Translocation of 78-kDa glucose-regulated protein (GRP78) from endoplasmic reticulum (ER) to plasma membrane represents a paradigm shift beyond its traditional function as an ER chaperone protein. Cell surface GRP78 (csGRP78) exerts novel signaling functions, and mechanisms underlying its cell surface expression are just emerging. Acquired tamoxifen resistance of breast cancer cells is accompanied with elevated level of csGRP78. Therefore, the tamoxifen-resistant MCF7 breast cancer cells (MCF7-LR) represents a clinically relevant model to study mechanisms of csGRP78 expression. We discovered that a proline-rich region (PRR) containing three consecutive prolines close to the COOH-terminus of GRP78 is important for its ability to form a complex with the partner protein, CD44v, as demonstrated by in vitro glutathione S-transferase pull-down assay. Proline to alanine mutations at the PRR compromised GRP78 expression level on the cell surface as evidenced by purification of biotinylated cell surface proteins. Reconstitution of MCF7-LR cells with the PRR mutant after knockdown of endogenous GRP78 diminished the capacity of GRP78 to stimulate STAT3 activation. The enforced expression of a short peptide bearing the PRR region of GRP78 led to reduction of CD44v and Cyclin D1 protein levels as well as cell viability, accompanied with increase in apoptotic signaling including cleaved Caspase-3 and PARP. These findings suggest that the COOH-terminal PRR of GRP78 is critical for its interaction with CD44v as well as its cell surface expression, and enforced expression of the short peptide bearing the PRR region may provide a new approach to lower the viability of tamoxifen-resistant breast cancer cells.

中文翻译：

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GRP78的COOH末端富含脯氨酸的区域是其细胞表面表达和抗他莫昔芬的乳腺癌细胞生存能力的关键调节剂。

78 kDa葡萄糖调节蛋白（GRP78）从内质网（ER）转移到质膜代表了一种范式转变，超越了其作为ER伴侣蛋白的传统功能。细胞表面GRP78（csGRP78）发挥了新的信号传导功能，其细胞表面表达的潜在机制也正在兴起。获得性乳腺癌细胞对他莫昔芬的耐药性伴随着csGRP78水平的升高。因此，耐他莫昔芬的MCF7乳腺癌细胞（MCF7-LR）代表了一种临床相关模型，用于研究csGRP78表达的机制。我们发现，一个富含脯氨酸的区域（PRR）靠近GRP78的COOH末端包含三个连续的脯氨酸，对于其与伴侣蛋白CD44v形成复合物的能力非常重要，如体外谷胱甘肽S-转移酶拉-向下测定。PRR上的丙氨酸突变脯氨酸会破坏细胞表面GRP78的表达水平，这是通过纯化生物素化细胞表面蛋白来证明的。内源性GRP78敲低后，用PRR突变体重建MCF7-LR细胞会降低GRP78刺激STAT3激活的能力。带有GRP78 PRR区的短肽的强制表达导致CD44v和Cyclin D1蛋白水平的降低以及细胞活力的降低，并伴随着凋亡信号的增加，包括Caspase-3和PARP的裂解。这些发现表明，GRP78的COOH末端PRR对于其与CD44v的相互作用以及其细胞表面表达至关重要，