title: Limited mutations in full-length tetrameric human alpha2-macroglobulin abrogate binding of platelet-derived growth factor-BB and transforming growth factor-beta1.
abstract: alpha2-Macroglobulin (alpha2M) inhibits diverse extracellular proteases, binds growth factors such as platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-beta1 (TGF-beta1), and carries beta-amyloid peptide. alpha2M may also trigger cell signaling by binding to the low density lipoprotein receptor-related protein (LRP-1) and/or other cell surface receptors. Based on studies with recombinant alpha2M fragments expressed in bacteria and synthetic peptides, we previously localized a growth factor-binding site near the center of the alpha2M subunit. However, because intact alpha2M forms a hollow cylinder structure, an alternative model for growth factor binding involves nonspecific entrapment within the alpha2M core. To distinguish between these two models, we engineered mutations in the putative growth factor binding sequence of full-length alpha2M. These mutations did not perturb the tetrameric structure of alpha2M, reaction with proteases, the thiol ester bonds, or binding to LRP-1. A single mutation (E730R) completely blocked binding of platelet-derived growth factor-BB to intact alpha2M. E730R did not alter TGF-beta1 binding; however, this mutation in combination with mutations at Glu714 and Asp719 eliminated the increase in TGF-beta1 binding associated with alpha2M conformational change. These studies demonstrate that growth factor binding to intact alpha2M is specific, involving a defined region of the alpha2M subunit. The exact sequences required for binding different growth factors may be non-identical, mimicking the model of the bait region in which different proteases target adjacent and sometimes overlapping sequences.
authors.affiliation: Department of Pathology, University of California San Diego, La Jolla, California 92093.
authors.forename: Sanja
authors.initials: S
authors.lastname: Arandjelovic
authors.affiliation: Department of Pathology, University of Virginia, Charlottesville, Virginia 22908.
authors.forename: Cristina L
authors.initials: CL
authors.lastname: Van Sant
authors.affiliation: Department of Pathology, University of California San Diego, La Jolla, California 92093. Electronic address: sgonias@ucsd.edu.
authors.forename: Steven L
authors.initials: SL
authors.lastname: Gonias
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000077214:Becaplermin; D006801:Humans; D020014:K562 Cells; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010982:Platelet-Derived Growth Factor / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D020574:Proto-Oncogene Proteins c-sis; D016212:Transforming Growth Factor beta / Q000737:chemistry* / Q000378:metabolism; D053773:Transforming Growth Factor beta1; D000511:alpha-Macroglobulins / Q000737:chemistry*
chemical_list: D010455:Peptides; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; C508960:TGFB1 protein, human; C508961:Tgfb1 protein, mouse; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D000511:alpha-Macroglobulins; D000077214:Becaplermin