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Sortilin and sorCS1 [sortilin-related Vps10p (vacuolar protein sorting/targeting protein 10) domain-containing receptor 1], both members of the Vps10p-D (Vps10p-domain) receptor family, are synthesized as precursor proteins and are converted into their mature form by enzymatic cleavage of a short N-terminal propeptide. SorCS1 does not bind its propeptide, but sortilin is able to bind not just its own propeptide, but also that of sorCS1. In the present study we show that the propeptide region of sorCS1 contains two separate sites for binding to sortilin and that only one of these sites is removed from human (as opposed to mouse) sorCS1 during processing. This leaves mature human sorCS1 with a sortilin-binding N-terminus, which allows formation of a complex between the two receptors in solution and on cell membranes. Furthermore, we find that the interaction with sorCS1 has a pronounced effect on sortilin's ability to mediate the cellular uptake of alternative ligands, and to hamper its facilitation of CNTF (ciliary neutrophic factor) signalling and the induction of phosphorylated STAT3 (signal transducer and activator of transcription 3). Thus the present study reveals a novel regulatory mechanism and suggest an entirely new role for sorCS1 as a modulator of sortilin function.

The type I transmembrane protein SorCS1 is a member of the Vps10p-domain receptor family comprised of Sortilin, SorLA and SorCS1, -2 and -3. Current information indicates that Sortilin and SorLA mediate intracellular protein trafficking and sorting, but little is known about the cellular functions of the SorCS subgroup. SorCS1 binds platelet-derived growth factor-BB (PDGF-BB) and is expressed in isoforms differing only in their cytoplasmic domains. Here, we identify two novel isoforms of mouse SorCS1 designated m-SorCS1c and -d. In situ hybridization revealed a combinatorial expression pattern of the variants in brain and embryonic tissues. We demonstrate that among the mouse variants, only SorCS1c mediates internalization and that the highly conserved SorCS1c is internalized through a canonical tyrosine-based motif. In contrast, human SorCS1a, whose cytoplasmic domain is completely different from mouse SorCS1a, is internalized through a DXXLL motif. We report that the human SorCS1a cytoplasmic domain interacts with the alphaC/sigma2 subunits of the adaptor protein (AP)-2 complex, and internalization of human SorCS1a and -c is mediated by AP-2. Our results suggest that the endocytic isoforms target internalized cargo to lysosomes but are not engaged in Golgi-endosomal transport to a significant degree.

Some endocytosis receptors related to the low-density lipoprotein receptor, including low-density lipoprotein receptor-related protein-1A, very-low-density lipoprotein receptor, and sorting protein-related receptor, bind protease-inhibitor complexes, including urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), and the uPA-PAI-1 complex. The unique capacity of these receptors for high-affinity binding of many structurally unrelated ligands renders mapping of receptor-binding surfaces of serpin and serine protease ligands a special challenge. We have mapped the receptor-binding area of the uPA-PAI-1 complex by site-directed mutagenesis. Substitution of a cluster of basic residues near the 37-loop and 60-loop of uPA reduced the receptor-binding affinity of the uPA-PAI-1 complex approximately twofold. Deletion of the N-terminal growth factor domain of uPA reduced the affinity 2-4-fold, depending on the receptor, and deletion of both the growth factor domain and the kringle reduced the affinity sevenfold. The binding affinity of the uPA-PAI-1 complex to the receptors was greatly reduced by substitution of basic and hydrophobic residues in alpha-helix D and alpha-helix E of PAI-1. The localization of the implicated residues in the 3D structures of uPA and PAI-1 shows that they form a continuous receptor-binding area spanning the serpin as well as the A-chain and the serine protease domain of uPA. Our results suggest that the 10-100-fold higher affinity of the uPA-PAI-1 complex compared with the free components depends on the bonus effect of bringing the binding areas on uPA and PAI-1 together on the same binding entity.

Several transmembrane molecules are cleaved at juxtamembrane extracellular sites leading to shedding of ectodomains. We analysed shedding of members of the Vps10p-D (Vps10p domain; where Vps is vacuolar protein sorting) family of neuronal type-I receptors with partially overlapping functions, and additional proteolytic events initiated by the shedding. When transfected into CHO (Chinese-hamster ovary) cells (CHO-K1), sorCS1a-sorCS1c isoforms were shed at high rates (approximately 0.61% x min(-1)) that were increased approx. 3-fold upon stimulation with phorbol ester. sorCS1c identified in the cultured neuroblastoma cell line SH-SY5Y was shed similarly. In CHO-K1 transfectants, constitutive and stimulated shedding of sorCS3 also occurred at high rates (0.29% and 1.03% x min(-1)). By comparison, constitutive and stimulated shedding of sorLA occurred at somewhat lower rates (0.07% and 0.48% x min(-1)), whereas sorCS2 and sortilin were shed at very low rates even when stimulated (approximately 0.01% x min(-1)). Except for sorCS2, shedding of the receptors was dramatically reduced in mutant CHO cells (CHO-M2) devoid of active TACE (tumour necrosis factor alpha-converting enzyme), demonstrating that this enzyme accounts for most sheddase activity. The release of sorCS1 and sorLA ectodomains initiated rapid cleavage of the membrane-tethered C-terminal stubs that accumulated only in the presence of gamma-secretase inhibitors. Purified shed sorLA bound several ligands similarly to the entire luminal domain of the receptor, including PDGF-BB (platelet-derived growth factor-BB) and amyloid-beta precursor protein. In addition, PDGF-BB also bound to the luminal domains of sorCS1 and sorCS3. The results suggest that ectodomains shed from a subset of Vps10p-D receptors can function as carrier proteins.

NAC, a 35-residue peptide derived from the neuronal protein alpha-synuclein/NAC precursor, is tightly associated with Abeta fibrils in Alzheimer's disease amyloid, and alpha-synuclein has recently been shown to bind Abeta in vitro. We have studied the interaction between Abeta and synucleins, aiming at determining segments in alpha-synuclein that can account for the binding, as well as identifying a possible interaction between Abeta and the beta-type synuclein. We report that Abeta binds to native and recombinant alpha-synuclein, and to beta-synuclein in an SDS-sensitive interaction (IC50 approx. 20 microM), as determined by chemical cross-linking and solid-phase binding assays. alpha-Synuclein and beta-synuclein were found to stimulate Abeta-aggregation in vitro to the same extent. The synucleins also displayed Abeta-inhibitable binding of NAC and they were capable of forming dimers. Using proteolytic fragmentation of alpha-synuclein and cross-linking to 125I-Abeta, we identified two consecutive binding domains (residues 1-56 and 57-97) by Edman degradation and mass spectrometric analysis, and a synthetic peptide comprising residues 32-57 possessed Abeta-binding activity. To test further the possible significance in pathology, alpha-synuclein was biotinylated and shown to bind specifically to amyloid plaques in a brain with Alzheimer's disease. It is proposed that the multiple Abeta-binding sites in alpha-synuclein are involved in the development of amyloid plaques.

