Expression and localization of Rab3D in rat parotid gland.
Expression and localization of Rab3D in rat parotid gland.
复制标题
Rab3D 在大鼠腮腺中的表达和定位。
DOI：
10.1016/s0167-4889(99)00052-x
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Ojakian,GK
中科院分区：
文献类型：
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作者：
Raffaniello,RD;Lin,J;Schwimmer,R;Ojakian,GK
Rab 3蛋白（同种型A、B、C和D）是低分子量GTP结合蛋白，被认为参与调节胞吐作用。本研究采用逆转录聚合酶链反应（RT PCR）、免疫印迹和免疫细胞化学方法检测大鼠腮腺Rab 3蛋白的表达和定位。采用RTPCR方法从腮腺组织RNA中扩增出约200 bp的PCR产物，并对该片段进行克隆和测序。从5个克隆中获得的核苷酸和推导的氨基酸序列与rab 3D相同。从腮腺腺泡制备的膜和胞质组分用四种Rab 3同种型中的每一种的特异性抗血清进行免疫印迹。用Rab 3D特异性抗血清在两种组分中检测到28 kDa蛋白，其中在膜组分中染色更强烈。通过免疫印迹法未检测到其他Rab 3亚型，这一结果与通过rtPCR获得的结果一致。Rab 3D富集在酶原颗粒膜和Triton X-114提取揭示，这种亚型主要是在腮腺脂质修饰。通过免疫荧光显微镜在腮腺冷冻切片上进行Rab 3D的定位。染色主要在腺泡细胞中观察到，并邻近腺泡腔。孵育分散的腺泡与异丙肾上腺素和P物质刺激淀粉酶分泌4倍和2倍以上的基础，分别。异丙肾上腺素，而不是P物质，诱导Rab 3D从细胞质到膜部分分散腮腺腺泡的重新分配。与这些发现一致，异丙肾上腺素注射到禁食大鼠也导致腮腺腺泡中膜相关Rab 3D增加。这些结果表明Rab 3D是：（1）在大鼠腮腺中表达的Rab 3的主要同种型;（2）定位于酶原颗粒膜;（3）参与调节腺泡细胞中的酶分泌。
Rab3 proteins (isoforms A, B, C and D) are low molecular weight GTP-binding proteins proposed to be involved in regulated exocytosis. In the present study, Rab3 protein expression and localization was examined in rat parotid gland by reverse transcription (rt) PCR, Western blotting and immunocytochemistry. An ∼200 bp PCR product was obtained from parotid RNA by rtPCR and this fragment was cloned and sequenced. Nucleotide and deduced amino acid sequences obtained from five clones were identical to rab3D. Membrane and cytosolic fractions prepared from parotid acini were immunoblotted with antisera specific for each of the four Rab3 isoforms. A 28 kDa protein was detected with Rab3D-specific antisera in both fractions with staining being more intense in the membrane fraction. No other Rab3 isoforms were detected by immunoblotting, a result consistent with those obtained by rtPCR. Rab3D was enriched in zymogen granule membranes and Triton X-114 extraction revealed that this isoform is predominantly lipid-modified in parotid. Localization of Rab3D was done on frozen sections of parotid gland by immunofluorescence microscopy. Staining was observed primarily in the acinar cells and was adjacent to the acinar lumen. Incubation of dispersed acini with isoproterenol and substance P stimulated amylase secretion 4- and 2-fold above basal, respectively. Isoproterenol, but not substance P, induced redistribution of Rab3D from the cytosol to the membrane fraction in dispersed parotid acini. Consistent with these findings, isoproterenol injections into fasted rats also resulted in increased membrane-associated Rab3D in the parotid acini. These results indicate that Rab3D is: (1) the major Rab3 isoform expressed in rat parotid gland; (2) localized to zymogen granule membranes; and (3) involved with regulated enzyme secretion in acinar cells.