The Low Density Lipoprotein Receptor-related Protein/α2-Macroglobulin Receptor Regulates Cell Surface Plasminogen Activator Activity on Human Trophoblast Cells

129

150

The low density lipoprotein receptor-related proteindeleted in tumor (LRP1B, initially referred to as LRP-DIT) was cloned and characterized as a candidate tumor suppressor. It is a new member of the low density lipoprotein receptor gene family. Its overall domain structure and large size (ϳ600 kDa) are similar to LRP and suggest that it is a multifunctional cell surface receptor. Herein, we characterize a series of ligands for the receptor using cell lines that stably express it as a domain IV minireceptor (mLRP1B4). Ligands of LRP including receptor-associated protein, urokinase plasminogen activator, tissue-type plasminogen activator, and plasminogen activator inhibitor type-1 each demonstrate binding, internalization, and degradation via mLRP1B4. Interestingly, the kinetics of ligand endocytosis is distinctly different from that of LRP, with LRP1B exhibiting a markedly diminished internalization rate. In addition, tissue expression analysis reveals that the LRP1B gene is expressed in brain, thyroid, and salivary gland. These studies thus extend the physiological roles of members of the LDL receptor family.Members of the low density lipoprotein receptor gene family play a wide variety of roles in normal cell function and development. For example, mutations of the low density lipoprotein receptor gene, the prototypic family member, result in the genetic disease familial hypercholesterolemia (1). Much attention has focused on other members of this gene family (including LRP, 1 megalin, apolipoprotein E receptor-2, very low density lipoprotein receptor) because of their recently recognized roles in development, cell signaling, and pathogenesis (2, 3). The low density lipoprotein receptor-related protein LRP1B (initially referred to as LRP-DIT (deleted in tumor)), a candidate tumor suppressor, is a new member of the giant receptor subgroup of this gene family. It is located at chromosome 2q21.2 and was isolated by positional cloning based on homozygous deletions detected in human cancer cell lines (4 -6). Mutation analysis revealed that the gene is frequently (45%) inactivated in human non-small cell lung cancer cell lines by intragenic homozygous deletions, point mutations, and aberrant transcripts missing internal portions. Loss of heterozygosity analysis also detected high allelic loss within the locus using two independent microsatellite polymorphism markers, suggesting that the gene is a candidate tumor suppressor (4). The low density lipoprotein receptor gene family previously contained two very large members, LRP (LRP1), a dimer of 515 and 85 kDa, and its closely related homolog, megalin (LRP2), a single species of ϳ600 kDa (2, 3). LRP1B is more closely related to LRP (with similarity of 59 and 52% identity at the cDNA and predicted amino acid levels, respectively) than to LRP2 (4). The cDNA of LRP1B is 16.5 kilobases, with an open reading frame of 13,797 base pairs, which encodes a protein of 4599 amino acids. Characterization of exon-intron boundaries determined that the genomic DNA of LRP1B contains 91 e...

Section: Discussionmentioning

confidence: 93%

“…LRP is also involved in signal transduction (12)(13)(14). Among its myriad functions, LRP modulates the urokinase plasminogen activator system (15)(16)(17)(18)(19)(20)(21). The close similarity in protein domain structure between LRP1B and LRP strongly suggests that the two receptors may share similarity in ligand interaction; i.e.…”

mentioning

confidence: 99%

The Putative Tumor Suppressor LRP1B, a Novel Member of the Low Density Lipoprotein (LDL) Receptor Family, Exhibits Both Overlapping and Distinct Properties with the LDL Receptor-related Protein

Liu

Obermoeller-McCormick

et al. 2001

129

150

“…6) and MDA-MB-231 (7) cells, as shown for uPA-PAI-1 complexes in other cell lines (51). This in turn facilitates the clearance of cell surface plasminogen-activating capability (52) and may possibly mediate cell signaling events (53).…”

