Mutation of lysine 1370 in full-length human alpha2-macroglobulin blocks binding to the low density lipoprotein receptor-related protein-1.
alpha2-Macroglobulin (alpha2M) 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 alpha2M 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 alpha2M-signaling receptor. We expressed full-length recombinant human alpha2M (r(alpha)2M) and mutants in which Lys(1370) or Lys(1374) was converted to alanine in K-562 cells. The r(alpha)2M 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-beta1 was unaltered. Mutation of Lys(1370) almost entirely inhibited specific binding of methylamine-activated r(alpha)2M to RAW 264.7 cells. Mutation of Lys(1374) had no effect. Binding of r(alpha)2M 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)2M is sufficient to block binding to LRP-1.
Arandjelovic
Sanja
S
Department of Pathology, University of California San Diego, 9500 Gilman Road, La Jolla, CA 92093-0612, USA.
Hall
Brian D
BD
Gonias
Steven L
SL
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D006801:Humans; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013329:Structure-Activity Relationship; D000511:alpha-Macroglobulins / Q000737:chemistry* / Q000378:metabolism*
D026503:Low Density Lipoprotein Receptor-Related Protein-1; D011994:Recombinant Proteins; D000511:alpha-Macroglobulins; D008239:Lysine