Abstract

Traditionally, GRP78 has been regarded as an endoplasmic reticulum (ER) lumenal protein due to its carboxyl KDEL retention motif. Recently, a subfraction of GRP78 is found to localize to the surface of specific cell types, serving as co-receptors and regulating signaling. However, the physiological relevance of cell surface GRP78 (sGRP78) expression in cancer and its functional interactions at the cell surface are just emerging. In this report, we combined biochemical, imaging and mutational approaches to address these issues. For detection of sGRP78, we utilized a mouse monoclonal antibody highly potent and specific for GRP78 or epitope-tagged GRP78, coupled with imaging and biochemical techniques that allowed detection of sGRP78 but not intracellular GRP78. Our studies revealed that breast and prostate cancer cells resistant to hormonal therapy actively promote GRP78 to the cell surface, which can be further elevated by a variety of ER stress-inducing conditions. We showed that sGRP78 forms complex with PI3K, and overexpression of sGRP78 promotes PIP3 formation, indicative of PI3K activation. We further discovered that an insertion mutant of GRP78 at its N-terminus domain, while retaining stable expression and the ability to translocate to the cell surface as the wild-type protein, exhibited reduced complex formation with p85 and production of PIP3. Thus, our studies provide a mechanistic explanation for the regulation of the PI3K/AKT signaling by sGRP78. Our findings suggest that targeting sGRP78 may suppress therapeutic resistance in cancer cells and offer a novel strategy to suppress PI3K activity.

Highlights

  • The 78 kDa glucose-regulated protein (GRP78), referred to as BiP/HSPA5, is a major endoplasmic reticulum (ER) chaperone with anti-apoptotic properties [1] and a master regulator of ER stress signaling [2,3]

  • From Western blot analysis of the biotinylated proteins and the total lysate, the levels of surface GRP78 (sGRP78) and total intracellular GRP78 were determined for each cell line, with EphB4 and NKA a1 serving as loading controls for cell surface proteins for breast cancer and prostate cancer cell lines, respectively. b-actin, a cytosolic protein, served as loading control for the total cell lysates

  • The discovery that GRP78 can localize to the cell surface under pathophysiologic conditions such as cancer, opens up new mechanisms whereby this protein may exert its pro-proliferative and anti-apoptotic function in cancer

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Summary

Introduction

The 78 kDa glucose-regulated protein (GRP78), referred to as BiP/HSPA5, is a major endoplasmic reticulum (ER) chaperone with anti-apoptotic properties [1] and a master regulator of ER stress signaling [2,3]. Understanding how GRP78 exerts its pleiotrophic effects on cell proliferation and survival is of major importance. Cell surface proteome profiling of tumor cells revealed a relative abundance of heat shock chaperones and glucose-regulated proteins, including GRP78 [17]. Preferential expression of GRP78 on the surface of tumor cells but not in normal organs enables specific tumor targeting, leading to tumor suppression without harmful effects on normal tissues [18,19,20,21]

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