Selective mutations in cloned and expressed alpha-macroglobulin receptor binding fragment alter binding to either the alpha2-macroglobulin signaling receptor or the low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor.
alpha2-Macroglobulin (alpha2M) activated with methylamine binds to two distinct cell-surface receptors: low density-lipoprotein receptor-related protein/alpha2M receptors and alpha2M signaling receptors. Binding to lipoprotein receptor-related protein/alpha2M receptor but not alpha2M signal receptor is inhibitable by another ligand, receptor-associated protein. Direct binding studies with a recombinant receptor binding fragment (RBF) from rat alpha1M and murine macrophages demonstrate two classes of binding sites of apparent Kd = 90 pM (1500 sites/cell) and 40 nM (60,400 sites/cell). Receptor-associated protein competes with RBF for binding to the lower but not the higher affinity site. Site-directed mutation of Lys-1374 (human numbering) in RBF to Arg or Ile residues almost completely abolishes signal transduction as compared to wild-type RBF. Direct binding studies with K1374R demonstrated no significant alteration in binding to the lower affinity site; however, binding to the high affinity site is reduced by 83%. Mutation of Lys-1370 to Ala resulted in a 4-5-fold increase in the Kd for binding to the lower affinity site with no significant alteration in binding to the high affinity site or signal transduction properties. Studies demonstrate comparable internalization and degradation of wild-type RBF and K1374R; however, internalization and degradation of K1370A is negligible. These studies suggest that regions around Lys-1370 and Lys-1374 are involved in lipoprotein receptor-related protein/alpha2M receptor and alpha2M signaling receptor binding, respectively.
Howard
G C
GC
Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Yamaguchi
Y
Y
Misra
U K
UK
Gawdi
G
G
Nelsen
A
A
DeCamp
D L
DL
Pizzo
S V
SV
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D006801:Humans; D007700:Kinetics; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008239:Lysine; D008264:Macrophages / Q000276:immunology; D008744:Methylamines; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D051381:Rats; D011971:Receptors, Immunologic / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*
D017931:DNA Primers; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008744:Methylamines; D010446:Peptide Fragments; D011971:Receptors, Immunologic; D011994:Recombinant Proteins; D000511:alpha-Macroglobulins; C027451:methylamine; D008239:Lysine