Cell Surface GRP78, a New Paradigm in Signal Transduction Biology

Cell Surface GRP78, a New Paradigm in Signal Transduction Biology presents a new paradigm that has emerged in the past decade with the discovery that various intracellular proteins may acquire new functions as cell surface receptors. Two very prominent examples are ATP synthase and GRP78. While the role of cell surface ATP synthase has been reviewed in various books, this book directs its attention to the story of cell surface GRP78.

• Edited by the researcher who identified cell surface expression of the molecular chaperone GRP78 as a major factor in prostate cancer and other malignancies
• Presents an in-depth treatment of the biological underpinnings of GRP78 and its connection to disease
• Provides four-color illustrations that facilitate the narrative

• Language: English
• Publisher: Academic Press
• Release date: Mar 20, 2018
• ISBN: 9780128123522

Book preview

States

Preface

Salvatore V. Pizzo

Why write this book now? At first glance, the topic may seem esoteric and just about one more signaling pathway. Yet this is a most unusual mechanism which repurposes an intracellular protein, the Glucose Regulated Protein of molecular weight 78,000 (GRP78), as a cell surface signaling receptor. There are precedents for such behavior, for example cell surface ATP synthase as the angiostatin receptor. However, cell surface GRP78 has unique properties. The intracellular version of the protein exists in the endoplasmic reticulum (ER) where it is a key player in the unfolded protein response, essential for cell survival under conditions of stress. Cells may be stressed by many factors such as hypoxia, disregulation of glucose metabolism, or injury to tissues requiring rapid division of cells to repair the damage. Under these conditions, protein synthesis ramps up and there is a risk of piling up misfolded proteins in the ER. GRP78 is crucial for driving proper folding of such proteins, and its ATP binding domain is essential for these functions. When GRP78 reaches the cell surface, this ATP binding domain is essential, allowing GRP78 to autophosphorylate and function like a classic tyrosine kinase-type receptor. To our knowledge, this represents unique behavior for a repurposed intracellular protein. Of particular interest, GRP78 as a cell surface receptor is generally associated with disordered cell biology. Other than a small fraction of the population of activated macrophages, it does not demonstrate signal transduction properties in normal cells. Cancer cells are the main arena where cell surface GRP78 functions as a pro-proliferative, promigratory, and antiapoptotic receptor. But, similar biology is seen in synovial cells from rheumatoid arthritis patients, and in damaged endothelial cells such as are seen in atherosclerosis. On the cell surface, GRP78 associates with many other proteins playing a regulatory dance. One such example to be discussed in detail in this book is its association with the procoagulant molecule tissue factor. Indeed, regulation of these complexes is almost certainly critical with respect to the association of cancer and thrombotic events.

Thus, we feel that this is a good time for such a book and we thank Elsevier for the opportunity.

June 2017

Chapter 1

An Historical Perspective

Cell Surface GRP78, a New Paradigm in Signal Transduction Biology

Salvatore V. Pizzo,    Duke University Medical Center, Durham, NC, United States

Abstract

This chapter will present a historical perspective with respect to the presence of Glucose Regulated Protein 78,000 (GRP78) on the cell surface. Its presence there was completely unexpected since this protein normally functions as a molecular chaperone within the endoplasmic reticulum. When it appears on the cell surface, most particularly of cancer cells, it functions as a cell surface receptor coupled to signaling cascades which are pro-proliferative, antiapoptotic, and promigratory. Studies have identified antibodies in the plasma of cancer patients which bind to the amino-terminal domain of GRP78 where natural ligands also bind, functioning as agonistics. These antibodies are markers suggesting a poor prognosis. By contrast, antibodies which bind to the carboxyl-terminal domain block cancer cell proliferation and migration while promoting apoptosis. Thus, cell surface-associated GRP78 offers a new target for cancer therapy, particularly employing monoclonal antibodies.