It has been reported that alpha 2-macroglobulin (alpha 2 M), known as a plasma protease inhibitor, promotes neurite outgrowth of cultured neurons from rat cerebral cortex. The neurons dissociated from 17-day embryonic (E17) rat cerebral cortex were cultured in the medium containing methylamine-modified alpha 2M labeled with fluorescein isothiocyanate (MA- alpha 2 M-FITC). The modified alpha 2 M was taken up by neurons and localized mainly at the base of neurites. The fluorescence intensity of internalized MA- alpha 2 M-FITC in the cell was correlated to the degree of neurites extension. An immunocytochemical study using anti-alpha 2 M receptor (alpha 2 MR) polyclonal antibody showed that the neurons cultured for a longer period were stained weaker than those cultured for a shorter period. Many alpha 2 MRs were expressed when the neurons were likely to be extending the neurites well. From the developmental study of rat cerebrum, the expression of alpha 2 MR reached a maximum at postnatal 2 days (P2) when the neurons started to exhibit active neurite extension. These observations led us to conclude that neurite promoting effect of alpha 2 M acts through alpha 2 M-alpha 2MR binding and/or the following uptake of alpha 2 M.

Next Section Abstract We have used a combination of biochemical and immunological methods to probe for proteins that interact with the cytoplasmic form of plasminogen activator inhibitor 2 (PAI-2) and to identify the structure in PAI-2 that mediates the binding. By affinity chromatography on immobilized PAI-2, we purified a collection of PAI-2-binding proteins. These proteins bound 125I-labeled PAI-2 in vitro (IC50, ∼10-100 nM) in a calcium-independent reaction that did not abrogate the proteinase inhibitory function of PAI-2. Annexin I was identified among the eluted proteins, and purified annexins I, II, IV, and V, but not III and VI, possessed 125I-labeled PAI-2 binding activity. Immune precipitation by anti-PAI-2 monoclonal and polyclonal antibodies of metabolically labeled melanoma cells treated with a cleavable cross-linker prior to analysis revealed three prominent proteins with apparent masses of 100, 70, and 50 kDa. We localized the protein binding domain in PAI-2 between amino acid residues 66 and 98, as determined by using a PAI-2 mutant lacking this domain and a synthetic peptide spanning this region. This region of PAI-2 corresponds to exon 3 of the gene sequence thought to be critical for PAI-2 functions.

The subcellular localization of the alpha 2-macroglobulin receptor, also known as the low density lipoprotein receptor-related protein (LRP), was studied in postmortem human brain tissue by light and electron microscopic immunocytochemistry. A specific monoclonal antibody (A2MR2) against the extracellular alpha-chain of the molecule was utilized. Light microscopically, LRP was detected strongly in neurons, weakly in some glial cells and discontinuously along capillary membranes. At the electron microscopic level, positive reaction products were found to be associated with plasma membranes, ribosomes, lysosomes and lipofuscin granules of neurons, glial cells and pericytes. The results suggest that LRP may have a function, particularly in neurons, of receptor-mediated endocytosis with subsequent lysosomal uptake and degradation of ligands such as alpha 2-macroglobulin proteinase complexes and apolipoprotein E.

The GPI-anchored urokinase plasminogen activator receptor (uPAR) does not internalize free urokinase (uPA). On the contrary, uPAR-bound complexes of uPA with its serpin inhibitors PAI-1 (plasminogen activator inhibitor type-1) or PN-1 (protease nexin-1) are readily internalized in several cell types. Here we address the question whether uPAR is internalized as well upon binding of uPA-serpin complexes. Both LB6 clone 19 cells, a mouse cell line transfected with the human uPAR cDNA, and the human U937 monocytic cell line, express in addition to uPAR also the endocytic alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP/alpha 2-MR) which is required to internalize uPAR-bound uPA-PAI-1 and uPA-PN-1 complexes. Downregulation of cell surface uPAR molecules in U937 cells was detected by cytofluorimetric analysis after uPA-PAI-1 and uPA-PN-1 incubation for 30 min at 37 degrees C; this effect was blocked by preincubation with the ligand of LRP/alpha 2-MR, RAP (LRP/alpha 2-MR-associated protein), known to block the binding of the uPA complexes to LRP/alpha 2-. MR. Downregulation correlated in time with the intracellular appearance of uPAR as assessed by confocal microscopy and immuno-electron microscopy. After 30 min incubation with uPA-PAI-1 or uPA-PN-1 (but not with free uPA), confocal microscopy showed that uPAR staining in permeabilized LB6 clone 19 cells moved from a mostly surface associated to a largely perinuclear position. This effect was inhibited by the LRP/alpha 2-MR RAP. Perinuclear uPAR did not represent newly synthesized nor a preexisting intracellular pool of uPAR, since this fluorescence pattern was not modified by treatment with the protein synthesis inhibitor cycloheximide, and since in LB6 clone 19 cells all of uPAR was expressed on the cell surface. Immuno-electron microscopy confirmed the plasma membrane to intracellular translocation of uPAR, and its dependence on LRP/alpha 2-MR in LB6 clone 19 cells only after binding to the uPA-PAI-1 complex. After 30 min incubation at 37 degrees C with uPA-PAI-1, 93% of the specific immunogold particles were present in cytoplasmic vacuoles vs 17.6% in the case of DFP-uPA. We conclude therefore that in the process of uPA-serpin internalization, uPAR itself is internalized, and that internalization requires the LRP/alpha 2-MR.

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.

A region containing sites for ligand binding was localized in the 4525-amino acid residue alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) by ligand- and immunoblotting of proteinase and CNBr digests of the purified human placental protein. 125I-Labeled rat alpha 1-macroglobulin light chain, urokinase-plasminogen activator inhibitor type-1 complex, and alpha 2MR-associated protein all bound to a 75-kDa CNBr-generated fragment (68 kDa after deglycosylation). In addition to the three ligands, the fragment bound a novel monoclonal antibody reacting in the region defined by amino acid residues 1165-1246 as determined by binding to recombinant fragments of alpha 2MR/LRP. The positions of methionine residues in alpha 2MR/LRP suggested that the ligand-binding CNBr fragment contained three disulfide-linked peptides comprising the residues 776-1399. This origin was confirmed by partial amino acid sequencing of the electroblotted fragment and polypeptides generated by reduction of the fragment. The identified region represents 13.6% of the molecular mass (nonglycosylated) of alpha 2MR/LRP and contains one of three large clusters of complement-type repeats.

