Epithelial inositol 1,4,5-trisphosphate receptors. Multiplicity of localization, solubility, and isoforms.
Epithelial inositol 1,4,5-trisphosphate receptors. Multiplicity of localization, solubility, and isoforms.
复制标题
上皮肌醇 1,4,5-三磷酸受体。
DOI：
--
复制
发表时间：
1994
影响因子：
4.8
通讯作者：
S. Nigam
中科院分区：
文献类型：
--
作者：
K. Bush;R. O. Stuart;S. H. Li;L. Moura;A. Sharp;C. Ross;S. Nigam
在早期对上皮组织（肝脏和胰腺）的亚细胞分离研究中，我们发现肌醇1,4,5-三磷酸受体（IP3R）与生物化学上不同的细胞膜有关，包括内质网（ER）和质膜(Sharp, a.h., Snyder, s.h., and Nigam, s.k. (1992) J. Biol。化学，267,7444-7449)。为了进一步表征上皮IP3Rs，我们现在使用培养的Madin-Darby犬肾（MDCK）细胞，这是一种经过充分研究的紧密极化上皮细胞类型。通过免疫印迹和免疫沉淀，使用抗血清特异性识别MDCK细胞中的IP3R的间接免疫荧光显示er样染色模式和基底外侧质膜样染色模式，后者在高度融合的单层中尤其明显。在成年大鼠肾小管切片中观察到类似的染色模式。有趣的是，虽然已知的基底外侧蛋白（Na+，K(+)- atp酶和葡萄糖转运蛋白）呈现连续的基底外侧染色模式，但IP3R却呈现不连续的（点状）。小泡蛋白（caveolin）的染色模式高度相似，表明IP3R的点状基底外侧质膜样染色模式反映了其定位于基底外侧小泡。非透性化和透性化MDCK细胞的生物素化、IP3R的免疫沉淀和链亲和素检测表明，虽然大多数IP3R定位于生物素无法进入的区室（即内质网），但一部分（10-20%）IP3R主要可以从基底外侧进入外部添加的生物素。这一结果与免疫细胞化学提示的双内质网和基底外侧腔泡定位一致，尽管它也不排除基底外侧质膜中存在一些IP3R。溶解度研究表明，IP3R比碱性蛋白、Na+、K(+)- atp酶和肝细胞粘附分子以及细胞骨架蛋白、锚蛋白和fodrin更不溶。在最不溶的部分中，IP3R与小泡蛋白一起被发现，进一步支持了部分细胞IP3R池与小泡相关的观点。由于IP3R在细胞内的多个定位可能反映了多个同种异构体的存在，因此使用针对IP3R三种同种异构体的特异性引物对第一链cDNA进行了聚合酶链反应扩增。IP3R的所有三种亚型均在均匀的MDCK细胞群体中表达。在同一细胞类型中存在不同的膜定位和多种IP3R亚型，这可以解释上皮细胞和其他细胞中肌醇1,4,5-三磷酸敏感Ca2+池中Ca2+释放的复杂时空模式。
In an earlier subcellular fractionation study of epithelial tissue (liver and pancreas), we demonstrated that the inositol 1,4,5-trisphosphate receptor (IP3R) is found in association with biochemically distinct cellular membranes, including the endoplasmic reticulum (ER) and plasma membrane (Sharp, A. H., Snyder, S. H., and Nigam, S. K. (1992) J. Biol. Chem. 267, 7444-7449). To further characterize epithelial IP3Rs, we have now employed cultured Madin-Darby canine kidney (MDCK) cells, a well studied tight polarized epithelial cell type. Indirect immunofluorescence with an antiserum which specifically recognizes IP3R in MDCK cells by immunoblotting and immunoprecipitation gave an ER-like staining pattern as well as a basolateral plasma membrane-like staining pattern, the latter being particularly evident in highly confluent monolayers. In sections of adult rat kidney tubules a similar staining pattern was observed. Interestingly, whereas known basolateral proteins (Na+,K(+)-ATPase and the facilitated glucose transporter) gave a continuous basolateral staining pattern, that seen for IP3R was discontinuous (punctate). A highly similar staining pattern was observed for the caveolar protein, caveolin, suggesting that the punctate basolateral plasma membrane-like staining pattern observed for IP3R reflects its localization to basolateral caveolae. Biotinylation of non-permeabilized and permeabilized MDCK cells, followed by immunoprecipitation of IP3R and detection with streptavidin, indicated that while most IP3R is localized to biotin-inaccessible compartments (i.e. ER), a fraction (10-20%) of IP3R was accessible to externally added biotin primarily from the basolateral side. This result is compatible with the dual ER and basolateral caveolar localization suggested by immunocytochemistry, although it does not exclude the presence of some IP3R in the basolateral plasma membrane as well. Solubility studies revealed IP3R to be considerably more insoluble than the basolateral proteins, Na+,K(+)-ATPase and the liver cell adhesion molecule, as well as the cytoskeletal proteins, ankyrin and fodrin. In the most insoluble fraction, IP3R was found along with caveolin, further supporting the notion that part of the cellular IP3R pool associates with caveolae. Since multiple localizations of IP3R within a cell might reflect the existence of multiple isoforms, polymerase chain reaction amplification of first strand cDNA with primers specific for the three isotypes of IP3R was performed. All three isoforms of IP3R were expressed in the homogeneous population of MDCK cells. The existence of distinct membrane localizations and multiple isoforms of IP3R within the same cell type suggests an explanation for the complex spatiotemporal patterns of Ca2+ release from inositol 1,4,5-trisphosphate-sensitive Ca2+ pools in epithelial and other cells.