Keywords

GRP78; signaling cascades; cell surface receptors; cancer cell proliferation; monoclonal antibody therapy

Outline

Discovery of Cell Surface GRP78

GRP78 and Cancer Cell Biology

A Most Complex Signaling Mechanism

References

Discovery of Cell Surface GRP78

This book will focus on the presence of the Glucose Regulated Protein 78,000 (GRP78) at the cell surface and its role in signal transduction. GRP78 is best known for its function as a molecular chaperone in the endoplasmic reticulum (ER; see Chapter 3: Cell Surface GRP78: Anchoring and Translocation Mechanisms and Therapeutic Potential in Cancer). As such, its presence on the cell surface was not anticipated. The story of cell surface GRP78 actually begins with the study of the proteinase inhibitor α2-macroglobulin (α2M). α2M is a broad-based proteinase inhibitor that first appeared some 600,000,000 years ago.¹ Its persistence over such a long period of time is somewhat perplexing since, in general, it appears to play a secondary role as an inhibitor of proteinases. Over the course of evolution, many much more specific inhibitors have appeared such as α1-proteinase inhibitor, antithrombin III, and tissue inhibitors of metalloproteinase. Nevertheless, no known case of a total α2M deficiency has been identified in humans.¹ These facts led us to propose that α2M is a sensor of proteolysis, rather than primarily an inhibitor of proteinases.² α2M is ideally suited for such a role, since it binds proteinases from all four mechanistic classes.¹ When activated, a thiolester bond in each of its four identical subunits ruptures and the proteinase undergoes a major conformational change.³,⁴ The molecule undergoes a 10% compacting of its structure exposing receptor recognition sites in each of its four subunits.⁵ The activated form is designated α2M* to distinguish it from the native molecule, α2M. It was recognized as early as the 1980s that receptors are present on a variety of cells in the body including macrophages and hepatocytes which cause rapid removal of α2M* from blood, t1/2=2–5 min.⁵,⁶ The first receptor identified as an α2M* binding site by Strickland and his colleagues was the lipoprotein receptor-related protein (LRP), a broad-based catabolic receptor which removes various proteins from the circulation.⁷ We argued that a second, signal transducing receptor must exist to explain how α2M could function as a sensor of proteolysis.² If α2M* is to function as such a sensor, cellular mechanism(s) must exist to transduce information to the signaling machinery of the cells which recognize the protein. That is to say, an α2M* signaling receptor (α2M*SR) must exist. There was one study demonstrating the ability of α2M* to stimulate prostaglandin E2 synthesis,⁸ but the search for a signaling receptor began in earnest in 1993. Detailed studies of α2M* and signal transduction became possible with the arrival of Dr Uma Misra in the Department of Pathology at the Duke Medical School. Misra was an expert in signal transduction biochemistry. He performed the initial studies employing elicited murine peritoneal macrophages. Misra was able to show that when treated with α2M* a percentage of these cells demonstrated increased intracellular calcium, inositol phosphates, and cyclic AMP.⁹ He subsequently demonstrated activation of phospholipase C, phospholipase A2, and protein kinase C in these cells.¹⁰ In 1994 he showed that the α2M signaling receptor must be distinct from LRP.¹¹ This work was extended by mutation studies of the carboxyl-terminal domain of α2M where the receptor binding site is located. Mutation of specific amino acid residues affected binding of the receptor binding domain to either LRP or the as yet unidentified α2M*SR.¹² Subsequent studies employing ¹²⁵I- α2M* demonstrated that activated murine peritoneal macrophages have two distinct classes of binding sites as shown by a biphasic Scatchard plot.¹³ One site was clearly LRP (Kd~1–10 nM, ~250,000 binding sites/cell) while the other was the α2M*SR (Kd~50–100 pM, ~10,000 binding sites/cell). By contrast, naïve murine peritoneal macrophages possess only LRP.¹⁴ In a number of subsequent publications, Misra dissected the α2M*-dependent signaling pathways (e.g., see Refs. 15–26). These pathways will be discussed in greater detail by Dr Udhayakumar Gopal in Chapter 2, The Endoplasmic Reticulum Chaperone GRP78 Also Functions as a Cell Surface Signaling Receptor. The major breakthrough with respect to identification of this receptor occurred in 2002 when we isolated and characterized the cell surface GRP78 as the α2M*SR.¹⁷ We had by this time undertaken studies of α2M*-mediated signal transduction in prostate cancer cells as well as activated macrophages (e.g., see Ref. 19).

GRP78 and Cancer Cell Biology

Scientific progress often occurs for odd reasons. Dr Misra is a devout Hindu and vegetarian. One day I asked him how he felt about sacrificing mice for his studies. He said that he always prayed for their little souls. This troubled me and (not for the first time) I urged him to switch to cancer cells. Perhaps they might also express α2M*SR on the cell surface. This seemed like a logical possibility to me since the pathways activated were pro-proliferative in nature. We had a number of prostate cancer cell lines frozen in storage including PC-3 cells and their derived offspring 1-LN cells. The latter were obtained from a lymph node metastasis. They demonstrate a much more aggressive phenotype than their parent