Localization of the alpha 2 macroglobulin receptor (alpha 2MR) was studied in postmortem human brain tissue of Alzheimer disease (AD) and age-matched control cases with a monoclonal antibody (A2MR alpha 2) to the receptor. In control cases alpha 2MR was detected in neurons, glia, and some capillaries. Neuronal staining was most conspicuous in the hippocampus and entorhinal cortex. In AD, alpha 2MR immunoreactivity was enhanced. The staining intensity of some neurons was increased, as was the number of positive glial cells. In addition, senile plaques, tangles, and dystrophic neurites were strongly stained. These results suggest that alpha 2MR is involved in AD pathology.

gp330, a large glycoprotein located in renal proximal tubules, has sequence similarities with the low-density lipoprotein receptor and the alpha 2-macroglobulin receptor/low-density lipoprotein receptor-related protein. The 40 KD human alpha 2-macroglobulin receptor-associated protein is a newly discovered heparin binding protein homologous to a major rat Heymann nephritis factor and exhibiting high affinity binding to the alpha 2-macroglobulin receptor. The present study shows by ligand blotting, light and electron microscopic autoradiography, and cytochemistry that gp330 located in coated apical membrane regions of the rat proximal tubule strongly binds the 40 KD protein. Furthermore, 45Ca2+ blotting experiments disclosed gp330 as a quantitatively important Ca2+ binding protein in renal cortex. Binding of 125I-labeled 40 KD protein to electroblotted gp330 and to coated apical membrane regions in sections of renal proximal tubules was abolished by excess unlabeled 40 KD protein, heparin, and EDTA. The endocytic properties of gp330 were investigated by in vivo microperfusion of rat proximal tubules. After 6 min, 125I-labeled 40 KD protein was mainly found in endocytic vacuoles and later accumulated in lysosomes. These data demonstrate that gp330 is a Ca2+ binding receptor for endocytosis of protein and is functionally related to the alpha 2-macroglobulin receptor/low-density lipoprotein receptor-related protein. Furthermore, our results demonstrate the usefulness of semi-thin and ultra-thin cryosections in studies of ligand binding and subcellular localization of receptors with autoradiographic techniques.

The hepatic alpha 2-macroglobulin receptor (alpha 2MR)/low density lipoprotein receptor-related protein (LRP) binds and endocytoses alpha 2-macroglobulin-proteinase complexes in plasma. In addition, it binds lipoproteins, a novel 40 kDa protein, and complexes between plasminogen activators and plasminogen activator inhibitor type-1. This study shows, for the first time, the tissue distribution of alpha 2MR/LRP as determined by immunohistochemistry with specific monoclonal antibodies. The analysis revealed alpha 2MR/LRP-expression in a restricted spectrum of cell types, including neurons and astrocytes in the central nervous system, epithelial cells of the gastrointestinal tract, smooth muscle cells, fibroblasts, Leydig cells in testis, granulosa cells in ovary, and dendritic interstitial cells of kidney. Monocyte-derived cells displayed marked alpha 2MR/LRP expression in the phagocytes of liver, lung and lymphoid tissues, but no or low expression in antigen-presenting cells including Langerhans' cells of the skin. The high abundance of alpha 2MR/LRP in certain cell types of most organs suggests two main routes for alpha 2MR/LRP-mediated ligand clearance: (1) systemic removal in liver of circulating ligands, and (2) non-hepatic interstitial removal in different organs, including the brain.

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 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.

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.

A Vps10p domain makes up the entire luminal part of Sortilin, and this type of domain is the hallmark of a new family of neuronal receptors that target a variety of ligands, including neurotrophins and neuropeptides. We have shown that two structural features of the Vps10p domain, the N-terminal propeptide and the C-terminal segment of ten conserved cysteines (10CC), are key elements in the function of Sortilin. The propeptide has two functions. (i) It binds the mature part of Sortilin and prevents ligands in the biosynthetic pathway from binding to the uncleaved proreceptor, and (ii) it facilitates receptor transport in early Golgi compartments by a mechanism that does not depend on its ability to prevent ligand binding. In contrast, other Vps10p domain receptors, such as SorLA and SorCS3, do not need their propeptide for normal and swift processing. The 10CC segment constitutes an exchangeable module containing five conserved disulfide bridges, and using module-shuffling and truncations, we have shown that the 10CC segment is a major ligand-binding region in Sortilin.

We previously isolated and sequenced murine sorCS1, a type 1 receptor containing a Vps10p-domain and a leucine-rich domain. We now show that human sorCS1 has three isoforms, sorCS1a–c, with completely different cytoplasmic tails and differential expression in tissues. The b tail shows high identity with that of murine sorCS1b, whereas the a and c tails have no reported counterparts. Like the Vps10p-domain receptor family members sortilin and sorLA, sorCS1 is synthesized as a proreceptor that is converted in late Golgi compartments by furin-mediated cleavage. Mature sorCS1 bound its own propeptide with low affinity but none of the ligands previously shown to interact with sortilin and sorLA. In transfected cells, about 10% of sorCS1a was expressed on the cell surface and proved capable of rapid endocytosis in complex with specific antibody, whereas sorCS1b presented a high cell surface expression but essentially no endocytosis, and sorCS1c was intermediate. This is an unusual example of an alternatively spliced single transmembrane receptor with completely different cytoplasmic domains that mediate different trafficking in cells.

The type-I ribosome-inactivating protein trichosanthin displays selective cytotoxicity, suggesting specific mechanisms for entry into cells. Here we show that trichosanthin binds specifically to the endocytic receptors LRP and megalin, and that binding as well as uptake into cells is inhibited by the receptor-associated protein (RAP). The results suggest that the known abortifacient and renotoxic actions of trichosanthin are caused by LRP-mediated uptake in trophoblasts and megalin-mediated uptake in proximal tubule epithelial cells, respectively.

Sortilin belongs to a growing family of multiligand type-1 receptors with homology to the yeast receptor Vps10p. Based on structural features and sortilin's intracellular predominance, we have proposed it to be a sorting receptor for ligands in the synthetic pathway as well as on the cell membrane. To test this hypothesis we examine here the cellular trafficking of chimeric receptors containing constructs of the sortilin tail. We report that sorting signals conforming to YXX and dileucine motifs mediate rapid endocytosis of sortilin chimeras, which subsequently travel to the trans-Golgi network, showing little or no recycling. Furthermore, we found that cation-independent mannose 6-phosphate receptor (MPR300)-sortilin chimeras, expressed in mannose 6-phosphate receptor knockout cells, were almost as efficient as MPR300 itself for transport of newly synthesized beta-hexosaminidase and beta-glucuronidase to lysosomes, and established that the sortilin tail contains potent signals for Golgi-endosome sorting. Finally, we provide evidence suggesting that sortilin is the first example of a mammalian receptor targeted by the recently described GGA family of cytosolic sorting proteins, which condition the Vps10p-mediated sorting of yeast carboxypeptidase Y.