Section: Discussionmentioning

confidence: 99%

The Urokinase/PAI-2 Complex

Croucher

Saunders

Ranson

2006

The efficient inactivation of urokinase plasminogen activator (uPA) by plasminogen activator inhibitor type 2 (PAI-2) at the surface of carcinoma cells is followed by rapid endocytosis of the uPA-PAI-2 complex. We now show that one pathway of this receptormediated endocytosis is mediated via the low density lipoprotein receptor-related protein (LRP) in prostate cancer cells. Detailed biochemical analyses using ligand binding assays and surface plasmon resonance revealed a novel and distinct interaction mechanism between native, human LRP and uPA-PAI-2. As reported previously for PAI-1, inhibition of uPA by PAI-2 significantly increased the affinity of the complex for LRP (K D of 36 nM for uPA-PAI-2 versus 200 nM for uPA). This interaction was maintained in the presence of uPAR, confirming the validity of this interaction at the cell surface. However, unlike PAI-1, no interaction was observed between LRP and PAI-2 in either the stressed or the relaxed conformation. This suggests that the uPA-PAI-2-LRP interaction is mediated by site(s) within the uPA molecule alone. Thus, as inhibition of uPA by PAI-2 resulted in accelerated clearance of uPA from the cell surface possibly via its increased affinity for LRP, this represents a mechanism through which PAI-2 can clear proteolytic activity from the cell surface. Furthermore, lack of a direct interaction between PAI-2 and LRP implies that downstream signaling events initiated by PAI-1 may not be activated by PAI-2.Inhibition of urokinase plasminogen activator (uPA) 4 proteolytic activity at the cell surface is an important step in the regulation of pericellular plasminogen activation (1-4). This process is facilitated by members of the serpin (serine protease inhibitor) superfamily, most notably by plasminogen activator inhibitors 1 (PAI-1) and 2 (PAI-2) (SerpinE1 and SerpinB2, respectively) (5-7). Although both are efficient uPA inhibitors, PAI-1 and PAI-2 are structurally and functionally quite distinct serpins, as recognized by their grouping into different serpin subfamily groups (6). For example, PAI-1 also has alternative non-uPA inhibitory activities that affect cell adhesion, intracellular signaling, and cell migration (8) that have not been demonstrated for PAI-2.In their classical inhibitory role, serpins interact with their target protease through an exposed peptide loop, the reactive center loop (6, 9). This acts as bait for the active site of the protease, leading to formation of an equimolar covalent complex. The formation of this complex results in extensive deformation of both the protease and the serpin (9), resulting in the conversion of the serpin from a "stressed" to a "relaxed" form. The relaxed conformation of PAI-2 and other serpins can also be induced by the insertion of a peptide that mimics the reactive center loop (10).uPA is a potent marker of metastatic capacity in multiple human tumors and makes an attractive therapeutic target (11-13). Recent work has shown that cytotoxins attached to the PAI-2 molecule can be specifically delivered to ...

“…LRP-mediated internalization of uPA⅐PAI-1⅐uPAR complexes results in the lysosomal degradation of uPA and PAI-1 and recycling of uPAR to the cell surface (4 -7). This process allows for the regeneration of unoccupied uPAR, immobilization of fresh uPA in the form of its zymogen, pro-uPA, and the eventual re-expression of active uPA at the cell surface (8,10). Failure to remove uPA⅐PAI-1complexes from the cell surface may diminish the cellular capacity for plasminogen activation and may impair cell migration/invasion mediated by uPAR (8 -10, 26).…”

Section: Discussionmentioning

confidence: 99%

Low Density Lipoprotein (LDL) Receptor-related Protein 1B Impairs Urokinase Receptor Regeneration on the Cell Surface and Inhibits Cell Migration

Knisely²,

Lü³

et al. 2002

The low density lipoprotein (LDL) receptor-related protein 1B (LRP1B) is a newly identified member of the LDL receptor family and is closely related to LRP. It was discovered as a putative tumor suppressor and is frequently inactivated in lung cancer cells. In the present study, we used an LRP1B minireceptor (mLRP1B4), which mimics the function and trafficking of LRP1B, to explore the roles of LRP1B on the plasminogen activation system. We found that mLRP1B4 and urokinase plasminogen activator receptor (uPAR) form immunoprecipitable complexes on the cell surface in the presence of complexes of uPA and its inhibitor, plasminogen activator inhibitor type-1 (PAI-1). However, compared with cells expressing the analogous LRP minireceptor (mLRP4), cells expressing mLRP1B4 display a substantially slower rate of uPA⅐PAI-1 complex internalization. Expression of mLRP1B4, or an mLRP4 mutant deficient in endocytosis, leads to an accumulation of uPAR at the cell surface and increased cell-associated uPA and PAI-1 when compared with cells expressing mLRP4. In addition, we found that expression of mLRP1B or the mLRP4 endocytosis mutant impairs the regeneration of unoccupied uPAR on the cell surface and that this correlates with a diminished rate of cell migration. Taken together, these results demonstrate that LRP1B can function as a negative regulator of uPAR regeneration and cell migration.The plasminogen activation system consists of a cascade of enzymes and plays a central role in many physiological processes requiring the degradation of basement membrane and components of the extracellular matrix. When the regulation of this system is disrupted, as occurs in the pathogenesis of cancer, malignant cells are able to invade surrounding tissue and metastasize to distant body regions (1-3). Urokinase plasminogen activator (uPA) 1 catalyzes the formation of plasmin from its inactive precursor, plasminogen. The activity of uPA is regulated by two proteins, the glycosylphosphatidylinositollinked uPA receptor (uPAR) and plasminogen activator inhibitor type 1 (PAI-1). The binding of uPA to uPAR at the cell surface greatly increases its catalytic rate. In contrast, uPA is inactivated by binding to PAI-1. When active uPA is bound to uPAR, it is not internalized but remains at the cell surface. However, when receptor-bound uPA is complexed to its inhibitor, PAI-1, the complex is rapidly internalized and degraded. The mechanism by which this occurs was unknown until it was determined that low density lipoprotein receptor-related protein (LRP) is responsible for this process (4). Following the internalization, uPAR and LRP recycle back to the cell surface, while uPA and PAI-1 are degraded in lysosomes (4 -7). The regeneration of unoccupied uPAR at the cell surface is thus critical for the maintenance of plasminogen activation and for regulation of cellular migration and invasion (8 -10).LRP1B is a recently discovered member of the LDLR family (11, 12). The LDLR family previously contained two large members, LRP (LRP1), a dimer of 515-and...