Distinct binding sites in the structure of alpha 2-macroglobulin mediate the interaction with beta-amyloid peptide and growth factors.
Alpha(2)-macroglobulin (alpha(2)M) and its receptor, low density lipoprotein receptor-related protein (LRP), function together to facilitate the cellular uptake and degradation of beta-amyloid peptide (Abeta). In this study, we demonstrate that Abeta binds selectively to alpha(2)M that has been induced to undergo conformational change by reaction with methylamine. Denatured alpha(2)M subunits, which were immobilized on polyvinylidene difluoride membranes, bound Abeta, suggesting that alpha(2)M tertiary and quaternary structure are not necessary. To determine whether a specific sequence in alpha(2)M is responsible for Abeta binding, we prepared and analyzed defined alpha(2)M fragments and glutathione S-transferase-alpha(2)M peptide fusion proteins. A single sequence, centered at amino acids (aa) 1314-1365, was identified as the only major Abeta-binding site. Importantly, Abeta did not bind to the previously characterized growth factor-binding site (aa 718-734). Although the Abeta binding sequence is adjacent to the binding site for LRP, the results of experiments with mutated fusion proteins indicate that the two sites are distinct. Furthermore, a saturating concentration of Abeta did not inhibit LRP-mediated clearance of alpha(2)M-MA in mice. Using various methods, we determined that the K(D) for the interaction of Abeta with its binding site in the individual alpha(2)M subunit is 0.7-2.4 microm. The capacity of alpha(2)M to bind Abeta and deliver it to LRP may be greater than that predicted by the K(D), because each alpha(2)M subunit may bind Abeta and the bound Abeta may multimerize. These studies suggest a model in which alpha(2)M has three protein interaction sites with distinct specificities, mediating the interaction with Abeta, growth factors, and LRP.
Mettenburg
Joseph M
JM
Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.
Webb
Donna J
DJ
Gonias
Steven L
SL
D016229:Amyloid beta-Peptides / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D006801:Humans; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D000511:alpha-Macroglobulins / Q000737:chemistry / Q000378:metabolism*
D016229:Amyloid beta-Peptides; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D000511:alpha-Macroglobulins