alpha-Synuclein has been implicated in the pathogenesis of several neurodegenerative disorders based on the direct linking of missense mutations in alpha-synuclein to autosomal dominant Parkinson's disease and its presence in Lewy-like lesions. To gain insight into alpha-synuclein functions, we have investigated whether it binds neuronal proteins and modulates their functional state. The microtubule-associated protein tau was identified as a ligand by alpha-synuclein affinity chromatography of human brain cytosol. Direct binding assays using (125)I-labeled human tau40 demonstrated a reversible binding with a IC(50) about 50 pM. The interacting domains were localized to the C terminus of alpha-synuclein and the microtubule binding region of tau as determined by protein fragmentation and the use of recombinant peptides. High concentrations of tubulin inhibited the binding between tau and alpha-synuclein. Functionally, alpha-synuclein stimulated the protein kinase A-catalyzed phosphorylation of tau serine residues 262 and 356 as determined using a phospho-epitope-specific antibody. We propose that alpha-synuclein modulates the phosphorylation of soluble axonal tau and thereby indirectly affects the stability of axonal microtubules.

We recently reported the isolation and sequencing of sortilin, a new putative sorting receptor that binds receptor-associated protein (RAP). The luminal N-terminus of sortilin comprises a consensus sequence for cleavage by furin, R41WRR44, which precedes a truncation originally found in sortilin isolated from human brain. We now show that the truncation results from cellular processing. Sortilin is synthesized as a proform which, in late Golgi compartments, is converted to the mature receptor by furin-mediated cleavage of a 44 residue N-terminal propeptide. We further demonstrate that the propeptide exhibits pH-dependent high affinity binding to fully processed sortilin, that the binding is competed for by RAP and the newly discovered sortilin ligand neurotensin, and that prevention of propeptide cleavage essentially prevents binding of RAP and neurotensin. The findings evidence that the propeptide sterically hinders ligands from gaining access to overlapping binding sites in prosortilin, and that cleavage and release of the propeptide preconditions sortilin for full functional activity. Although proteolytic processing is involved in the maturation of several receptors, the described exposure of previously concealed ligand-binding sites after furin-mediated cleavage of propeptide represents a novel mechanism in receptor activation.

The complete amino acid sequence of human placental oxytocinase (placental leucine aminopeptidase) has been determined by cDNA cloning and sequencing. Oxytocinase is a type II integral membrane protein of 1025 amino acid residues, consisting of an acidic intracellular region of 110 amino acids followed by a hydrophobic transmembrane segment of 22 residues and 893 extracellular residues containing the characteristic Zn2+ coordination sequence element His-Glu-Xaa-Xaa-His-(18 residues)-Glu found in gluzincins. Two sets of cDNA clones with different 5′-ends were isolated and suggested to represent different spliced products of 3.6 kb (mature mRNA) and 12 kb, respectively. Oxytocinase mRNA is present in large amounts in placenta, heart and skeletal muscle and in small amounts in brain, kidney, liver and pancreas. A conserved sequence element, the GAMEN motif, which distinguishes the aminopeptidase family among gluzincins from other gluzincins, has been identified.

The GPI-anchored urokinase plasminogen activator receptor (uPAR) does not internalize free urokinase (uPA) but readily internalizes and degrades uPA:serpin complexes in a process that requires the alpha2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha2MR-LRP). This process is accompanied by the internalization of uPAR which renders it resistant to phosphatidylinositol-specific phospholipase C (PI-PLC). In this paper we show that during internalization of uPA:serpins at 37 degrees C, analysed by FACScan, immunofluorescence and immunoelectron microscopy, an initial decrease of cell surface uPAR was observed, followed by its reappearance at later times. This effect was not due to redistribution of previously intracellular receptors, nor to the surface expression of newly synthesized uPAR. Recycling was directly demonstrated in cell surface-biotinylated, uPA:PAI-1-exposed cells in which biotinylated uPAR was first internalized and subsequently recycled back to the surface upon incubation at 37 degrees C. In fact, uPAR was resistant to PI-PLC after the 4 degrees C binding of uPA:PAI-1 to biotinylated cells, but upon incubation at 37 degrees C PI-PLC-sensitive biotinylated uPAR reappeared at the cell surface. Binding of uPA:PAI-1 by uPAR, while essential to initiate the whole process, was, however, dispensable at later stages as both internalization and recycling of uPAR could be observed also after dissociation of the bound ligand from the cell surface.

The expression of tissue transglutaminase (tTG) was studied during the formation of the normal human placenta and in molar pregnancies and choriocarcinoma, in order to correlate its expression with the functional characteristics of the recognized trophoblast cell types. tTG expression was found to be developmentally regulated. Before 6-7 weeks' gestation, only the chorionic villous cytotrophoblast expresses tTG. Thereafter the overlying syncytiotrophoblast becomes positive. tTG expression is gradually downregulated in the intermediate trophoblast cells emerging from the tips of the chorionic villi invading the uterine tissue. In the decidual wall, the intermediate trophoblast does not express tTG, whereas scattered syncytial cells, the placental bed giant cells, express tTG. Villi from complete hydatidiform mole (CHM) show tTG expression in both the cyto- and the syncytiotrophoblast. The intermediate trophoblast cells from CHM show heterogeneous tTG expression, with a majority of negative cells, whereas extravillous syncytia always express tTG. In choriocarcinoma, the tumour cells show heterogeneous tTG expression, with a majority of positive cells. Analysis of tTG protein and mRNA in placental extracts by Western and Northern blotting did not provide evidence for expression of the truncated form of tTG found in some cell types. The regulated expression of tTG in the normal placenta suggests that the enzyme is involved in important trophoblastic functions and may participate in the control of invasion.

Udgivelsesdato: 28. feb. 1997 Lipoprotein lipase (LpL) can mediate cellular uptake of chylomicron and VLDL remnants via binding to heparan sulfate proteoglycans (HSPG) and the endocytic alpha2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha2MR/LRP). Whereas it is established that the C-terminal folding domain binds to alpha2MR/LRP, it remains uncertain whether it binds to heparin and to HSPG. To identify segments important for binding to alpha2MR/LRP and to clarify possible binding to heparin, we produced constructs of the human C-terminal folding domain, LpL-(313-448), and of the fragment LpL-(347-448) in Escherichia coli. In addition to binding to alpha2MR/LRP, LpL-(313-448) displayed binding to heparin with an affinity similar to that of the LpL monomer, whereas it bound poorly to lipoprotein particles. Moreover, LpL-(313-448) displayed heparin sensitive binding to normal, but not to HSPG deficient Chinese hamster ovary cells. LpL-(313-448) and LpL-(347-448) showed similar affinities for binding to both purified alpha2MR/LRP and to heparin. Deletion of LpL residues 380-384 abolished the binding to LRP, whereas binding to heparin was unperturbed. The binding to both heparin and alpha2MR/LRP was essentially abolished following deletion of residues 404-430, and pretreatment of CHO cells with the peptide comprising aa 402-423 inhibited the binding of LpL-(313-448). We conclude that the C-terminal folding domain of human LpL has a site for binding to heparin and to HSPG, presumably involving amino acids within residues 404-430. Two segments of the domain are necessary for efficient binding to alpha2MR/LRP, one comprising residues 380-384 and another overlapping the segment important for binding to heparin.

