Analysis of ligand recognition by the purified alpha 2-macroglobulin receptor (low density lipoprotein receptor-related protein). Evidence that high affinity of alpha 2-macroglobulin-proteinase complex is achieved by binding to adjacent receptors.
The molecular basis for binding of alpha-macroglobulin-proteinase complexes to the human two-chain 500/85-kDa (alpha/beta) alpha 2-macroglobulin (alpha 2M) receptor (alpha 2MR)/low density lipoprotein receptor-related protein was analyzed. Ligand blotting experiments showed that a 40-kDa protein, present in the affinity-purified alpha 2MR preparation, is bound to the alpha 2MR alpha-chain and released by heparin. Removal of the 40-kDa protein resulted in a 3-5-fold increase in binding of alpha 2M-trypsin. Nitrocellulose-immobilized pure two-chain alpha 2MR was incubated with human alpha 2M-trypsin, containing four identical subunits, and two monovalent ligands: rat alpha 1-inhibitor-3-chymotrypsin and the 18-kDa receptor binding fragment of the alpha 2M subunit. Binding of alpha 2M-trypsin to the alpha-chain of immobilized alpha 2MR was composed of a high (Kd = 40 pM at 4 degrees C) and a low (Kd = 2 nM) affinity component. alpha 1-Inhibitor-3-chymotrypsin bound to the same sites but with one component (Kd = 0.4 nM). Competition-inhibition experiments and dissociation experiments, using ligands with different valences, as well as experiments with alpha 2MR immobilized at different densities, led to the following model. The low (Kd = 2 nM) affinity of alpha 2M-proteinase is prevalent when only one of the four domains binds to alpha 2MR, i.e. when the receptor density is low or when neighboring receptors are occupied. The high (Kd = 40 pM) affinity is achieved by binding of at least two domains to adjacent receptors.
Moestrup
S K
SK
Institute of Physiology, University of Aarhus, Denmark.
Gliemann
J
J
D000818:Animals; D002918:Chymotrypsin / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008024:Ligands; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D046911:Macromolecular Substances; D008970:Molecular Weight; D011485:Protein Binding; D051381:Rats; D011940:Receptor Aggregation; D011971:Receptors, Immunologic / Q000737:chemistry / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000378:metabolism*
D008024:Ligands; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D046911:Macromolecular Substances; D011971:Receptors, Immunologic; D000511:alpha-Macroglobulins; D002918:Chymotrypsin; D014357:Trypsin