Renal LRP2 expression in man and chicken is estrogen-responsive.

In mammals, low-density lipoprotein receptor-related protein-2 (LRP2) is an endocytic receptor that binds multiple ligands and is essential for a wide range of physiological processes. To gain new insights into the biology of this complex protein, we have initiated the molecular characterization of the LRP2 homolog from an oviparous species, the chicken (Gallus gallus). The galline LRP2 cDNA encodes a membrane protein of 4658 residues. Overall, the galline and human proteins are 73% identical, indicating that the avian gene has been well conserved over 300 million years. Unexpectedly, LRP2 transcript and protein levels in the kidney of females and estrogen-treated roosters were significantly higher than those in untreated males. The estrogen-responsiveness of avian LRP2 may be related to the dramatic differences in lipoprotein metabolism between mature roosters and laying hens. Newly identified potential estrogen-responsive elements (ERE) in the human and galline LRP2 gene, and additional Sp1 sites present in the promoter of the chicken gene, are compatible with both direct estrogen induction via the classical ligand-induced ERE pathway and the indirect transcription factor crosstalk pathway engaging the Sp1 sites. In agreement with this assumption, estrogen induction of LRP2 was observed not only in primary cultured chicken kidney cells, but also human kidney cell lines. These findings point to novel regulatory features of the LRP2 gene resulting in sex-specific receptor expression.

LDL receptor family: isolation, production, and ligand binding analysis.

Members of the low density lipoprotein receptor gene family have recently received particular attention because of their involvement not only in lipoprotein transport, but also in signal transduction pathways. The main characteristic feature of this protein group is their cysteine-rich ligand binding domain, which is able to bind many unrelated proteins, such as apolipoproteins, proteases, and protease/inhibitor complexes, signaling molecules such as reelin, and several other groups of proteins. The main challenges of studying these proteins in vitro are their extremely high content of disulfide bridges and the detergent-sensibility of their classical ligands, i.e, lipoproteins. Here, we describe generally applicable procedures for the analysis of these receptors. We present an outline of established methodology for their isolation and visualization, the production of recombinant fragments, in particular of soluble ligand binding domains, and we describe standard procedures for the analysis of the functionality of the receptors and recombinant receptor ligand binding fragments, respectively.

A model for modulation of leptin activity by association with clusterin.

Transport, biological action, and clearance of leptin are subject to modulation by plasma components responsible for the formation of the so-called "bound" fraction of serum leptin. Candidates for modulators have been identified previously, but mechanisms for their action, and thus their physiological roles, have remained unclear. Here we have obtained evidence for a role of serum-borne clusterin in leptin biology and have delineated a possible mechanism for its action. We demonstrate complex formation between clusterin and leptin by several approaches and show that the binary complex retains the ability to transduce the leptin signal via binding to the leptin receptor and activation of the Janus kinase/signal transducer and activator of transcription pathway. The interaction of leptin with clusterin does not require additional serum components. Furthermore, and importantly for modulation of the bioactivity of leptin, uptake of leptin present in the complex can be mediated by members of the low density lipoprotein (LDL) receptor family, i.e., apolipoprotein receptor type-2 and the very LDL receptor, which here are shown to efficiently endocytose both free and leptin-associated clusterin. Thus, bioavailability of leptin at a given tissue site may be determined by the levels of clusterin and/or by the relative distribution of certain relatives of the LDL receptor vis-à-vis active leptin receptors.

Receptor-mediated chicken oocyte growth: differential expression of endophilin isoforms in developing follicles.

Receptor-mediated endocytosis of yolk precursors via clathrin-coated structures is the key mechanism underlying rapid chicken oocyte growth. In defining oocyte-specific components of clathrin-mediated events, we have to date identified oocyte-specific yolk transport receptors, but little is known about the oocytes' supporting endocytic machinery. Important proteins implicated in clathrin-mediated endocytosis and recycling are the endophilins, which thus far have been studied primarily in synaptic vesicle formation; in the present study, as a different highly active endocytic system, we exploit rapidly growing chicken oocytes. Molecular characterization of the chicken endophilins I, II, and III revealed that their mammalian counterparts have been highly conserved. All chicken endophilins interact via their SH3 domain with the avian dynamin and synaptojanin homologues and, thus, share key functional properties of mammalian endophilins. The genes show different expression patterns: As in mammals, expression is low to undetectable in the liver and high in the brain; in ovarian follicles harboring oocytes that are rapidly growing via receptor-mediated endocytosis, levels of endophilins II and III, but not of endophilin I, are high. Immunohistochemical analysis of follicles demonstrated that endophilin II is mainly present in the theca interna but that endophilin III predominates within the oocyte proper. Moreover, in a chicken strain with impaired oocyte growth and absence of egg-laying because of a genetic defect in the receptor for yolk endocytosis, endophilin III is diminished in oocytes, whereas endophilin III levels in the brain and endophilin II localization to theca cells are unaltered. Thus, the present study reveals that the endophilins differentially contribute to oocyte endocytosis and development.

A secreted soluble form of ApoE receptor 2 acts as a dominant-negative receptor and inhibits Reelin signaling.

Specialized neurons throughout the developing central nervous system secrete Reelin, which binds to ApoE receptor 2 (ApoER2) and very low density lipoprotein receptor (VLDLR), triggering a signal cascade that guides neurons to their correct position. Binding of Reelin to ApoER2 and VLDLR induces phosphorylation of Dab1, which binds to the intracellular domains of both receptors. Due to differential splicing, several isoforms of ApoER2 differing in their ligand-binding and intracellular domains exist. One isoform harbors four binding repeats plus an adjacent short 13 amino acid insertion containing a furin cleavage site. It is not known whether furin processing of this ApoER2 variant actually takes place and, if so, whether the produced fragment is secreted. Here we demonstrate that cleavage of this ApoER2 variant does indeed take place, and that the resulting receptor fragment consisting of the entire ligand-binding domain is secreted as soluble polypeptide. This receptor fragment inhibits Reelin signaling in primary neurons, indicating that it can act in a dominant-negative fashion in the regulation of Reelin signaling during embryonic brain development.