Basolateral targeting and efficient consumption of transforming growth factor-alpha when expressed in Madin-Darby canine kidney cells.
Basolateral targeting and efficient consumption of transforming growth factor-alpha when expressed in Madin-Darby canine kidney cells.
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发表时间：
1994-06
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通讯作者：
Peter J. Dempsey;R. J. Coffey
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作者：
Peter J. Dempsey;R. J. Coffey
为了研究转化生长因子-α前体（pro-TGF-α）在极化上皮细胞中的运输，将野生型和膜固定的人pro-TGF-α引入Madin-Darby犬肾（MDCK）细胞中。我们表明，野生型pro-TGF-α的合成和加工正常释放成熟的5.6 kDa的TGF-α到基础培养基，而膜固定的pro-TGF-α保持细胞相关。抗体（mAb-528）受体阻断实验证明了基底外侧表皮生长因子受体对基底释放的TGF-α的有效消耗，表明TGF-α可以在这些极化上皮细胞中以自分泌方式起作用。生物化学分析显示，pro-TGF-α在基底外侧表面上表达为17-或30-kDa种类;两种pro-TGF-α构建体的17-kDa形式具有> 20：1的基底外侧/顶端比率。通过共聚焦显微镜，将膜固定的pro-TGF-α免疫定位于侧膜表面。在结合细胞表面免疫沉淀的脉冲追踪实验中，我们证明了新合成的野生型和膜固定的pro-TGF-α分别以94%和96%的效率直接递送到基底外侧表面。这些结果也提供了直接的证据pro-TGF-α在基底外侧膜表面的顺序切割。因此，pro-TGF-α在细胞内被分选，并矢量靶向这些极化上皮细胞中的基底外侧膜结构域。MDCK细胞系提供了一种理想的体外模型，以检查在极化上皮细胞中运输pro-TGF-α和其他表皮生长因子样生长因子的分子基础及其与基底外侧表皮生长因子受体的潜在相互作用。
To investigate the trafficking of transforming growth factor-alpha precursor (pro-TGF-alpha) in polarized epithelial cells, wild type and membrane-fixed human pro-TGF-alpha were introduced into Madin-Darby canine kidney (MDCK) cells. We show that wild type pro-TGF-alpha was synthesized and processed normally to release mature 5.6-kDa TGF-alpha into the basal medium while membrane-fixed pro-TGF-alpha remained cell-associated. Antibody (mAb-528) receptor blockade experiments demonstrated the efficient consumption of basally released TGF-alpha by basolateral epidermal growth factor receptors, indicating that TGF-alpha can act in an autocrine manner in these polarized epithelial cells. Biochemical analysis showed pro-TGF-alpha was expressed on the basolateral surface as either a 17- or 30-kDa species; the 17-kDa forms of both pro-TGF-alpha constructs had basolateral/apical ratios of > 20:1. By confocal microscopy, membrane-fixed pro-TGF-alpha was immunolocalized to lateral membrane surfaces. In pulse-chase experiments combined with cell surface immunoprecipitation, we demonstrated that newly synthesized wild type and membrane-fixed pro-TGF-alpha are delivered directly to the basolateral surface with 94 and 96% efficiency, respectively. These results also provide direct evidence for sequential cleavage of pro-TGF-alpha at the basolateral membrane surface. Thus, pro-TGF-alpha is sorted intracellularly and vectorially targeted to the basolateral membrane domain in these polarized epithelial cells. The MDCK cell line provides an ideal in vitro model to examine the molecular basis for trafficking of pro-TGF-alpha and other epidermal growth factor-like growth factors in polarized epithelial cells and their potential interactions with basolateral epidermal growth factor receptors.