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A recombinant version of the receptor binding domain (RBDv) of human alpha 2-macroglobulin (alpha 2M) has been expressed in E. coli and refolded using a novel iterative procedure. RBDv (Val1299-Ala1451) is extended by 15 residues at the N-terminal side of the Lys1313-Glu papain cleavage site in human alpha 2M. RBDv contains the intra-chain bridge Cys1329-Cys1444 and is soluble and monomeric. Competition experiments with 125I-labelled methylamine-treated alpha 2M reveal that RBDv binds to the placental receptor for transformed alpha 2M with a Kd of 8 nM, i.e. the binding affinity of RBDv is of the same order of magnitude as the intrinsic affinity for binding of one domain in transformed alpha 2M to one receptor molecule.

Recent findings have elucidated the mechanism for clearance from the extracellular space of the two types of plasminogen activators, urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA), and their type-1 inhibitor (PAI-1). Activator/PAI-1 complexes and uncomplexed t-PA bind to the multi-ligand receptors alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR) and epithelial glycoprotein 330 (gp330). These receptors mediate endocytosis and degradation of u-PA/PAI-1 complex bound to the glycosyl phosphatidyl inositol-anchored urokinase receptor (u-PAR) on cell surfaces, and participate, in cooperation with other receptors, in hepatic clearance of activator/PAI-1 complexes and uncomplexed t-PA from blood plasma. The alpha 2MR- and gp330-mediated endocytosis of a ligand (u-PA/PAI-1 complex) initially bound to another receptor (u-PAR) is a novel kind of interaction between membrane receptors. Binding to alpha 2MR and gp330 is a novel kind of molecular recognition of serine proteinases and serpins.

Flourescence-activated cell scanning analysis of human blood cells revealed novel urokinase receptors in large granular lymphocytes and a small subset of T-cells (CD3+). Culturing of T-cells with interleukin-2 to generate CD3+ lymphokine-activated killer cells caused a large increase in urokinase binding, suggesting that the urokinase receptor is an activation antigen. The receptor in lymphocytes was similar to that in monocytes with regard to size, affinity and ligand specificity, but did not mediate degration of urokinase-inhibitor complexes. It is suggested that lymphocyte-bound pro-urokinase is activated, e.g. by the human T-cell specific serine proteinase. HuTSP-1, and thereby starts a cascade of plasminogen activation important for extravasation of the cells.

The human placental receptor (alpha 2MR) for alpha 2-macroglobulin-proteinase complexes contains 3 polypeptides of approx. 500 kDa, 85 kDa, and 40 kDa. N-terminal sequence analysis of the 500 kDa and 85 kDa polypeptides, analysis of a random selection of peptides convering 536 residues from these polypeptides, and analysis of a 1772 bp cDNA encoding part of the 500 kDa polypeptide provide evidence that the 500 kDa and 85 kDa chains are the alpha- and beta-subunits, respectively, of a recently cloned hepatic membrane protein, termed the low density lipoprotein receptor related protein (LRP) (Herz, J., Hamann, U., Rogne, S., Myklebost, O., Gausepohl, H. and Stanley, K.K. (1988) EMBO J. 7, 4119-4127; Herz, J., Kowal, R.C., Goldstein, J.L. and Brown, M.S. (1990) EMBO J. 9, 1769-1776). N-terminal sequence analysis of the 40 kDa polypeptide shows that it is of distinct genetic origin. It is suggested that LRP is the functional receptor for alpha 2-macroglobulin-proteinase complexes (alpha 2MR) and in addition may have as yet unsettled functions in lipoprotein metabolism.

Carbohydrates were characterized in the human placental α2-macroglobulin receptor and its associated protein. Carbohydrates, largely N-linked, contributed to about 18% of the size of the receptor α-chain and to about 25% of the β-chain. The 40 kDa receptor-associated Protein also contained carbohydrate. The α- and β-chains contained a wide variety of carbohydrates as judged by binding of lectins. Monosaceharide-competing inhibition of α2M-methylamine binding by WGA suggested a functional significance of sugars in binding of ligands to the α-chain.

