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Expression and Characterization of a Putative High Affinity Human Soluble Leptin Receptor

Neurocrine Biosciences, Inc., San Diego, California 92121

Address all correspondence and requests for reprints to: Errol B. De Souza, Neurocrine Biosciences, Inc., 3050 Science Park Road, San Diego, California 92121.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Leptin, a circulating 16-kDa protein secreted by adipocytes, decreases body weight by reducing food intake and enhancing energy utilization. Leptin receptors that share homology to the glycoprotein gp130 have been recently cloned. In addition, differentially spliced leptin receptor messenger RNAs have been identified. Functional mutations in either the leptin or leptin receptor gene cause obesity. In the present study, expression of the full length human leptin receptor complementary DNA encoding the long cytoplasmic domain of leptin receptor in COS7 cells resulted in high affinity membrane binding of ¹²⁵I-leptin (K_i 200 pM); no detectable binding was present in the medium. In addition, we expressed the extracellular domain of human leptin receptor in COS7 cells and identified a soluble leptin receptor in the conditioned medium that binds human and mouse leptin with high affinity comparable with the full length membrane receptor. Transfected COS7 cells expressing the soluble leptin receptor also demonstrated modest specific ¹²⁵I-leptin binding in whole cells, presumably due to association of the soluble leptin receptor to cell membrane proteins. Data from cross-linking studies identified two specific bands in the ¹²⁵I-leptin/soluble leptin receptor complex with molecular masses of approximately 130–150 kDa and 300 kDa. The 130–150 kDa molecular mass was confirmed in Western blot analysis and Coomassie staining of the purified soluble receptor and probably represents the glycosylated form of the receptor. The 300-kDa band most likely represents a homodimer of the soluble leptin receptor complex because HPLC gel filtration analysis of the ¹²⁵I-leptin/soluble leptin receptor complex identified a single peak corresponding to a molecular mass of approximately 340 kDa. The soluble leptin receptor antagonized ¹²⁵I-leptin binding to the membrane receptor, suggesting its potential utility as a functional tool for determining the role of endogenous leptin.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
LEPTIN, a secreted 16-kDa protein of the obese (ob) gene, is primarily produced by adipocytes (1). Leptin regulates body weight by decreasing food intake and increasing energy utilization (2). Its primary role in body weight regulation is further substantiated by the observation that a functional mutation of the leptin gene causes obesity in homozygous mice (ob/ob) (3). Leptin functions through leptin receptors that have been recently cloned in a variety of species including human (4), mouse (5, 6), and rat (7). Leptin receptors share homology to the glycoprotein gp130, which is a common subunit of receptors such as interleukin-6 (IL-6), cilliary neurotrophic factor (CNTF), and leukemia inhibitory factor (LIF), to name a few (8). In addition, three differentially spliced leptin receptor messenger RNA (mRNA) variants have recently been identified. They include a putative soluble leptin receptor, a membrane bound receptor with a short cytoplasmic domain with no signal transduction function, and a membrane bound receptor with a long cytoplasmic domain with signal transduction ability (5, 6). Janus kinase (Jak) and signal tranducers and activators of transcription (STAT) interaction motifs are predicted in the long cytoplasmic domain of the leptin receptor, and recent studies have demonstrated that this form of the receptor activates STAT-3, STAT-5, and STAT-6 (9, 10); leptin receptor with a short cytoplasmic domain did not activate the STAT proteins (10). A single point mutation in the leptin receptor gene resulted in abnormal splicing of leptin receptor mRNA and prevented the expression of the leptin receptor with a long cytoplasmic domain (5, 6, 11). This mutation was proposed to confer a type II diabetes phenotype because mice with the homozygous mutation (db/db) lack expression of the leptin receptor with the long cytoplasmic signaling domain (5, 6, 11).

