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A model for modulation of leptin activity by association with clusterin¹

Institute of Medical Biochemistry, Department of Molecular Genetics, BioCenter and University Vienna, A-1030 Vienna, Austria

²Correspondence: Institute of Medical Biochemistry, Department of Molecular Genetics, Dr. Bohr Gasse 9/2, A-1030 Vienna, Austria. E-mail: wjs{at}mol.univie.ac.at

SPECIFIC AIMS

Our aim was to identify plasma components that act as modulators of the transport, biological action, and clearance of leptin. Having obtained multiple evidence for a function of clusterin (apolipoprotein J) in leptin biology, we sought to delineate a possible mechanism for its action.

PRINCIPAL FINDINGS

1. Pull-down experiments using recombinant leptin-GST fusion protein with human serum resulted in the identification of an 80 kDa protein under nonreducing conditions, which was not visible under reducing conditions.
Immunoblotting of the coprecipitated serum proteins with anti-clusterin antibodies identified the 80 kDa protein as the heterodimer of the two disulfide-bonded 40 kDa subunits of clusterin (also termed apolipoprotein J). GST-leptin coprecipitated clusterin not only from human, but also from murine plasma. Pull-down experiments with GST alone did not yield proteins from either plasma.

2. To investigate whether a complex between clusterin and leptin could be formed in serum, we expressed murine leptin as carboxyl-terminally HA- and his-tagged recombinant protein.
After incubation of leptin with serum, the proteins were separated by FPLC on Sephacryl S-300 and representative fractions were analyzed for content of leptin, apo A-I (the main protein component of HDL), clusterin, and soluble leptin receptor (sLEPR). When leptin alone was subjected to chromatography, it was immunodetected only in the low molecular weight fraction, but when preincubated with serum, leptin’s position shifted to the peak containing high molecular weight components. This fraction also contained clusterin and apo A-I. Thus, to shed light on the nature of the high M_r complex and to determine whether leptin’s interaction with clusterin required additional components (e.g., apo A-I), the two recombinant proteins clusterin-myc-his and leptin-HA-his were incubated in the absence of serum. Subsequent analysis by coimmunoprecipitation and immunodetection clearly demonstrated the formation of the binary complex.

3. To test for biological activity of the recombinant leptin and the leptin–clusterin complex, STAT3 phosphorylation triggered by the protein(s) was analyzed in PC12Cl8 cells, a PC12-derived cell line stably expressing the long isoform of the murine leptin receptor (Fig. 1) .
STAT3 phosphorylation in these cells was stimulated in leptin-dependent fashion, as observed after incubation with recombinant leptin (Fig. 1A ) and with the high molecular weight chromatography fraction obtained by incubation of leptin with serum (see 2.) (Fig. 1B ). We could show that the fraction containing sLEPR was able to stimulate STAT3 phosphorylation, suggesting that the receptor fragment binds bioactive leptin despite the failure to detect it by immunoblotting (Fig. 1B , lane 3, and Fig. 1C ).

View larger version (24K): [in this window] [in a new window]   Figure 1. Recombinant murine leptin and leptin–clusterin complex bind to the leptin receptor and stimulate STAT3 phosphorylation. PC12Cl8 cells were serum depleted for 4 h. A) Cells were supplemented with 100 ng/mL recombinant GST-leptin (lane 1), medium alone (lane 2), or HA- and his-tagged leptin (lane 3). B) The incubation media were supplemented with 25 µL of FPLC fractions (lane 1, high Mr peak; lane 2, no addition; lane 3, sLEPR-containing fraction). After 15 min the cell lysates (20 µg/lane) were subjected to SDS-PAGE and immunoblotting with rabbit anti-STAT3 (top panel) or rabbit anti-phospho STAT3 (Tyr 705) (bottom panel), followed by HRP-coupled goat anti-rabbit IgG and ECL. C) The FPLC fraction containing sLEPR (100 µL) was incubated with (lane 1) or without (lane 2) 1 µg HA- and his-tagged leptin; immunoprecipitation was performed using anti-HA monoclonal antibody and protein G-agarose. The bead-bound proteins were eluted with SDS sample buffer, and analyzed by immunoblotting with anti-sLR monoclonal antibody. The positions of migration of Mr standards (kDa) are indicated.

