Molecular Cloning of a Novel Lipocalin-1 Interacting Human Cell Membrane Receptor Using Phage Display

Abstract

Human lipocalin-1 (Lcn-1, also called tear lipocalin), a member of the lipocalin structural superfamily, is produced by a number of glands and tissues and is known to bind an unusually large array of hydrophobic ligands. Apart from its specific function in stabilizing the lipid film of human tear fluid, it is suggested to act as a physiological scavenger of potentially harmful lipophilic compounds, in general. To characterize proteins involved in the reception, detoxification, or degradation of these ligands, a cDNA phage-display library from human pituitary gland was constructed and screened for proteins interacting with Lcn-1. Using this method an Lcn-1 interacting phage was isolated that expressed a novel human protein. Molecular cloning and analysis of the entire cDNA indicated that it encodes a 55-kDa protein, lipocalin-1 interacting membrane receptor (LIMR), with nine putative transmembrane domains. The cell membrane location of this protein was confirmed by immunocytochemistry and Western blot analysis of membrane fractions of human NT2 cells. Independent biochemical investigations using a recombinant N-terminal fragment of LIMR also demonstrated a specific interaction with Lcn-1 in vitro. Based on these data, we suggest LIMR to be a receptor of Lcn-1 ligands. These findings constitute the first report of cloning of a lipocalin interacting, plasma membranelocated receptor, in general. In addition, a sequence comparison supports the biological relevance of this novel membrane protein, because genes with significant nucleotide sequence similarity are present in Takifugu rubripes, Drosophila melanogaster, Caenorhabditis elegans, Mus musculus, Bos taurus, and Sus scrofa. According to data derived from the human genome sequencing project, the LIMR-encoding gene has to be mapped on human chromosome 12, and its intron/exon organization could be established. The entire LIMR-encoding gene consists of about 13.7 kilobases in length and contains 16 introns with a length between 91 and 3438 base pairs.The protein superfamily of lipocalins consists of small, mainly secretory proteins defined on the basis of conserved amino acid sequence motifs and their common structure. Functionally they share several properties including the ability to bind/transport a remarkable array of small hydrophobic molecules, the formation of macromolecular complexes, and the binding to specific cell surface receptors (1-3). Whereas a large number of various lipophilic ligands able to bind to lipocalins are known, only limited data are available concerning the identity of lipocalin receptors. There is clear evidence of a specific receptor for plasma retinol-binding protein (RBP) 1 (4 -7) and more indirect evidence for receptors for ␣1-microglobulin (8), major urinary protein (9), ␤-lactoglobulin (10), olfactory-binding protein (11), ␣1-acid glycoprotein (12), and glycodelin (13), but with the exception of megalin, which seems to be an endocytic receptor for a variety of soluble macromolecules including several lipocalins, no specific lipocal...

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

“…LIMR nificant similarity to known proteins (11). However, we found a number of thus far uncharacterized genes with statistically significant deduced amino acid similarities (Fig.…”

“…However, with the exception of megalin, none of these cell receptors has been isolated or characterized on a molecular level. We have recently isolated a novel human cell membrane protein, LIMR, which interacts with Lcn-1 (11). Lcn-1 is a lipocalin member produced by a number of human secretory glands and tissues that is known to bind a variety of ligands, including fatty acids, fatty alcohols, cholesterol, retinol, retinoic acid, phosphatidylcholine, and arachidonic acid and its peroxidation products (12,13).…”

“…Because it is known that both LIMR and Lcn-1 are expressed in mammary gland (11,18), we searched for human breast cells expressing LIMR. It is evident from immunostaining using LIMR antibodies that human T-47D cells (19) express LIMR (Fig.…”

“…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.…”

“…We have recently identified and characterized LIMR, 1 a novel human 57-kDa cell membrane protein (11), which interacts with Lcn-1, a lipocalin member produced by a number of secretoric glands and tissues and known to bind a variety of lipophilic compounds (12)(13)(14). Because we found expression of LIMR in the human NT2 cell line (11), we used an antisense gene knockout technology to investigate the role of this receptor in the mechanism of ligand delivery in more detail.…”