be reversibly methylated. The prenylcysteine analog N-acetyl-S-geranylgeranyl-cysteine (AGGC) (50 microM), a competitive inhibitor of prenylcysteine methyl transferases, introduced into streptolysin-O permeabilized HIT-T15 cells doubled the rate of basal (0.1 microM Ca2+) and of stimulated (10 microM Ca2+ or 100 microM GTP gamma S) insulin secretion in a reversible and ATP-dependent manner. N-acetyl-S-farnesylcysteine (AFC) was less potent while N-acetyl-S-geranyl-cysteine was inactive. Prenylcysteine action on exocytosis did not involve inhibition of G-protein methylation, since (1) the methyl ester derivative of AFC, an inefficient inhibitor of methyltransferases in HIT-T15 cell fractions, was as potent as AGGC in stimulating exocytosis; (2) S-adenosyl-homocysteine, a general inhibitor of methylation reactions, did not alter basal or GTP gamma S-triggered secretion while inhibiting Ca(2+)-induced insulin release. The binding of G-proteins to Rab/GDP-dissociation inhibitor, Rab3A/GTPase activating protein or rabphilin-3A was not affected by the prenylcysteine analogs. AGGC or AFC had the same effect on insulin release as a synthetic peptide mimicking the amino acid residues 52-67 of the G-protein Rab3A (Rab3AL). Moreover, the action on secretion of the combination of Rab3AL and prenylcysteines was not additive. We propose that the prenylcysteines and the Rab3AL peptide influence exocytosis by affecting the association of Rab3A with different proteins of the exocytotic machinery of insulin-secreting cells.
大多数鸟嘌呤核苷酸结合蛋白（G蛋白）都有一个经S-异戊二烯化修饰的C端半胱氨酸，其羧基可发生可逆甲基化。异戊二烯基半胱氨酸类似物N-乙酰基-S-香叶基香叶基半胱氨酸（AGGC）（50微摩尔）是异戊二烯基半胱氨酸甲基转移酶的竞争性抑制剂，将其引入经链球菌溶血素O通透处理的HIT-T15细胞后，基础状态（0.1微摩尔钙离子）和刺激状态（10微摩尔钙离子或100微摩尔GTPγS）下胰岛素分泌速率以可逆且依赖ATP的方式增加了一倍。N-乙酰基-S-法尼基半胱氨酸（AFC）的作用较弱，而N-乙酰基-S-香叶基半胱氨酸则无活性。异戊二烯基半胱氨酸对胞吐作用的影响并不涉及对G蛋白甲基化的抑制，原因如下：（1）AFC的甲酯衍生物，即HIT-T15细胞组分中甲基转移酶的低效抑制剂，在刺激胞吐作用方面与AGGC效力相当；（2）S-腺苷同型半胱氨酸，一种甲基化反应的通用抑制剂，在抑制钙离子诱导的胰岛素释放的同时，并未改变基础状态或GTPγS触发的分泌。G蛋白与Rab/GDP解离抑制剂、Rab3A/GTP酶激活蛋白或rabphilin-3A的结合不受异戊二烯基半胱氨酸类似物的影响。AGGC或AFC对胰岛素释放的作用与模拟G蛋白Rab3A氨基酸残基52 - 67的合成肽（Rab3AL）相同。此外，Rab3AL和异戊二烯基半胱氨酸组合对分泌的作用并非相加性的。我们提出，异戊二烯基半胱氨酸和Rab3AL肽通过影响Rab3A与胰岛素分泌细胞胞吐机制中不同蛋白质的结合来影响胞吐作用。
