The Role of Grp 78 in α2-Macroglobulin-induced Signal Transduction

Abstract

The low density lipoprotein receptor-related protein (LRP) is a scavenger receptor that binds to many proteins, some of which trigger signal transduction. Receptor-recognized forms of ␣ 2 -Macroglobulin (␣ 2 M*) bind to LRP, but the pattern of signal transduction differs significantly from that observed with other LRP ligands. For example, neither Ni 2؉ nor the receptor-associated protein, which blocks binding of all known ligands to LRP, block ␣ 2 M*-induced signal transduction. In the current study, we employed ␣ 2 -macroglobulin (␣ 2 M)-agarose column chromatography to purify cell surface membrane binding proteins from 1-LN human prostate cancer cells and murine macrophages. The predominant binding protein purified from 1-LN prostate cancer cells was Grp 78 with small amounts of LRP, a fact that is consistent with our previous observations that there is little LRP present on the surface of these cells. The ratio of LRP:Grp 78 is much higher in macrophages. Flow cytometry was employed to demonstrate the presence of Grp 78 on the cell surface of 1-LN cells. Purified Grp 78 binds to ␣ 2 M* with high affinity (K d ϳ150 pM). A monoclonal antibody directed against Grp 78 both abolished ␣ 2 M*-induced signal transduction and co-precipitated LRP. Ligand blotting with ␣ 2 M* showed binding to both Grp 78 and LRP heavy chains in these preparations. Use of RNA interference to silence LRP expression had no effect on ␣ 2 M*-mediated signaling. We conclude that Grp 78 is essential for ␣ 2 M*-induced signal transduction and that a "co-receptor" relationship exists with LRP like that seen with several other ligands and receptors such as the uPA/uPAR (urinary type plasminogen activator or urokinase/uPA receptor) system.1 is a plasma proteinase inhibitor with broad specificity. Upon binding to proteinases, it undergoes a major conformational change that exposes receptor recognition sites on the molecule (1). This activated form of ␣ 2 M is designated ␣ 2 M*. Though difficult to reconcile, accumulating evidence has demonstrated that ␣ 2 M*-induced signaling occurs via a receptor that is functionally unique from the previously characterized ␣ 2 M* receptor, the low density lipoprotein receptor-related protein (LRP). LRP is a scavenger receptor (1) that binds to a variety of proteins, many of which trigger signal transduction (2-5). This pathway requires the activation of a pertussis toxin-sensitive G protein (2-5). The receptor-associated protein (RAP) that blocks the binding of all known ligands to LRP is also an antagonist for this signaling pathway (2-3). Whereas ␣ 2 M* binds to LRP, when cells are exposed to ␣ 2 M* a distinct set of signaling events is observed that differ from those induced by other ligands for this receptor (2-6). These include activation of a different G protein and the lack of antagonism by RAP or Ni 2ϩ (2-7). In addition, binding studies with a variety of cells in culture demonstrate two classes of binding sites, one of very high affinity (K d ϳ100 pM and 1600 sites/cell) and one of lower affinity (K d ...

Binding of Activated α2-Macroglobulin to Its Cell Surface Receptor GRP78 in 1-LN Prostate Cancer Cells Regulates PAK-2-dependent Activation of LIMK

“…The expression of GRP78 on the surface of PC3, DU145, and LnCap cells was either absent or minimal (17,18). By contrast, the highest levels that we have detected are on the surface of 1-LN cells, which were employed to purify ␣ 2 M* signaling receptor and identify it as GRP78 (20). Preincubation of 1-LN cells with antibodies against GRP78, silencing of the expression of the GRP78 gene and silencing of the expression of MTJ-1, with which GRP78 associates, greatly attenuated ␣ 2 M*-induced mitogenic signaling and cellular responses (16,17,19,35).…”

“…These events require the presence of a small number of sites (ϳ1500/cells) demonstrating very high ligand affinity (K d 50 -100 pM) for cellular binding of ␣ 2 M* or its receptor binding domain. This second receptor, initially termed the ␣ 2 M* signaling receptor, was later isolated from murine peritoneal macrophages and 1-LN human prostate cancer cells and identified as cell surface-associated GPR78, a heat shock protein of the HSP70 family (20). This molecular chaperone has been highly characterized for its ability to promote cell survival during endoplasmic reticulum (ER) stress (see reviews in Refs.…”

“…Binding of ␣ 2 M* to macrophages (14,15), rheumatoid synovial fibroblasts (16), and 1-LN prostate cancer cells triggers increases in the levels of intracellular inositol 1,3,4-trisphosphate and cytosolic-free calcium [Ca 2ϩ ] i and is followed by activation of components of the Ras/MAPK and PI 3-kinase signaling cascades (17)(18)(19)(20). As a consequence of these events, ␣ 2 M* up-regulates DNA synthesis and cellular proliferation (17)(18)(19)(20). Based on these and other observations, we hypothesized that ␣ 2 M* functions like a growth factor, and its receptor functions as a growth factor receptor (13).…”

“…The activated form of ␣ 2 M (␣ 2 M*) is also produced by direct reaction of internal thiol esters present in each of its four identical subunits with small amines or ammonia (13). Binding of ␣ 2 M* to macrophages (14,15), rheumatoid synovial fibroblasts (16), and 1-LN prostate cancer cells triggers increases in the levels of intracellular inositol 1,3,4-trisphosphate and cytosolic-free calcium [Ca 2ϩ ] i and is followed by activation of components of the Ras/MAPK and PI 3-kinase signaling cascades (17)(18)(19)(20). As a consequence of these events, ␣ 2 M* up-regulates DNA synthesis and cellular proliferation (17)(18)(19)(20).…”

The Urokinase/PAI-2 Complex

“…Binding of uPA-PAI-2 to Immobilized LRP-Ligand blotting was undertaken for the initial characterization of uPA-PAI-2 binding to LRP as this procedure is often used to investigate the interaction between LRP and potential ligands (39,44,45). Purified LRP (1 g) was dotted onto nitrocellulose and dried for 30 min at 37°C.…”