Jørgen Gliemann

The sortilin cytoplasmic tail conveys Golgi–endosome transport and binds the VHS domain of the GGA2 sorting protein

Sortilin belongs to a growing family of multiligand type‐1 receptors with homology to the yeast receptor Vps10p. Based on structural features and sortilins 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 YXXΦ 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 β‐hexosaminidase and β‐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.

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 ∼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.

Evidence that epithelial glycoprotein 330/megalin mediates uptake of polybasic drugs.

Glycoprotein 330 (gp330) is an endocytic receptor expressed in the renal proximal tubules and some other absorptive epithelia, e.g., in the inner ear. The present study shows that the antifibrinolytic polypeptide, aprotinin, and the nephro- and ototoxic antibiotics, aminoglycosides, and polymyxin B compete for binding of 125I-urokinase-plasminogen activator inhibitor type-1 complexes to purified rabbit gp330. Half maximal inhibition was measured at 4 microM for aprotinin, 50 microM for gentamicin, and 0.5 microM for polymyxin B. Drug binding to gp330 was validated by equilibrium dialysis of [3H] gentamicin-gp330 incubations and binding/uptake studies in rat proximal tubules and gp330-expressing L2 carcinoma cells. Analyses of mutant aprotinins expressed in Saccharomyces cerevisiae revealed that basic residues are essential for the binding to gp330 and renal uptake. The polybasic drugs also antagonized ligand binding to the human alpha 2-macroglobulin receptor. However, the rapid glomerular filtration of the drugs suggests kidney gp330 to be the quantitatively most important target. In conclusion, a novel role of gp330 as a drug receptor is demonstrated. The new insight into the mechanism of epithelial uptake of polybasic drugs might provide a basis for future design of drugs with reduced toxicity.

Recycling of the urokinase receptor upon internalization of the uPA:serpin complexes

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 α2‐macroglobulin receptor/low density lipoprotein receptor‐related protein (α2MR‐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°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°C. In fact, uPAR was resistant to PI‐PLC after the 4°C binding of uPA:PAI‐1 to biotinylated cells, but upon incubation at 37°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.

Evidence that the newly cloned low‐density‐lipoprotein receptor related protein (LRP) is the α2‐macroglobulin receptor

The human placental receptor (α2MR) for α2‐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 covering 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 α‐ and β‐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 α2‐macroglobulin‐proteinase complexes (α2MR) and in addition may have as yet unsettled functions in lipoprotein metabolism.

Cubilin dysfunction causes abnormal metabolism of the steroid hormone 25(OH) vitamin D 3

Steroid hormones are central regulators of a variety of biological processes. According to the free hormone hypothesis, steroids enter target cells by passive diffusion. However, recently we demonstrated that 25(OH) vitamin D3 complexed to its plasma carrier, the vitamin D-binding protein, enters renal proximal tubules by receptor-mediated endocytosis. Knockout mice lacking the endocytic receptor megalin lose 25(OH) vitamin D3 in the urine and develop bone disease. Here, we report that cubilin, a membrane-associated protein colocalizing with megalin, facilitates the endocytic process by sequestering steroid–carrier complexes on the cellular surface before megalin-mediated internalization of the cubilin-bound ligand. Dogs with an inherited disorder affecting cubilin biosynthesis exhibit abnormal vitamin D metabolism. Similarly, human patients with mutations causing cubilin dysfunction exhibit urinary excretion of 25(OH) vitamin D3. This observation identifies spontaneous mutations in an endocytic receptor pathway affecting cellular uptake and metabolism of a steroid hormone.

Molecular characterization of a novel human hybrid-type receptor that binds the alpha2-macroglobulin receptor-associated protein

The 39-40-kDa receptor-associated protein (RAP) binds to the members of the low density lipoprotein receptor gene family and functions as a specialized endoplasmic reticulum/Golgi chaperone. Using RAP affinity chromatography, we have purified a novel ∼250-kDa brain protein and isolated the corresponding cDNA. The gene, designated SORL1, maps to chromosome 11q 23/24 and encodes a 2214-residue type 1 receptor containing a furin cleavage site immediately preceding the N terminus determined in the purified protein. The receptor, designated sorLA-1, has a short cytoplasmic tail containing a tyrosine-based internalization signal and a large external part containing (from the N-terminal): 1) a segment homologous to domains in the yeast vacuolar protein sorting 10 protein, Vps10p, that binds carboxypeptidase Y, 2) five tandemly arranged YWTD repeats and a cluster of 11 class A repeats characteristic of the low density lipoprotein receptor gene family receptors, and 3) six tandemly arranged fibronectin type III repeats also found in certain neural adhesion proteins. sorLA-1 may therefore be classified as a hybrid receptor. Northern blotting revealed specific mRNA transcripts in brain, spinal cord, and testis but not in several major organs. Both RAP and an antibody against a synthetic peptide derived from a sequence determined in the mature protein detected sorLA-1 in crude human brain extracts. The domain structure suggests that sorLA-1 is an endocytic receptor possibly implicated in the uptake of lipoproteins and of proteases.

Sortilin/neurotensin receptor-3 binds and mediates degradation of lipoprotein lipase.

Lipoprotein lipase and the receptor-associated protein (RAP) bind to overlapping sites on the low density lipoprotein receptor-related protein/α2-macroglobulin receptor (LRP). We have investigated if lipoprotein lipase interacts with the RAP binding but structurally distinct receptor sortilin/neurotensin receptor-3. We show, by chemical cross-linking and surface plasmon resonance analysis, that soluble sortilin binds lipoprotein lipase with an affinity similar to that of LRP. The binding was inhibited by heparin and RAP and by the newly discovered sortilin ligand neurotensin. In 35S-labeled 3T3-L1 adipocytes treated with the cross-linker dithiobis(succinimidyl propionate), lipoprotein lipase-containing complexes were isolated by anti-sortilin antibodies. To elucidate function in cells, sortilin-negative Chinese hamster ovary cells were transfected with full-length sortilin and shown to express about 8% of the receptors on the cell surface. These cells degraded125I-labeled lipoprotein lipase much faster than the wild-type cells. The degradation was inhibited by unlabeled lipoprotein lipase, indicating a saturable pathway, and by RAP and heparin. Moreover, inhibition by the weak base chloroquine suggested that degradation occurs in an acidic vesicle compartment. The results demonstrate that sortilin is a multifunctional receptor that binds lipoprotein lipase and, when expressed on the cell surface, mediates its endocytosis and degradation.

Propeptide cleavage conditions sortilin/neurotensin receptor‐3 for ligand binding

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.

Receptor‐mediated endocytosis of plasminogen activators and activator/inhibitor complexes

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 multiligand receptors α2 macroglubulin receptor/low density lipoprotein receptor‐related protein (α2MR) 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 α2MR‐ 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 α2MR and gp330 is a novel kind of molecular recognition of serine proteinases and serpins.