Uptake of rat and human alpha 2-macroglobulin-trypsin complexes was measured in rat hepatocytes, rat and human adipocytes and human fibroblasts. Uptake and degradation of 125I-labelled rat complex were about one-third of that of the human complex in the various isolated cell types. In rat hepatocytes, the apparent Km for cell association of the rat complex was about 16 nM as compared to about 6 nM for the human complex. The Vmax values were similar, about 1 X 10(4) molecules X cell-1 X min-1. Thus, rat alpha 2-macroglobulin (an acute-phase protein) complexed with trypsin follows the same pathways of uptake as the human homologue, although with a somewhat lower affinity for the uptake system.

We report that the Vps10p domain receptor sorLA binds the adaptor proteins GGA1 and -2, which take part in Golgi-endosome sorting. The GGAs bind with differential requirements via three critical residues in the C-terminal segment of the sorLA cytoplasmic tail. Unlike in sortilin and the mannose 6-phosphate receptors, the GGA-binding segment in sorLA contains neither an acidic cluster nor a dileucine. Our results support the concept of sorLA as a potential sorting receptor and suggest that key residues in sorLA and sortilin conform to a new type of motif (Ψ-Ψ-X-X-∅) defining minimum requirements for GGA binding to cytoplasmic receptor domains. © 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

The cellular binding and uptake was studied for α 1 -inhibitor 3 , a monomeric 200 kDa proteinase inhibitor present in rat plasma. After intravenous injection in the rat the inhibitor disappeared from the circulation with a half-time of 2.5 min when complexed with chymotrypsin, whereas the half-time for uncomplexed inhibitor was more than 60 min. 6 min after the injection of labelled complex, 83% was in the liver and 2.5% in the spleen. In vitro experiments at 4°C with isolated hepatocytes and peritoneal macrophages showed binding to the previously described receptors which bind and internalize the tetrameric rat and human α 2 -macroglobulin-proteinase complexes. The binding affinities were similar for the two types of complexes and binding was followed by uptake and degradation of the labelled complex when the cells were warmed to 37°C. The binding of uncomplexed α 1 -inhibitor 3 was low and did not increase following treatment with methylamine in spite of cleavage of the internal thiol ester. α 1 -Inhibitor 3 -methylamine was changed to the receptor binding form when treated with chymotrypsin which caused the cleavage of at least one peptide bond in the bait region.

Digestion of methylamine-treated alpha 2-macroglobulin (alpha 2M X MA) with catalytic amounts of papain at pH 4.5 has been investigated. Cleavage of Lys(1313)-Glu resulted in two major products, which could be separated by gel chromatography: a large disulfide bridged fragment set nearly the size of intact alpha 2M X MA, and an 18 kDa fragment, constituting the carboxy-terminal domain of alpha 2M. This domain contained the receptor recognition site, exposed as a result of cleavage of the internal beta-cysteinyl-gamma-glutamyl thiol esters in alpha 2M. Compared with alpha 2M-trypsin complex the apparent affinity for binding to rat hepatocyte receptors was 0.1 and 2% at 4 and 37 degrees C, respectively. The receptor-binding domain presumably forms a compact globular beta-barrel-type structure, stable at pH 2.5-9.0. Chemical modification experiments suggest that receptor binding is contributed by a determinant formed by the precise folding of the polypeptide chain.

Transport of the nonmetabolizable hexose analogue 3-O-methyl-D-glucose (30MG) was measured in human polymorphonuclear leukocytes at 37 degrees C, pH 7.4. 3OMG at very low concentration (0.05 mM) equilibrated with the intracellular water with a rate constant of about 0.08 s-1. Transport of 3OMG in the presence of 20 microM cytochalasin B and transport of L-glucose were insignificant. Countertransport of 14C-labelled 3OMG was demonstrated. Exchange of 3OMG between the extracellular and intracellular water showed saturation with a Km of about 4 mM. Thus, the transport of 3OMG is mediated almost exclusively by facilitated diffusion.