Abstract
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.
Highlights
From the Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710 and the ‡Department of Pharmacology, University of Texas-Southwestern Medical Center, Dallas, Texas 75235
Binding to lipoprotein receptor-related protein/␣2M receptor but not ␣2M signal receptor is inhibitable by another ligand, receptor-associated protein
Direct binding studies with K1374R demonstrated no significant alteration in binding to the lower affinity site; binding to the high affinity site is reduced by 83%
Summary
␣2-Macroglobulin Signaling Receptor or the Low Density Lipoprotein Receptor-related Protein/␣2-Macroglobulin Receptor*. Each ␣2M subunit contains an internal -cysteinyl␥-glutamyl thiol ester which can undergo direct nucleophilic attack by methylamine or ammonia This reaction causes a conformational change in the inhibitor exposing the receptor recognition sites. The low density lipoprotein receptor-related protein/␣2M receptor (LRP/␣2MR) has been identified as an ␣2M receptor (for review, see Ref. 3) This multivalent receptor binds many ligands besides ␣2M-proteinase or -methylamine including receptor-associated protein (RAP), apolipoprotein E, lactoferrin, lipoprotein lipase, Pseudomonas exotoxin A, plasminogen activators alone or in complex with inhibitors, and potentially other serpin-proteinase complexes. Most of these ligands do not compete for binding with each other; RAP is able to block binding of all known ligands to LRP/␣2MR. Site-directed mutagenesis together with secondary structure prediction was employed to probe regions of RBF which might be important for binding to LRP/␣2MR and ␣2MSR
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