The widespread deposition of amyloid plaques is one of the hallmarks of Alzheimer disease (AD). A recently described component of amyloid plaques is the 35-residue peptide, non-Aβ component of AD amyloid, which is derived from a larger intracellular neuronal constituent, α-synuclein. We demonstrate that transglutaminase catalyses the formation of the covalent non-Aβ component of AD amyloid polymers in vitro as well as polymers with β-amyloid peptide, the major constituent of AD plaques. The transglutaminase-reactive amino acid residues in the non-Aβ component of AD amyloid were identified as Gln79 and Lys80. Lys80 is localized in a consensus motif Lys-Thr-Lys-Glu-Gly-Val, which is conserved in the synuclein gene family. Thus transglutaminase might be involved in the formation of insoluble amyloid deposits and participate in the modification of other members of the synuclein family.

The β-amyloid peptide (βA4), derived from a larger amyloid precursor protein, is the principal component of senile plaques in Alzheimer's disease. Here we report that the full-length (1-40) synthetic βA4 peptide, containing one glutamine and two lysine residues, is able to form homopolymers in a transglutaminase-mediated reaction. Moreover, transglutaminase catalysed the formation of heteropolymers in reactions of βA4 with α2M receptor, a constituent of amyloid plaques, and with extracellular matrix proteins. Incorporation of site-specific probes followed by enzymatic digestion and sequencing of tracer-containing fractions demonstrated that both Lys16, Lys28 and Gin15 in βA4 were susceptible to cross-linking by transglutaminase.

The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) binds several ligands, including complex between the two chain urokinase-type plasminogen activator (uPA) and type-1 plasminogen activator inhibitor (PAI-1), and the single chain zymogen pro-urokinase (pro-uPA). We have used truncated variants of uPA and PAI-1 as well as Fab fragments of monoclonal antibodies with known epitopes to identify regions in the uPA.PAI-1 complex and in pro-uPA involved in binding to alpha 2MR/LRP.uPA.PAI-1 complex bound with high affinity (EC50 about 0.4 nM) via contacts in the PAI-1 moiety as well as the uPA serine proteinase domain and the uPAA chain. Pro-uPA bound with lower affinity (EC50 about 10 nM), and efficient binding to alpha 2MR/LRP was dependent on contact with both the A chain and the serine proteinase domain. We analyzed the effect of complex formation with the urokinase receptor since this is the primary target for binding of uPA.PAI-1 and pro-uPA at the cell surface, and since it has been demonstrated that urokinase receptor-bound uPA.PAI-1 complex is internalized following interaction with alpha 2 MR/LRP (Nykjaer, A., Petersen, C. M., Moller, B., Jensen, P.H., Moestrup, S.K., Holtet, T.L., Etzerodt M., Thogersen, H.C., Munch, M., Andreasen, P.A., and Gliemann, J. (1992) J. Biol. Chem. 267, 14543-14546). Soluble recombinant urokinase receptor blocked the binding of pro-uPA to alpha 2MR/LRP but caused only a slight reduction in the affinity for binding of uPA.PAI-1. Moreover, pro-uPA bound to the urokinase receptor at the cell surface was not internalized and degraded unless activated to uPA and complexed with PAI-1. We conclude that pro-uPA is protected against degradation via alpha 2MR/LRP when bound to uPAR due to shielding of a binding contact in the A chain, whereas the affinity of uPAR-bound uPA.PAI-1 complex for binding to alpha 2MR/LRP remains sufficient to allow rapid internalization and degradation.

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.

The alpha 2-macroglobulin receptor was recently purified from rat liver and human placenta. Three different monoclonal antibodies have now been raised against the human receptor and expression of the 440-kDa receptor protein is demonstrated in human placenta, fibroblasts, liver, and monocytes by immunoblot analysis. Flow cytometric studies showed that anti-alpha 2-macroglobulin receptor monoclonal antibodies bind to 90-100% of the blood monocyte population and not to other blood cells. This defines the alpha 2-macroglobulin receptor as a monocyte differentiation antigen, different from any of the classified leucocyte cluster determinants. Electron microscopic gold immunocytochemistry revealed the subcellular distribution of the receptor in human cultured monocytes and fibroblasts. In these cells, 18-33% of the gold particles were found on the outside of the plasma membrane, and in fibroblasts, especially, in coated invaginations. The intracellular receptors were mainly distributed in vesicles and tubular structures.

The receptor for alpha 2-macroglobulin-proteinase complexes (alpha 2MR) was purified recently, and its binding of ligand was shown to depend on calcium ions (Moestrup, S. K., and Gliemann, J. (1989) J. Biol. Chem. 264, 15574-15577). This paper shows that the 440-kDa human placental alpha 2MR is a cysteine-rich glycoprotein with high affinity calcium binding sites important for receptor conformation; and the relationship between Ca2+ concentration and receptor function is presented. Autoradiography showed 45Ca2+ binding to the 440-kDa alpha 2MR blotted onto nitrocellulose from a sodium dodecyl sulfate-polyacrylamide gel. alpha 2MR immobilized on nitrocellulose in the absence of sodium dodecyl sulfate bound 45Ca2+ in the presence of 5 mM Mg2+, and 2-3 microM unlabeled Ca2+ was required to displace half of the bound 45Ca2+. The calcium concentration dependence showed upward concave Scatchard plots, and the number of binding sites was estimated to be approximately eight/alpha 2MR molecule. Binding of calcium did not change in the pH range 6.5-8.0 but decreased at lower pH values. Addition of Ca2+ to the medium was necessary for receptor binding of the alpha 2-macroglobulin-trypsin complex, and half of the maximal binding capacity was obtained with about 16 micrograms Ca2+ at pH 7.8. The requirement for calcium was increased at lower pH values, and half of the maximal 125I-alpha 2M-trypsin binding was obtained with about 30-40 microM Ca2+ at pH 7.0. Monoclonal antibodies were produced against alpha 2MR, and one of them distinguished between the Ca2(+)-occupied and nonoccupied forms. Like Ca2+, Sr2+ and Ba2+ elicited ligand binding affinity and competed for binding with 45Ca2+ in the order Ca2+ greater than Sr2+ greater than Ba2+. In conclusion, calcium ions bind specifically to alpha 2MR with high affinity, and it is likely that several sites on the alpha 2MR molecule have to be occupied to elicit the conformation recognizing the ligand.

