title: The monomeric receptor binding domain of tetrameric α2-macroglobulin binds to cell surface GRP78 triggering equivalent activation of signaling cascades.
abstract: α2-Macroglobulin (α2M) is a broad spectrum proteinase inhibitor that when activated by proteinases (α2M*) 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 [Mr ∼ 78000 (GRP78)]. The latter is a very high affinity receptor (Kd = 50-100 pM) whose ligation triggers pro-proliferative and anti-apoptotic signaling cascades. Despite its four binding sites, Scatchard analysis of binding of α2M* to cells does not yield a cooperative plot. We, therefore, hypothesize that a monomeric cloned and expressed α2M 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 α2M*. Akt and mTORC1 activation and signaling are also comparably upregulated by α2M*, RBD, or mutant K1370A. Antibodies directed against the carboxyl-terminal domain of GRP78 are antagonists that block α2M*-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 α2M at pH values from 5.7 to 7.0 causes production of free RBD and RBD-containing fragments. Thus, while α2M* ligates only one GRP78 receptor molecule per α2M*, it may potentially serve as a reservoir for release of up to four binding fragments per molecule.
authors.affiliation: Department of Pathology, Duke University Medical Center, Durham, NC 27710, USA.
authors.forename: Uma Kant
authors.initials: UK
authors.lastname: Misra
authors.forename: Sturgis
authors.initials: S
authors.lastname: Payne
authors.forename: Salvatore Vincent
authors.initials: SV
authors.lastname: Pizzo
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D045744:Cell Line, Tumor; D000091342:Endoplasmic Reticulum Chaperone BiP; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D020935:MAP Kinase Signaling System; D000076222:Mechanistic Target of Rapamycin Complex 1; D046912:Multiprotein Complexes; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017430:Prostate-Specific Antigen / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D054730:Protein Interaction Domains and Motifs; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D014158:Transcription, Genetic; D015854:Up-Regulation; D000511:alpha-Macroglobulins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D046912:Multiprotein Complexes; D000511:alpha-Macroglobulins; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D017430:Prostate-Specific Antigen