Mannose 6-phosphate/insulin-like growth factor-II receptor targets the urokinase receptor to lysosomes via a novel binding interaction.

The urokinase-type plasminogen activator receptor (uPAR) plays an important role on the cell surface in mediating extracellular degradative processes and formation of active TGF-beta, and in nonproteolytic events such as cell adhesion, migration, and transmembrane signaling. We have searched for mechanisms that determine the cellular location of uPAR and may participate in its disposal. When using purified receptor preparations, we find that uPAR binds to the cation-independent, mannose 6-phosphate/insulin-like growth factor-II (IGF-II) receptor (CIMPR) with an affinity in the low micromolar range, but not to the 46-kD, cation-dependent, mannose 6-phosphate receptor (CDMPR). The binding is not perturbed by uPA and appears to involve domains DII + DIII of the uPAR protein moiety, but not the glycosylphosphatidylinositol anchor. The binding occurs at site(s) on the CIMPR different from those engaged in binding of mannose 6-phosphate epitopes or IGF-II. To evaluate the significance of the binding, immunofluorescence and immunoelectron microscopy studies were performed in transfected cells, and the results show that wild-type CIMPR, but not CIMPR lacking an intact sorting signal, modulates the subcellular distribution of uPAR and is capable of directing it to lysosomes. We conclude that a site within CIMPR, distinct from its previously known ligand binding sites, binds uPAR and modulates its subcellular distribution.

Uptake of neutral alpha- and beta-amino acids by human proximal tubular cells.

The transport characteristics of amino acids in primary cell cultures from the proximal tubule of human adults (AHKE cells) were examined, using alpha-aminoisobutyric acid (AIB) and beta-alanine as representatives of alpha- and beta-amino acids, respectively. The Na(+)-gradient dependent influx of AIB occurred by a single, saturable transport system, whereas the Na(+)-gradient dependent uptake data for beta-alanine could be described in terms of two-independent transport components as well as one-transport one-leak model with identical kinetic constants for the high-affinity system. Competition experiments revealed that all the neutral amino acids tested reduced the uptake of AIB, whereas there was no effect of taurine, L-aspartic acid, and L-arginine. By contrast, the influx of beta-alanine was only drastically reduced by beta-amino acids, whereas the inhibition by neutral alpha-amino acids was relatively low. Nor did L-arginine and L-aspartic acid affect the uptake of beta-alanine into AHKE cells. Comparison with the results obtained for normal (NHKE) and immortalized (IHKE) embryonic cells suggested an unaltered expression of the types of transport carriers for neutral alpha- and beta-amino acids in the embryonic and AHKE cells. However, the uptake capacity of the above-mentioned transport proteins was relatively smaller in the embryonic kidney compared with the adult human kidney, which may explain, at least partly, the phenomenon of physiologic amino aciduria in neonates.

A comparative study on the uptake of alpha-aminoisobutyric acid by normal and immortalized human embryonic kidney cells from proximal tubule.

We investigated whether an immortalized human kidney epithelial cell line (IHKE), compared with normal embryonic cells (NHKE), can be used as a representative system with which to characterize the transport of neutral amino acids in the proximal tubule of the human kidney. The IHKE cell line, immortalized by treatment with NiSO4, exhibited microvilli and enzyme markers specific for highly specialized tubule cells. The Na(+)-dependent uptake of alpha-aminoisobutyric acid (AIB) by IHKE and NHKE cells occurred by means of a single transport system with identical half-saturation constants, but the capacity for uptake was higher in the immortalized cells. Proton-dependent influx of AIB was also mediated by a single transport component with similar uptake characteristics in both types of cells. Imposition of an H(+)-gradient to a Na(+)-gradient reduced the sodium dependent uptake of AIB with the exception of short incubation time (1 min), where addition of a proton gradient produced a marked increase in the Na(+)-dependent influx of AIB in NHKE but not in IHKE cells. Competition experiments revealed that the Na(+)-dependent uptake at 50 microM AIB was reduced by neutral alpha-amino acids in the two cell lines. L-Glutamate, L-aspartate, L-arginine and the beta-amino acid taurine had no effect. Only in the IHKE cell line did addition of 5 mM L-lysine produce a slight inhibition. Except for L-proline all of the neutral and acidic amino acids tested reduced the H(+)-dependent uptake of AIB in the IHKE cell line. By contrast, addition of L-aspartate did not influence the transport of AIB in NHKE cells. L-Arginine, but not L-lysine decreased the influx in both cell lines. We conclude that the IHKE cell line has retained the capability to accumulate AIB by transport protein(s) similar to those present for neutral alpha-amino acids in NHKE cells.

