Antisense Down-regulation of Lipocalin-interacting Membrane Receptor Expression Inhibits Cellular Internalization of Lipocalin-1 in Human NT2 Cells

Abstract

There is increasing experimental evidence demonstrating that many lipocalins bind to specific cell surface receptors. However, whereas the binding of lipocalins to their lipophilic ligands has now been characterized in much detail, there is a lack of knowledge about the nature of lipocalin receptors, the physiological role of receptor binding, and the molecular mechanism of ligand delivery. We previously identified a novel human membrane protein (lipocalin-1-interacting membrane receptor (LIMR)), which interacts with lipocalin-1 (Wojnar, P., Lechner, M., Merschak, P., and Redl, B. (2001) J. Biol. Chem. 276, 20206 -20212). In the present study, we investigated the physiological role of LIMR and found this protein to be essential for mediating internalization of lipocalin-1 (Lcn-1) in NT2 cells, leading to its degradation. Whereas control NT2 cells rapidly internalized 125 I-Lcn-1 or fluorescein isothiocyanate-labeled Lcn-1, NT2 cells that were made LIMR deficient by cDNA antisense expression greatly accumulated Lcn-1 in the culture medium but did not internalize it. Because sequence and structure analysis indicated that proteins similar to LIMR are present in several organisms and at least two closely related orthologues are found in human and mouse, we suggest LIMR to be the prototype of a new family of endocytic receptors, which are topographically characterized by nine putative transmembrane domains and a characteristic large central cytoplasmic loop.Lipocalins were found to be important extracellular carriers of lipophilic compounds in vertebrates, invertebrates, plants, and bacteria (1-4). There is increasing evidence that this group of proteins is involved in a variety of physiological processes including retinoid, fatty acid, and pheromone signaling; immunomodulation; inflammation; detoxification; modulation of growth and metabolism; tissue development; apoptosis; and even behavior processes (1, 5-7). Whereas the structural basis of lipocalin-ligand binding is now well understood (8), there is a major lack of knowledge regarding the mechanisms by which lipocalins exert their biological effects. It is well accepted that many, if not all, of these proteins are able to bind to specific cell receptors (9), although only two of these receptors have been identified thus far (10, 11). Due to limited data concerning the structure of the lipocalin receptors themselves, the molecular mechanisms beyond this receptor binding are very unclear at the moment. One hypothesis is that the holo-lipocalin releases its ligand upon receptor binding, and this ligand diffuses through the cell membrane to interact with an intracellular fatty acid-binding protein or an intracellular receptor. There is also some evidence that lipocalins undergo internalization by receptor-mediated endocytosis. Another plausible mechanism might be that the lipocalin-receptor interaction creates a direct signal inducing various physiological processes (9).We have recently identified and characterized LIMR, 1 a novel human 57-kDa cell membrane protein ...

Inhibition of MUC4 Expression Suppresses Pancreatic Tumor Cell Growth and Metastasis

“…Control cell clones were also generated by stably transfecting empty vector and partial length sense-RNA construct to minimize the effect, if any, because of the foreign DNA integration into the host genome, expression of nonhost transcripts, and clone selection conditions. Antisense-RNA-based gene silencing strategy has been used efficiently to study the gene functions in human cells (31,32); however, present work is the first report on MUC4 mucin down-regulation using this approach.…”

Expression of Neutrophil Gelatinase-associated Lipocalin Regulates Epithelial Morphogenesis in Vitro

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Human Tear Lipocalin Exhibits Antimicrobial Activity by Scavenging Microbial Siderophores

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