The monomeric receptor binding domain of tetrameric α_2- macroglobulin binds to cell surface GRP78 triggering equivalent activation of signaling cascades

摘要

α_2-Macroglobulin (α_2M) is a broad spectrum proteinase inhibitor that when activated by proteinases (α_2M*) undergoes a major conformational change exposing receptor recognition sites in each of its four subunits. These complexes bind to two distinct receptors, namely, the low-density lipoprotein receptor-related protein (LRP) and cell surface glucose-regulated protein [M_r ～ 78000 (GRP78)]. The latter is a very high affinity receptor (K_d = 50-100 pM) whose ligation triggers pro-proliferative and anti-apoptotic signaling cascades. Despite its four binding sites, Scatchard analysis of binding of α_2M* to cells does not yield a cooperative plot. We, therefore, hypothesize that a monomeric cloned and expressed α_2M receptor binding domain (RBD) should trigger comparable signaling events. Indeed, RBD or its K1370A mutant that binds to GRP78 but cannot bind to LRP regulates DNA and protein synthesis by human prostate cancer cells in a manner comparable to that of α_2M*. Akt and mTORC1 activation and signaling are also comparably upregulated by α_2M*, RBD, or mutant K1370A. Antibodies directed against the carboxyl-terminal domain of GRP78 are antagonists that block α_2M*-mediated effects on pro-proliferative and anti-apoptotic signaling cascades and protein and DNA synthesis. The effects of RBD and its mutant were similarly blocked by these antibodies. Finally, proteolysis of α_2M at pH values from 5.7 to 7.0 causes production of free RBD and RBD-containing fragments. Thus, while α_2M* ligates only one GRP78 receptor molecule per α_2M*, it may potentially serve as a reservoir for release of up to four binding fragments per molecule.