Comparative study of the uptake of L-cysteine and L-cystine in the renal proximal tubule.

1. The transport mechanisms of L-cysteine and L-cystine by luminal membrane vesicles isolated from either the proximal convoluted part (pars convoluta) or the proximal straight part (pars recta) of rabbit proximal tubuli were examined. 2. The uptake of L-cysteine in pars convoluta is characterized by a single sodium-dependent transport system (Km = 0.58 mM), whereas the sodiumdependent influx of L-cysteine in pars recta proceeds via transport systems with different affinities (Km1 = 0.03 mM, Km2 = 5.84 mM). An H(+)-gradient enhanced the uptake of L-cysteine in pars recta and only in the presence of a Na(+)-gradient. 3. The presence of a Na(+) gradient stimulated the influx of L-cystine in both parts of the nephron, but to a lesser extent than for L-cysteine. In addition a considerable amount of binding of L-cystine to membrane vesicles was observed in both pars convoluta and pars recta. 4. Stoichiometric studies indicated a coupling ratio of 1 Na(+): 1 amino acid for the transport components involved in the uptake of L-cysteine and L-cystine along the proximal tubule. 5. Competition experiments demonstrated that neutralα-amino acids inhibited the influx of L-cysteine in the proximal tubule. Basic and acidic amino acids had no effect on uptake of L-cysteine in pars convoluta, whereas a slight inhibitory effect of L-lysine and L-arginine was noted in pars recta. 6. Both basic and neutral amino acids, but not L-glutamate inhibited the uptake of L-cystine in pars convoluta. This was in general also the case in pars recta. However, addition of L-proline did not influenced the uptake of L-cystine, and L-phenylalanine, L-asparagine, L-glutamine, L-leucine and L-methionine only inhibited at a high concentration (5 mM).

Enzymatically active Ca2+ ATPase from sarcoplasmic reticulum membranes, solubilized by nonionic detergents. Role of lipid for aggregation of the protein.

The present study provides data on the properties of Ca2+-dependent Atpase of sarcoplasmic reticulum in states intermediary between the fully detergent-solubilized and vesicular form. After solubilization of ATPase vesicles by dodecyloctaoxyethylene glycol monoether (C12E8), the protein is mainly present as a monomer exhibiting enzymatic activity. Gel chromatography in presence or absence of Tween 80 gives rise to formation of oligomers of various size and smaller amounts of monomeric ATPase. Only the oligomeric species retain enzymatic activity (half-life, 3 to 4 days), while the gel chromatographic monomer is enzymatically inactive. Teteramers or trimers of ATPase, containing approximately 22 mol of phospholipid/mol of ATPase, are the smallest enzymatically active units after gel chromatography. Formation of larger sized particles and vesicles of ATPase appears to depend on the presence of sufficient lipid to make a cohesion between the tetrameric or trimeric units. The protein appears to be partially deaggregated by a relatively high Tween 80 concentration in the eluant (0.5 mg/ml) and under these conditions, phospholipid binding is reduced to a low level (approximately 11 mol/mol of protein). The data indicate that any bonds between ATPase polypeptide chains are easily disrupted by detergent and that lipid also may play a role in mediating contact between individual polypeptide chains in the tetrameric or trimeric units. Phospholipid analysis and exchange experiments indicate that the phospholipid left on ATPase after solubilization has a similar composition to that of the whole membrane. The binding of Tween 80 by soluble ATPase above the critical micellar concentration is 0.23 to 0.29 g/g of protein. The inactive monomer of ATPase binds phospholipid and Tween 80 to about the same extent, but has a slightly different circular dichroism spectrum, than oligomeric ATPase.

A carboxyl-terminal fragment of lipoprotein lipase binds to the low density lipoprotein receptor-related protein and inhibits lipase-mediated uptake of lipoprotein in cells.