4. To test whether the clusterin–leptin complex binds to members of the LDL receptor gene family, we first analyzed binding of clusterin to the recombinant ligand binding domains of the murine ApoE receptor type 2 (ApoER2) and of the human VLDL receptor (VLDLR) by ligand blotting.
Based on the demonstration of specific binding of clusterin to LDLR relatives in vitro by this approach, we showed that these receptors also mediated the internalization of clusterin as well as of the clusterin–leptin complex, but not of leptin alone (Fig. 2 A)into cells. In fact, the data showed that receptor-mediated uptake of leptin via ApoER2 and VLDLR was driven by the binding of clusterin to these receptors, coupled to the association of leptin with clusterin. The cellular uptake was inhibited by RAP, a pan-inhibitor of ligand interaction with LDLR relatives (Fig. 2B ), and was not observed in cells lacking either of these receptors.

View larger version (30K): [in this window] [in a new window]   Figure 2. Leptin uptake by 293 cells stably expressing apoER2 is driven by clusterin. A) 293 cells stably expressing full-length murine apoER2 (293-ApoER2; panels 1, 2, 4, 5), or mock cells (panels 3, 6) were incubated with 5 µg/mL myc- and his-tagged clusterin, 5 µg/mL HA- and his-tagged leptin, or a mixture of both. The proteins were visualized with anti-myc (for clusterin; panels 1, 3, 4, 6) or anti-HA (for leptin; panels 2, 5) mAbs, followed by Oregon Green 488 labeled anti-mouse antibodies. B) 293-ApoER2 cells were incubated with a mixture of myc- and his-tagged clusterin and HA- and his-tagged leptin (conc. as in panel A) alone or in the presence of 50 µg/mL RAP as indicated. The proteins were visualized by doubly staining with goat anti-clusterin polyclonal IgG or anti-HA mAb, Alexa Fluor 568-labeled anti-goat IgG (to visualize clusterin), and Alexa Fluor 488-labeled anti-mouse IgG (to detect leptin).

CONCLUSION AND SIGNIFICANCE

The current literature describes so-called "free" and "bound" fractions of serum leptin; the distribution of total serum leptin among these two fractions has been reported to alter the hormone’s efficacy, but detailed studies on underlying mechanism(s) are lacking. We hypothesized that 1) the ratio of bound to free leptin, 2) the nature of the serum-borne binding partner(s), and 3) the fate(s) of the free vs. the bound leptin should be important predictive parameters for leptin activity in vivo. Thus, the aim of this study was to identify a binding partner(s) of leptin in serum, study the biological activity of the complex formed, and shed light on the mechanism by which such binding would modulate leptin’s biological activity.

The current studies show by a variety of approaches with both native and recombinant proteins that leptin forms a complex with clusterin (also termed apolipoprotein J), a protein whose function(s) has not been clearly defined. In addition to the identification of clusterin as a novel binding protein for leptin, we show that the binary complex is able to transduce the leptin signal by binding to the leptin receptor and activating the JAK/STAT pathway and/or to be endocytosed via binding and uptake by members of the LDL receptor family that recognize the clusterin moiety of the complex (Fig. 3 ).

View larger version (64K): [in this window] [in a new window]   Figure 3. Schematic diagram depicting the proposed model for clusterin action in leptin biology. Signaling (left side): leptin and/or leptin–clusterin complexes bind to the leptin receptor (LEPR), triggering ligand binding-induced JAK/STAT3 phosphorylation reactions and subsequent induction of target gene transcription in the nucleus. Uptake (right side): clusterin and/or clusterin–leptin complexes bind to LDL receptor relatives (LRs), are endocytosed, likely followed by degradation of the ligands. In balance, the two processes facilitate a fine-tuning of leptin’s biological action.

The significance of high molecular weight forms of leptin in serum has been investigated extensively before, but any mechanism that might explain the relationship between the free-to-bound leptin ratio and leptin’s bioactivity has not yet emerged. The current studies identifying clusterin as partner for circulating leptin present a mechanism underlying the in vivo modulation of leptin action by serum proteins. The capacity of clusterin/leptin to bind to receptors that mediate clearance and to a receptor that transduces signaling facilitates the fine-tuning of leptin’s action by levels of the two receptor types at any site and by the concentrations of free leptin and/or clusterin.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1106fje;

This Article Full Text (PDF) All Versions of this Article: 17/11/1505 02-1106fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by BAJARI, T. M. Articles by SCHNEIDER, W. J. Search for Related Content PubMed PubMed Citation Articles by BAJARI, T. M. Articles by SCHNEIDER, W. J.