Mutation of lysine 1370 in full-length human α2-macroglobulin blocks binding to the low density lipoprotein receptor-related protein-1

alpha(2)-Macroglobulin (alpha(2)M) regulates cell physiology by binding to cellular receptors; however, residues that contribute to receptor-binding have not been elucidated in the full-length protein. In alpha(2)M fragments, expressed in bacteria, Lys(1370) and Lys(1374) are critical for binding to the low density lipoprotein receptor-related protein-1 (LRP-1) and a distinct alpha(2)M-signaling receptor. We expressed full-length recombinant human alpha(2)M (r alpha(2)M) and mutants in which Lys1370 or Lys(1374) was converted to alanine in K-562 cells. The r alpha(2)M species demonstrated intact structure and function, as determined by subunit size, intersubunit disulfide bonds, reaction with trypsin or methylamine, and ability to undergo conformational change. Binding of transforming growth factor-beta 1 was unaltered. Mutation of Lys(1370) almost entirely inhibited specific binding of methylamine-activated r alpha(2)M to RAW 264.7 cells. Mutation of Lys(1374) had no effect. Binding of r alpha(2)M to RAW 264.7 cells was blocked by receptor-associated protein, indicating an essential role for LRP-1. These studies demonstrate that a single mutation in full-length r alpha(2)M is sufficient to block binding to LRP-1. (c) 2005 Elsevier Inc. All rights reserved.