It has previously been shown that lipoprotein lipase can mediate uptake of remnant lipoprotein particles via binding to the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP). Binding of lipoprotein lipase, and of triglyceride-rich lipoproteins associated with the lipase, to LRP depends on an intact carboxyl-terminal folding domain of the lipase (Nykjaer, A., Bengtsson-Olivecrona, G., Lookene, A., Moestrup, S. K., Petersen, C. M., Weber, W., Beisiegel, W., and Gliemann, J. (1993) J. Biol. Chem. 268, 15048-15055). Here we show that the site for binding to the receptor is within residues 380-425 of the bovine and residues 378-423 of the human lipoprotein lipase. We demonstrate that a carboxyl-terminal fragment of human lipoprotein lipase (residues 378-448), expressed as fusion protein in Escherichia coli, binds to purified and cellular LRP but not to lipoproteins. Binding of the fragment to purified LRP was blocked by heparin. In addition, the fragment inhibited the binding of lipase and the lipase-mediated binding of lipoproteins to the purified receptor. The fragment exhibited reduced binding to proteoglycan-deficient cells. Moreover, the fragment inhibited the uptake of lipoproteins in cells mediated by the lipase via binding to heparan sulfate proteoglycans and LRP. We conclude that the fragment contains the site for binding to LRP and a candidate site for interaction with heparan sulfate proteoglycans, whereas binding to lipoproteins is inefficient. The fragment can therefore inhibit the lipase-mediated lipoprotein uptake, a process that may promote the development of atherosclerosis when occurring in cells of the arterial wall.

Epithelial glycoprotein-330 mediates endocytosis of plasminogen activator-plasminogen activator inhibitor type-1 complexes.

Epithelial glycoprotein 330 (gp330) is structurally similar to the multifunctional alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP), gp330 and alpha 2MR/LRP bind Ca2+ with high affinity, and both receptors bind and mediate endocytosis of alpha 2MR-associated protein (RAP). In the present report, we describe that affinity-purified gp330 from rabbit renal cortex binds plasminogen activator inhibitor type-1 (PAI-1) complexed with urokinase-type plasminogen activator (uPA). alpha 2M-methylamine, which binds with high affinity to alpha 2MR/LRP, did not bind to gp330. The apparent Kd for binding of uPA.PAI-1 complexes was about 0.8 nM at 4 degrees C. The binding was calcium-dependent and inhibited by recombinant RAP (rRAP) and tissue type plasminogen activator-PAI-1 complexes. Thin sections of rabbit renal proximal tubules bound 125I-labeled uPA.PAI-1 and rRAP in the apical part of proximal tubules corresponding to the localization of gp330. The binding of 125I-uPA.PAI-1 complexes in tubules was abolished by excess unlabeled rRAP, and a rRAP-inhibitable endocytosis and degradation of labeled uPA.PAI-1 complexes was demonstrated by perfusion of isolated rabbit proximal tubules. The results establish an endocytotic function of gp330 and suggest that gp330 is an important component of the fibrinolytic system in gp330-containing epithelial as found in, for example, kidney and lung.

Molecular identification of a novel candidate sorting receptor purified from human brain by receptor-associated protein affinity chromatography.

Receptor-associated protein (RAP) is an endoplasmic reticulum/Golgi protein involved in the processing of receptors of the low density lipoprotein receptor family. A approximately 95-kDa membrane glycoprotein, designated gp95/sortilin, was purified from human brain extracts by RAP affinity chromatography and cloned in a human cDNA library. The gene maps to chromosome 1p and encodes an 833-amino acid type I receptor containing an N-terminal furin cleavage site immediately preceding the N terminus determined in the purified protein. Gp95/sortilin is expressed in several tissues including brain, spinal cord, and testis. Gp95/sortilin is not related to the low density lipoprotein receptor family but shows intriguing homologies to established sorting receptors: a 140-amino acid lumenal segment of sortilin representing a hitherto unrecognized type of extracellular module shows extensive homology to corresponding segments in each of the two lumenal domains of yeast Vps10p, and the extreme C terminus of the cytoplasmic tail of sortilin contains the casein kinase phosphorylation consensus site and an adjacent dileucine sorting motif that mediate assembly protein-1 binding and lysosomal sorting of the mannose-6-phosphate receptors. Expression of a chimeric receptor containing the cytoplasmic tail of gp95/sortilin demonstrates evidence that the tail conveys colocalization with the cation-independent mannose6-phosphate receptor in endosomes and the Golgi compartment.