Cell membrane-localised GRP78

Increased expression of GRP78 caused by the UPR may lead to cell surface GRP78 (sGRP78) localisation [194,197,200,201]. This form of GRP78 is mainly found in cancer cells [194,233-244], where it has been partially studied. It is also found in trophoblastic cells [92,93,245]. No modifications in the amino acid sequence have been observed in sGRP78.

53 For that reason, it has been proposed that a glycosylation-masking KDEL signal [197], or a saturation in the KDEL retention system may be the cause behind the relocalisation of GRP78 to the cell surface [197,200]. The mechanisms in charge of translocating the protein from the ER to the cell surface are not completely understood; however, it has been reported that prostate apoptosis response protein-4 (Par-4) is involved in this process [245-247]. The cellular surface location of GRP78 can be achieved in three different ways: TM GRP78, association of GRP78 with a TM protein or association of GRP78 with glycosylphosphatidylinositol (GPI) [248]. The TM GRP78 localisation in the cell membrane displays a four-pass TM (4TM) conformation, with the N-terminus and C-terminus in the extracellular matrix (Figure 19) [197].

Figure 19. Cell surface GRP78 conformation. Transmembrane four-pass GRP78. GRP78 can insert into the cell membrane. Association of GRP78 with a transmembrane protein. The association of GRP78 with a transmembrane protein allows it to be anchored to the cell membrane and transmit the signal into the cytoplasm.

Association of GRP78 with glycosylphosphatidylinositol (GPI). GRP78 can be associated with GPI at the cell surface.

54 sGRP78 acts as a promiscuous receptor, since it can interact with several extracellular ligands [239,241,243-247,249-255] and be a co-receptor for viral entry [256-258]. Additionally, TM sGRP78 associates with cell surface-anchored proteins and integral membrane proteins, allowing signal transduction triggered by the ligand interaction [199,248].

In cancer cells, several proteins interact with sGRP78, among them activated α2 -macroglobulin (α2M*) [239,241,249,250], CRIPTO [243,244,251,252], integrin β-1 [253], Par-4 [245-247] and human plasminogen Kringle 5 (K5) [254,255].

1.2.2.5.1 α

M* interaction

The α-macroglobulins are a family of abundant plasma proteins composed of α1M, α2M, complement components and pregnancy zone protein (PZP) [259]. α2M is one of the major plasma proteinase inhibitors, which inhibits the four classes of proteinases and is the most studied member of this family [259-265].

α2M is transcribed as a monomer of 180 kDa, which folds into its native form and interacts with three identical α2M subunits, forming a homotetramer of 720 kDa, the subunits of which are bound by S-S bonds [259,264-267]. The active form of this glycoprotein is named α2M*, and in order to perform its different functions it needs to be activated by proteinases [268].

Nevertheless, monomers and dimers of α2M have been observed, even if their functions have not yet been elucidated [269,270].

To understand the different functions that α2M* can perform, it is necessary to consider its structure and its different domains. Characterisation of the α2M* demonstrated presence of a bait region-containing macroglobulin domain, a thiol ester-containing domain and an RBD.

55 The first two domains are implicated in the intrinsic proteinase inhibitor function of α2M*, while the third is involved in α2M* receptor binding [259,260,262-265,268,271,272].

Binding of α2M* and sGRP78 has been reported to activate different cellular mechanisms.

For instance, in sperm cells, the interaction of α2M* and sGRP78 promotes actin reorganisation, facilitating sperm motility [273]. The motility of macrophages is also affected by sGRP78–α2M* binding, but in this case, the mechanisms involved are dependent on the activation of p21-activated protein kinase 2 (PAK2) [274]. In prostate cancer cells, PAK2 is also activated by the interaction between α2M* and sGRP78, increasing the phosphorylation of Lin11, Isl-1 and Mec-3 (LIM) kinase and cytoskeletal organisation, conferring additional motility to prostate cancer cells and increasing their metastatic potential [239]. In prostate cancer cells, the binding of α2M* to sGRP78 also regulates cell growth by activating the Ras, the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)-dependent signalling pathway and the c-Jun N-terminal kinase (JNK) signalling cascade (Figure 20) [241,249]. α2M* is additionally able to promote cellular proliferation by activating extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38 MAPK) (Figure 20) [241]. Moreover, sGRP78 and α2M* binding affect cell survival by activating protein kinase B (also known as Akt) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) signalling in prostate cancer cells (Figure 20) [241]. Finally, in prostate cancer, the GRP78–α2M interaction induces the UPR by increasing GRP78, eIF2α, activating transcription factor 4 (ATF4) and ATF6 expression (Figure 20) [241].