Udgivelsesdato: 1990-May-22 Receptors for the 54 kDa plasminogen activator urokinase were characterized in freshly isolated and 5-14 day cultured human monocytes. The half saturation constant was about 55 pM in freshly isolated monocytes at 4 degrees C and 140 pM at 37 degrees C. Diisopropylfluorophosphate-inactivated urokinase was bound with the same affinity as catalytically active urokinase. Binding per cell of 2-5 pM urokinase increased progressively during cell culture with a concomitant decrease in the apparent affinity. By 14 days, binding had increased 5-7-fold and the half-saturation constant had increased to 500 pM at 4 degrees C, indicating a large increase in the binding capacity. Affinity cross-linking of labelled urokinase to receptors showed a 110 kDa complex in both freshly isolated and cultured monocytes. When cells with labelled urokinase (prebound at 4 degrees C) were incubated at 37 degrees C, about 80% of the urokinase dissociated as the intact molecule, whereas about 20% was degraded to iodide and iodotyrosine. Electron microscopic autoradiography of cultured monocytes incubated at 4 degrees C showed a marked heterogeneity between cells with regard to bound urokinase. Autoradiographic grains were mainly seen over the plasma membrane in areas rich in microvilli and invaginations. Transfer of the cells to 37 degrees C caused no major alteration in the distribution of grains. Thus, freshly prepared monocytes have urokinase receptors (approx. 55 kDa) of high affinity. Development to macrophage-like cells in culture causes a decrease in affinity and a large increase in capacity. The receptors are confined mainly to certain areas of the plasma membrane. Internalization and degradation of the ligand occurs only to a minor extent.

Rat adipocytes were incubated with 15 nM insulin in different buffers at 37°C. The cells were washed and reincubated at 16°C in the presence of 18 pM A14-[125I]monoiodoinsulin to determine the insulin receptor concentration. After incubation for 2 h in Tris buffer the binding decreased to about 30 %, whereas no decrease was found after incubation in Hepes, phosphate or bicarbonate buffers. Binding of tracer insulin reached a constant level by 45 min in Hepes buffer at 37°C, whereas it continued to increase in Tris buffer. Washout of tracer insulin after incubation in Tris buffer at 37°C showed a large, slowly dissociable fraction. It is suggested that the rapid down regulation of insulin receptors in vitro is an artifact of the Tris buffer and that the phenomenon is due to a slowly reversible occupancy of a receptor pool with unlabelled insulin.

The pH dependence of binding of [TyrA14-125I]monoiodoinsulin to isolated rat adipocytes was investigated at 37 degrees C. Although the amount of tracer bound at steady state at pH 7.8 was 6 times that at pH 6.8, the fractional degradation of receptor-bound iodoinsulin remained the same. The decreased binding at acidic pH could be accounted for by a decrease in the affinity. The pH-induced change in affinity was similar for iodoinsulin and insulin. The half-time of dissociation was not changed in the pH range 7.0 to 7.8. The presence of 25 nM insulin in the wash out medium caused a small increase in the dissociation of tracer independent of pH. Kinetic experiments revealed that the decreased affinity at acidic pH was mainly due to a decrease in the association rate constant. The decreased binding at acidic pH was reflected in a shift to the right of the insulin dose-response curve for initial rate of uptake of 3-O-[methyl-14C]glucose and the rate of conversion of glucose to lipids. It is concluded that the degradation of bound iodoinsulin is independent of pH (6.8 to 8.8), that the decreased binding at acidic pH is due to a decreased association rate constant, and that cells in acidic milieu are relatively insulin-resistant.

Cultured human lymphocytes of the IM-9 line were incubated with ( 125 I)monoiodoinsulin for 3 to 150 min at 37 °C and the location of the bound insulin was determined by quantitative electron microscope autoradiography. The radioactivity was primarily associated with the plasmamembrane although a few cells showed a limited endocytic uptake. The intracellular radioactivity was not more than 3–4 per cent of the total cell-associated radioactivity at any time and no preferential association to the nucleus could be detected.

The distribution spaces at equilibrium for 3H2O, [14C]urea and s-O-[14C]-methylglucose were measured in white fat cells using centrifugation through silicone oil at 2500 X g; no significant differences were observed. L-[14C]Glucose added immediately before the centrifugation was used as a marker for the extracellular water space. The calculated intracellular water content of the cells after the centrifugation through oil (e.g. 3H2O space minus L-[14C]glucose space) is an unbiased measure of the water content of the fat cells in suspension as judged by the following criteria: (1) The intracellular distribution space for 3-O-[14C]'methylglucose at equilibrium (methylglucose space minus L-glucose space) was not different from that calculated from a methylglucose wash-out curve. (2) The intracellular content of L-[14C]glucose (half time of efflux about 60 min) in cells preloaded during incubation of the tissue with collagenase was not different in cells recovered by (a) centrifugation through oil at 2500 X g, (b) centrifugation through oil at 600 X g, (c) centrifugation at 600 X g in the absence of oil and (d) filtration on Millipore filters. The intracellular content of water determined on cells from single rats weighing 120-150 g was 2.75 +/- 0.55 mul/100 mul fat cells (+/- S.D., n = 30). The intracellular content of potassium, determined on cells from the same rats, was 252 +/- 62 nmols/100 mul fat cells (+/- S.D., n = 30). The concentration of potassium in the intracellular water was calculated as 104 +/- 15 mM (+/- S.D., n = 30).

The cell association and degradation of insulin and alpha 2-macroglobulin-trypsin complex were measured in rat adipocytes with or without various inhibitors in the attempt to clarify whether the two ligands were taken up by the same or by different pathways. Several inhibitors, and particularly those of membrane traffic, lysosomal function and transglutaminase activity, affected the two ligands differently. Thus, chloroquine (100 microM) reduced both the uptake of alpha 2-macroglobulin X trypsin and its receptor-mediated degradation by about 70%. In contrast, the uptake of insulin was increased 2-3-times and the receptor-mediated degradation was only slightly reduced. Methylamine (10 mM) and ammonium chloride (10 mM) reduced degradation of alpha 2-macroglobulin X trypsin markedly without affecting that of insulin. Leupeptin (100 microM) increased uptake and reduced degradation of alpha 2-macroglobulin X trypsin without affecting insulin. Dansylcadaverine (500 microM) almost abolished uptake and degradation of alpha 2-macroglobulin X trypsin but had little effect on insulin. Moreover, uptake and degradation of alpha 2-macroglobulin X trypsin was much more sensitive than insulin to the action of metabolic inhibitors such as dinitrophenol and cyanide. The results show that the two ligands are taken up by functionally different systems. In addition, they support the hypothesis that lysosomes play a relatively minor role in the receptor-mediated degradation of insulin.

