Secretagogue-induced translocation of CRHSP-28 within an early apical endosomal compartment in acinar cells
Secretagogue-induced translocation of CRHSP-28 within an early apical endosomal compartment in acinar cells
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DOI：
10.1152/ajpgi.00033.2004
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发表时间：
2004-07-01
影响因子：
4.5
通讯作者：
Groblewski, GE
中科院分区：
文献类型：
--
作者：
Thomas, DDH;Weng, N;Groblewski, GE
Ca2+调节热稳定蛋白（CRHSP-28）是TPD52蛋白家族的一员，已被证明可调节胰腺腺泡细胞中Ca2+依赖性分泌活性。分离小叶的免疫荧光显微镜显示，CRHSP-28定位于腺泡的核上根尖室，并在CCK八肽（CCK-8）刺激后2分钟内立即积聚在根尖膜下方。双免疫荧光显微镜显示，CRHSP-28的内体定位与早期内体抗原-1 （EEA-1）在整个顶胞浆的囊泡结构上强烈重叠，但与基底侧来源的内体中存在的转铁蛋白受体只有很少的重叠。CRHSP-28与反式高尔基网络标记物-38在腺泡核上区域也有明显的重叠。有趣的是，用brefeldin A处理小叶可逆地破坏了顶端细胞质中CRHSP-28和EEA-1的囊泡定位。cck -8诱导的CRHSP-28在腺泡亚根尖区域的积累并未因肌动蛋白丝破坏剂latrunculin b抑制根尖内吞噬而改变。免疫电镜证实，CRHSP-28与根尖细胞质中低电子密度的不规则囊泡结构的限制膜有关，该膜在EEA-1染色中呈阳性。沿着酶原颗粒的限制膜也观察到稀疏但显著的CRHSP-28免疫反应性。与免疫荧光数据一致，在CCK-8刺激下，发现CRHSP-28聚集在核内体上，并位于细胞顶端以下的酶原颗粒之间。这些数据表明，CRHSP-28存在于腺泡细胞的内吞区和胞外区，并受到促分泌剂刺激的强烈调节。
Ca2+-regulated heat-stable protein (CRHSP-28) is a member of the TPD52 protein family that has been shown to regulate Ca2+-dependent secretory activity in pancreatic acinar cells. Immunofluorescence microscopy of isolated lobules demonstrated that CRHSP-28 is localized to a supranuclear apical compartment in acini and accumulates immediately below the apical membrane within 2 min of CCK octapeptide (CCK-8) stimulation. Dual-immunofluorescence microscopy demonstrated an endosomal localization of CRHSP-28 that strongly overlapped with early endosomal antigen-1 (EEA-1) on vesicular structures throughout the apical cytoplasm but showed only minimal overlap with the transferrin receptor, which is present in basolaterally derived endosomes. Significant overlapping of CRHSP-28 with the trans-Golgi network marker-38 was also noted in supranuclear regions of acini. Interestingly, treatment of lobules with brefeldin A reversibly disrupted the vesicular localization of CRHSP-28 and EEA-1 within the apical cytoplasm. The CCK-8-induced accumulation of CRHSP-28 in subapical regions of acini was not altered by inhibition of apical endocytosis with the actin filament-disrupting agent latrunculin B. Immunoelectron microscopy confirmed that CRHSP-28 is associated with the limiting membrane of irregularly shaped vesicular structures of low electron density in the apical cytoplasm that are positive for EEA-1 staining. Sparse, but significant, CRHSP-28 immunoreactivity was also observed along the limiting membrane of zymogen granules. Consistent with immunofluorescence data, CRHSP-28 was found to accumulate in clusters on endosomes and positioned between zymogen granules below the cell apex on CCK-8 stimulation. These data indicate that CRHSP-28 is present within endocytic and exocytic compartments of acinar cells and is acutely regulated by secretagogue stimulation.