56

Figure 20. Effects of the α2-macroglobulin (α2M) interaction with cell surface GRP78. The interaction of GRP78 with α2M affects cell motility through activation of p21-activated protein kinase 2 (PAK2) and LIM kinase in macrophages and prostate cancer cells. Cell growth was also affected by the GRP78–α2M interaction due to activation of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)-dependent signalling pathway and the c-Jun N-terminal kinase (JNK) in prostate cancer cells. Proliferation of prostate cancer cells was also altered after GRP78 interacted with α2M due to activation of p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal-regulated kinase 1/2 (ERK1/2). Prostate cancer cell survival was also affected by nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) and protein kinase B (also known as Akt) activation upon α2M binding to GRP78. Finally, the interaction between cell surface-localised GRP78 and α2M leads to activation of the unfolded protein response (UPR) in prostate cancer cells.

1.2.2.5.2 CRIPTO interaction

CRIPTO is a secreted GPI-anchored signalling protein involved in cancer and development [275]. CRIPTO binding to sGRP78 modulates transforming growth factor-β (TGF-β) signalling and promotes cell growth [243]. Additionally, the CRIPTO/sGRP78 interaction induces the Src, MAPK and PI3K molecular pathways, stimulating proliferation, migration and plasticity (Figure 21) [244,252].

57

Figure 21. Effects of cell surface-localised GRP78 interacting with CRIPTO, integrin β-1, prostate apoptosis response protein-4 (Par-4), Kringle 5 (K5) and viruses. CRIPTO. The binding of CRIPTO with cell surface-localised GRP78 activates cell growth through transforming growth factor-β (TGF-β) and Smad2/3 activation. It also increases cell migration by inducing activation of c-Src and the mitogen-activated protein kinase (MAPK) pathway, together with the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and protein kinase B (also known as Akt) pathways. This last pathway also impacts cell survival and proliferation. Integrin β-1. The interaction of GRP78 with integrin β-1 enhances focal adhesion kinase (FAK) activation, increasing cell proliferation. Par-4. The interaction of secreted Par-4 and GRP78 induces activation of the Fas-associated protein with death domain (FADD)/caspase-8/caspase-3 signalling pathway and promotes apoptosis. K5. The GRP78/K5 interaction activates the caspase-7 signalling pathway, leading to apoptosis. Viruses. Cell surface-localised GRP78 can act as a co-receptor for virus internalisation.

1.2.2.5.3 Integrin β-1 interaction

Integrin β-1 is a cell surface receptor, which normally associates with different cell membrane proteins to act as a receptor [276]. Integrin β-1 can associate with sGRP78 and regulate the focal adhesion kinase (FAK) signalling pathway, resulting in cell proliferation (Figure 21) [253].

58

1.2.2.5.4 Par-4 interaction

Par-4 is a 40 kDa tumour suppressor protein, generally located in the cytoplasm [246,277]

but has been reported to be secreted as well [246]. The interaction of secreted Par-4 and sGRP78 induces the Fas-associated protein with death domain (FADD)/caspase-8/caspase-3 signalling pathway and promotes apoptosis (Figure 21) [246].

1.2.2.5.5 Kringle 5 interaction

Kringle 5 (K5) is an autonomous protein domain that originates from human fibrinogen degradation [278]. Kringle 5 is capable of binding to sGRP78 [254,255]. The sGRP78/K5 interaction activates the caspase-7 signalling pathway, leading the cell towards apoptosis [254,255]. Moreover, sGRP78/K5 binding promotes anti-angiogenic activity and inhibits migration and proliferation of endothelial cells (Figure 21) [254].

Finally, sGRP78 can associate with major histocompatibility complex class I (MHC-I) molecules on the cell surface and act as a co-receptor for virus internalisation [256-258], including the dengue viruses or Coxsackie virus A9 (Figure 21) [256].