2-Deoxyglucose uptake (3 min) and 3-O- methylglucose transport (2 s) was measured in rat adipocytes preincubated with 5 μM epinephrine plus adenosine deaminase as described by Green (Green, A. (1983) FEBS Lett. 152, 261–264). 2-Deoxyglucose uptake was about 95% depressed in insulin-treated, but not in ‘basal’, cells preincubated with epinephrine plus adenosine deaminase for 60 min in broad agreement with Green's report. However, this depression was caused by a decrease in sugar phosphorylation rather than transport. In similarly incubated cells, transport of 3-O- methylglucose , a sugar analogue not phosphorylated in the adipocytes, was not affected by catecholamine plus adenosine deaminase. However, a decrease in transport of about 60% was observed both in the absence and the presence of insulin when the albumin concentration was high enough and the cell concentration low enough to prevent accumulation of free fatty acids in the medium. In addition, the insulin sensitivity with regard to hexose transport was markedly reduced. Transport was approximately doubled in cells incubated with 5 μM epinephrine in the absence of adenosine deaminase. Thus, epineprine at a high concentration stimulates hexose transport in the absence of adenosine deaminase (presence of adenosine) whereas it inhibits both basal and insulin-stimulated transport in the presence of adenosine deaminase (absence of adenosine).

1. The uptake and the washout of 45Ca2+ and 32Pi is described in free fat-cells and whole epididymal fat-pads from fed rats. 2. In isolated fat-cells, the uptake of 45Ca2+ proceeds with an initial rapid phase of about 1 min duration, followed by a slower subsequent accumulation. In contrast with the rapid phase, the slow phase is inhibited by 2,4-dinitrophenol, warfarin, oligomycin and verapamil, shows saturation, and presumably represents transport across the plasma membrane. 3. The washout of 45Ca2+ from preloaded cells consists of a rapid (1 min) initial phase and a slow phase which is non-monoexponential, suggesting that the radioactive isotope is released from several cellular pools. 4. When Pi is omitted from the incubation medium, the slow phase of 45Ca uptake is almost abolished, and the washout of 45Ca from preloaded fat-cells is markedly accelerated. At elevated extracellular concentrations of Pi (2,4-6.2mM), the uptake of 45Ca is stimulated by 2-10-fold, and the release of the radioactive isotope from preloaded cells is inhibited. In whole epididymal fat-pads, variations in the extracellular concentration of Pi have no detectable effect on the uptake or the washout of 45Ca. 5. In isolated fat-cells, the accumulation of 32Pi is inhibited by 2,4-dinitrophenol or the omission of glucose from the incubation medium. In a Ca2+-depleted buffer, the uptake of 32Pi is diminished, and hyperosmolarity, which stimulates 45Ca uptake, also accelerates the accumulation of 32Pi. 6. It is concluded that in free fat-cells, the uptake and release of Ca2+ and Pi take place by closely interrelated processes, which are dependent on mitochondrial energy production.

The transport of trace (a concentration much less than Km for transport) 2-deoxyglucose (deoxyglucose) and 3-O-methylglucose (methylglucose) into rat adipocytes was measured using 1- to 2-s pulses at 37 degrees C in the absence of glucose and in the presence of glucose with and without pre-exposure to glucose. Pre-exposure of insulin-stimulated adipocytes to 10 mM glucose for 30 min resulted in an acceleration of trace deoxyglucose transport above that seen in the absence of glucose. In contrast, when glucose was added together with deoxyglucose, an inhibition of transport was observed. There was no effect up to 5-min pre-exposure to 10 mM glucose, but acceleration appeared thereafter and reached a steady state by 30 min, i.e. a 3-fold higher transport rate than that seen when deoxyglucose and glucose were added together. Glucose (3.5 mM) appears to be a threshold concentration for this effect. A smaller effect was seen in the absence of insulin (1.5-fold) using 10 mM glucose and in the presence of insulin using 10 mM mannose (2.7-fold). No effect was seen using 10 mM galactose, fructose, pyruvate-lactate, deoxyglucose, and methylglucose. There was no effect of glucose pre-exposure on insulin-stimulated methylglucose transport. We propose that entry occurs across two independent barriers in series separated by an aqueous pore, that transport across the second barrier is more rate limiting for deoxyglucose than for methylglucose, and than metabolites of glucose decrease the resistance of the second barrier to deoxyglucose transport.

alpha 2-Macroglobulin-trypsin complex (alpha 2M.T) and alpha 2M-methylamine bind in a Ca2+-dependent way to a 400- to 500-kDa receptor in rat and human liver membranes (Gliemann, J., Davidsen, O., and Moestrup, S. K. (1989) Biochim. Biophys. Acta 980, 326-332). Here we report the preparation of alpha 2M receptors from rat liver membranes solubilized in 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonic acid (CHAPS) dihydrate and incubated with Sepharose-immobilized alpha 2M-methylamine. The receptor preparation eluted with EDTA (pH 6.0) contained a protein larger than the 360-kDa alpha 2M (nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and some minor contaminants. The reduced large protein was about 440 kDa using reduced laminin (heavy chain: 400 kDa) as a standard. About 10 micrograms of receptor protein was obtained from 100 mg of liver membranes. The receptor preparation immobilized on nitrocellulose sheets bound 125I-alpha 2M.T, and the binding activity co-eluted with the 440-kDa protein. 125I-Labeled rat alpha 1-inhibitor-3 (alpha 1I3), a 200-kDa analogue of the alpha 2M subunit which binds to the alpha 2M receptors, was cross-linked to the 440-kDa protein. The receptor preparation was iodinated, and the 125I-labeled 440-kDa protein was isolated. It showed Ca2+-dependent saturable binding to alpha 2M-methylamine. In conclusion, we have purified the major hepatic alpha 2M receptor as an approximately 440-kDa single chain protein.

