Cholecystokinin-regulated exocytosis in rat pancreatic acinar cells is inhibited by a C-terminus truncated mutant of SNAP-23
Cholecystokinin-regulated exocytosis in rat pancreatic acinar cells is inhibited by a C-terminus truncated mutant of SNAP-23
复制标题
DOI：
10.1097/00006676-200108000-00002
复制
发表时间：
2001-08-01
期刊：
影响因子：
2.9
通讯作者：
Gaisano, HY
中科院分区：
文献类型：
--
作者：
Huang, XH;Sheu, L;Gaisano, HY
简介：胞吐作用被认为是由囊泡和质膜的融合引起的，所述融合是由囊泡上的囊泡相关膜蛋白（VAMP）家族的蛋白质与质膜上的25 kd（SNAP-25）家族的突触融合蛋白和突触体相关蛋白的成员之间形成反式复合物引起的。在胰腺腺泡细胞中，23 kd的突触体相关蛋白（SNAP-23）是胰腺腺泡细胞中表达的主要SNAP-25同种型，但其在腺泡细胞胞吐中的作用尚未确定。为了检测SNAP-23在腺泡细胞中调节胞吐作用中的作用，我们将SNAP-23、SNAP-25、SNAP-25亚克隆到腺病毒载体中，和显性失活突变体，其中对应于肉毒杆菌神经毒素A切割位点的C-末端结构域缺失。通过亚细胞分级分离用这些腺病毒培养的大鼠胰腺腺泡的Hi，oh-效率感染显示过表达的SNAP-23，SNAP-25，并且它们的截短突变体蛋白均匀地靶向酶原颗粒和质膜。为了最大限度地刺激这些感染腺泡的顶端胞吐作用，我们使用了胆囊收缩素苯乙酯类似物（CCK-OPE），它在高剂量下不会抑制最大水平的分泌。CCK-OPE刺激的腺病毒-巨细胞病毒（AdCMV）-SNAP-23或AdCMV-SNAP-25感染的腺泡的淀粉酶释放达到与空载体感染的腺泡相同的程度。相反，CCK-OPE诱导的AdCMV-SNAP-23 Delta C8和AdCMV-SNAP-25（1-197）感染腺泡的酶分泌分别被抑制60%和40%。突变体SNAP-23和SNAP-25蛋白与SNAP-23相同地靶向相同的膜区室，这表明分泌的抑制是它们与内源性SNAP-23竞争的结果。当AdCMV-SNAP-25 Delta C8-或AdCMV-SNAP-25（1-197）感染腺泡后，与野生型ShiAP-23或SNAP-25共感染腺泡，突变蛋白的这种抑制作用被部分逆转或挽救。结论：SNAP-23在CCK诱导的腺泡细胞胞吐中起作用。
Introduction: Exocytosis is thought to result from the fusion of vesicle and plasma membranes caused by the formation of a trans-complex between proteins of the vesicle-associated membrane protein (VAMP) family on the vesicle with members of the syntaxin and synaptosomal-associated protein of 25 kd (SNAP-25) families on the plasma membrane. In the pancreatic acinar cell, synaptosomal-associated protein of 23 kd (SNAP-23) is the major SNAP-25 isoform expressed in pancreatic acinar cells, but its role in acinar cell exocytosis has not been determined.Aims: To examine the role of SNAP-23 in regulated exocytosis in acinar cells, we subcloned into adenoviral vectors SNAP-23, SNAP-25, and dominant negative mutants in which the C-terminal domains corresponding to the botulinum neurotoxin A cleavage sites are deleted.Methodology and Results: Hi,oh-efficiency infection of rat pancreatic acini in culture with these adenoviruses by subcellular fractionation showed that the overexpressed SNAP-23, SNAP-25, and their truncated mutant proteins were uniformly targeted to the zymogen granules and plasma membrane. To maximally stimulate apical exocytosis from these infected acini, we used the cholecystokinin-phenylethyl ester analog (CCK-OPE), which does not show inhibition of secretion from maximal levels at high doses. CCK-OPE-stimulated amylase release from adenovirus-cytomegalo virus (AdCMV)-SNAP-23 or AdCMV-SNAP-25-infected acini to the same extent as from acini infected with the empty vector. In contrast, CCK-OPE-evoked enzyme secretion from AdCMV-SNAP-23 Delta C8-and AdCMV-SNAP-25(1-197)-infected acini were inhibited by 60% and 40%, respectively. The identical targeting of the mutant SNAP-23 and SNAP-25 proteins to the same membrane compartments as SNAP-23 suggests that the inhibition of secretion was a result of their competition against endogenous SNAP-23. This is supported by the fact that this inhibition by the mutant proteins was partially reversed or rescued when the AdCMV-SNAP-25 Delta C8- or AdCMV-SNAP-25(1-197)-infected acini were co-infected with wild-type ShiAP-23 or SNAP-25.Conclusion: From these results, we conclude that SNAP-23 plays a role in CCK-evoked regulated exocytosis in the acinar cells.