Hepatocytes were isolated by application of the two-step collagenase technique to pieces of human liver. 125I-labelled alpha 2-macroglobulin-trypsin complex bound to hepatocytes at 4 degrees C with a half time of approximately 4.5 h. At near equilibrium half of the receptors were saturated at an alpha 2-macroglobulin-trypsin complex concentration of about 60 pmol 1(-1) and the Scatchard plot was linear. Dissociation of the labelled complex was slow (T1/2 = 24 h) at low receptor occupancies. At high receptor occupancies dissociation was biphasic with a rate constant (K-1) for the initial rapid phase of about 2.4 x 10(-2) min-1. Labelled alpha 2-macroglobulin-trypsin complex bound at 4 degrees C was rapidly internalized at 37 degrees C (T1/2 = 1.9 min), and in 3.5 h approximately 10% of the label was released into the medium in a trichloroacetic acid-soluble form. At 37 degrees C, 125I alpha 2-macroglobulin-trypsin was taken up by hepatocytes and trichloroacetic acid soluble radioactivity appeared in the medium following a sigmoidal curve. Similar results were obtained with 125I-pregnancy zone protein-chymotrypsin complex. At 4 degrees C, hepatocytes bound nearly equal amounts of labelled alpha 2-macroglobulin-trypsin and pregnancy zone protein-chymotrypsin complex, and a large excess (100 nmol 1(-1) of one of the macroglobulins could almost completely abolish binding of trace amounts (5-20 pmol 1(-1] of the other. The present findings strongly suggest that the hepatocyte is of major importance for removal of alpha 2-macroglobulin- and pregnancy zone protein-proteinase complex in humans, in agreement with previous results in rats and mice.

Rats were given intravenous injections of 125I-labelled human alpha 2-macroglobulin X trypsin. The half-time of disappearance of radioactivity from arterial blood was 2 min. External counting showed that radioactivity in the liver was maximal by 10 min and then decreased slowly. 87% of the injected dose was recovered in the liver by 10 min. Light- and electron microscopic autoradiography carried out on samples of liver fixed with glutaraldehyde 3 min or 30 min after the injection showed that 85-90% of the grains were over the hepatocytes and 4-9% were over the Kupffer cells. Thus, uptake into hepatocytes, and not into Kupffer cells as believed previously, appears to account for the major part of the uptake of alpha 2-macroglobulin X trypsin by the liver and thereby for its rapid removal from the blood.

• The binding of glucagon and its effect on adenosine 3′,5′-monophosphate (cyclic AMP) accumulation and glycerol release was assessed in isolated rat fat cells at 37°C. • The dissociation constant of the displaceable (saturable, specific) binding was about 1.5 nM. The rate constant of dissociation ( k −1 ) varied from 8 8.6 · 10 −4 s −1 to 3.3 · 10 −3 s −1 , i.e. T 1 2 varied from 800 s to 200 s. Within an experiment, k −1 was the same when cells with prebound 125 I-labelled glucagon were suspended in medium containing either no glucagon or 1 μM of unlabelled glucagon. Negative homotropic cooperativity therefore seemed absent. The half-time of association of 70 pM 125 I-labelled glucagon was of the same order of magnitude as that of dissociation. • The conversion of prelabelled ATP to labelled cyclic AMP was enhanced by glucagon with a time lag of less than 20 s. The maximal accumulation was reached at 2–5 min both in the absence and in the presence of 2.5 mM theophylline. The concentration of glucagon causing half-maximal accumulation of cyclic AMP at 5 min was about 2 nM both in the absence and in the presence of theophylline. Glucagon stimulated glycerol release half-maximally at a concentration of about 2 nM in 1 h incubations. • The des-His 1 -glucagon exhibited about half of the maximal effect of glucagon on cyclic AMP accumulation even though it was able to inhibit binding of 125 I-labelled glucagon to the same extent as glucagon. Half-maximal displacement and half-maximal effect were obtained with about 200 nM of des-His 1 -glucagon. • The results are compatible with the following model for the action of glucagon on adipocytes. The adenylyl cyclase is stimulated in a dose-dependent fashion at the low receptor occupancies obtained 20 s after the addition of glucagon in submaximally stimulating concentrations. The increase in cyclic AMP concentration is antagonized so that the maximal accumulation with a given concentration of glucagon is obtained after a few minutes even though the receptor occupancy continues to increase. The histidine residue is necessary for maximal activation of the adenylyl cyclase.

Isolated rat adipocytes were incubated with 15 nM [3-3H]glucose or 100 nM [U-14C]glucose with or without insulin and in the absence or presence of unlabelled glucose. Following a 2 h incubation with 15 nM [3-3H]glucose, about two thirds of the cell-associated 3H-labelled metabolic products were hydrophilic largely anionic intermediates and about one third was lipids. The equivalent values were 40 and 60%, respectively, when using 100 nM [U-14C]glucose. The only 14C-labelled metabolite escaping to the incubation medium was 14CO2, which accounted for about 15% of the rate of metabolism. Therefore, the rate of incorporation of 100 nM [U-14C]glucose into the cell-associated metabolites was quite a good measure of its net influx rate. The conversion of the two tracers to the sum of the metabolic products in cells treated with a maximally stimulating insulin concentration remained constant with glucose concentrations up to about 100 microM and then decreased progressively. The incorporation of radioactivity into the different metabolites varied markedly over the glucose concentration range 0-100 microM, presumably due to the saturation of different metabolic pools at different glucose concentrations. This variation was much less in cells not stimulated with insulin. Consequently, the maximal effect of insulin on the incorporation of the tracers into a given metabolite (e.g., labelled lipids) varied over the entire glucose concentration range. In addition, the apparent sensitivity (ED50) with respect to the incorporation into a given metabolite was also dependent on the glucose concentration.

125I-Labelled alpha 2-macroglobulin-trypsin complex (125I-labelled alpha 2-macroglobulin X trypsin) was associated to isolated rat adipocytes and hepatocytes with a half-time of about 60 min at 37 degrees C. The association of 0.5 micrograms/ml 125I-labelled alpha 2-macroglobulin X trypsin was inhibited by unlabelled alpha 2-macroglobulin X trypsin with a half-inhibition constant of about 8 micrograms/ml (11 nM). 125I-Labelled alpha 2-macroglobulin became cell-associated to a smaller extent (10-40% of that of alpha 2-macroglobulin X trypsin) and the half-inhibition constant was about 35 micrograms/ml in adipocytes. The cell association of 125I-labelled alpha 2-macroglobulin X trypsin was markedly inhibited by dansylcadaverine, bacitracin, omission of Ca2+ from the medium or pretreatment of the cells with trypsin. After incubation for 180 min more than 60% of the cell-associated 125I-labelled alpha 2-macroglobulin X trypsin was not removed by treatment of the cells with trypsin-EDTA and represented probably internalized material. 125I-Labelled alpha 2-macroglobulin X trypsin was degraded to trichloroacetic acid-soluble fragments by suspensions of both cell types but only to a negligible extent by incubation media preincubated with these cells. The rate of degradation of 0.5 micrograms/ml 125I-labelled alpha 2-macroglobulin was approx. 40% of that of 125I-labelled alpha 2-macroglobulin X trypsin. Degradation of 125I-labelled alpha 2-macroglobulin X trypsin was abolished by a high concentration (0.5 mg/ml) of alpha 2-macroglobulin X trypsin. It is concluded that alpha 2-macroglobulin X trypsin by a specific and saturable mechanism is bound to, internalized and degraded by isolated rat adipocytes and hepatocytes.