While the molecular and signaling characteristics of the different splice variants of the leptin receptor have been determined, their receptor binding characteristics remain to be fully elucidated. Furthermore, the structural requirements of leptin receptors which share homology to gp130, a common subunit of cytokine receptors that require two subunits to bind their respective ligands with high affinity, remain to be determined. In the present study, we have expressed the full length complementary DNA (cDNA) encoding the long cytoplasmic domain or the extracellular domain of the human leptin receptor in COS7 cells and characterized the membrane and soluble receptor leptin binding properties with respect to affinity, molecular mass of the complex, and ability of the soluble receptor to functionally neutralize the binding of the ligand.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Leptin preparation and radioiodination
Human recombinant leptin was expressed and purified from Escherichia coli. In brief, the human leptin cDNA encoding amino acids 22–167 with an extra methionine at the N-terminus was cloned into the prokaryotic expression vector pET-21-d (Novagen, Madison, WI) and expressed in BL21(DE3) (Stratagene, La Jolla, CA). Isopropyl-1-thio-ß-D-galactopyranoside (Fisher Scientific, Tustin, CA)-induced expression of human leptin caused the formation of leptin inclusion bodies that were first purified and then dissolved in 8 M urea, 100 mM Tris-HCl. The dissolved human leptin was diluted into 50 mM sodium citrate, 5 mM EDTA at pH 3.0, and 20 ml of SP-Sepharose (Pharmacia Biotech, Inc., Piscataway, NJ) was added; the mixture was incubated for 15 min with gentle stirring. The SP-Sepharose beads were centrifuged at low speed (1000 x g) and loaded onto a column. The column was extensively washed first with phosphate buffer (10 mM) at pH 5.0, then with 50 ml of phosphate buffer at pH 6.5 and eluted with phosphate buffer at pH 8.0. In general, one liter of cell culture medium gave a yield of approximately 30–50 mg of recombinant human leptin with a purity > 95%.

Mouse recombinant leptin was purchased from R & D System and radioiodinated using the iodogen (Pierce, Rockford, IL) method. Iodine-125-labeled mouse leptin was separated from the free iodine by passing through a G-15 gel (Pharmacia Biotech Inc.) filtration column. The resultant ¹²⁵I-leptin was then run on a SDS-PAGE and exposed to x-ray film to determine the purity of labeling and specific activity. A single radioactive band with the appropriate molecular weight (16 kDa) was detected. The specific activity of ¹²⁵I-leptin was approximately 2000 Ci/mmol.

Cloning and expression of human leptin receptors
The coding sequence of the full-length human leptin receptor cDNA was amplified from human hypothalamus cDNA using PCR with two primers: P1: 5' ATA CGT ACT CTG CAG CTT CTC TGA AGT AAG ATG ATT TGT C 3'; and P2: 5' ATA TAT AAG CTT GAA GGT TTC TTC AGT GAA ATT ACA CAG T 3'.

The putative human soluble leptin receptor cDNA coding sequence was constructed by PCR amplification of the extracellular domain coding sequence of the human leptin receptor with the P1 primer described above and P3: 5' AGA GAG AAG CTT TCA GGA TCC AAT TAT CTT TGG TTT TCC CAC TCC T 3'. The resultant cDNA encoded the 824 amino acids of the extracellular domain of human leptin receptor.

The cloned cDNAs were confirmed by DNA sequencing and then inserted in a eukaryotic expression vector pCMV-sport1 (GIBCO-BRL, Gaithersburg, MD). For the soluble leptin receptor, a FLAG peptide coding sequence was added to the C-terminal coding sequence of the cDNA. The human leptin receptor cDNAs in expression vectors were transfected into monkey kidney COS7 cells using LipofectAMINE (GIBCO-BRL) as described by the manufacturer.

Radioligand binding assays
¹²⁵I-Leptin binding to whole cell membranes was carried out as follows: One day following leptin receptor transfection, COS7 cells were trypsinized and seeded either in 6-well (3.5 cm; 5 x 10⁵ cells/well) or in 24-well (1.5 cm; 1 x 10⁵ cells/well) tissue culture plates. Three days after transfection, the cell culture medium was aspirated and the cells were first washed once with ice-cold PBS then overlaid with either 2 ml (6-well plates) or 1 ml (24-well plates) of ice-cold binding medium containing 50 pM of ¹²⁵I-leptin, 1% BSA, 20 mM HEPES, pH 7.2, and 0.02% sodium azide in DMEM either in the presence or absence of unlabeled leptin (either human or mouse) as competitors. The cells were incubated in the binding mixture described above for 5 h at 4 C, and the binding medium was aspirated; the cells on the dishes were then washed three times with ice-cold PBS. The cells were dissolved with 4 M guanidine-HCl, and bound ¹²⁵I-leptin was counted in a -counter (Packard, Downers Grove, IL).

¹²⁵I-Leptin binding to the soluble leptin receptor was carried out as follows: ¹²⁵I-Leptin was added to the cell culture supernatant from transfected COS7 cells at a final concentration of 50 pM. Different concentrations of unlabeled recombinant leptin (either human or mouse) were added to the binding mixture for competition studies. The binding mixture was incubated for 4 h at 4 C on a rocking platform. Next, 20 µl of anti-FLAG M2 antibody-coupled affinity gel (VWR, West Chester, PA) was added to the binding mixture and incubated overnight at 4 C on a rocking platform. The M2 affinity gel was spun down, the supernatant was aspirated, and the pellet was counted for bound ¹²⁵I-leptin in a -counter.

Molecular mass determination
The molecular mass of the leptin/soluble receptor complex was determined using the radioligand/receptor affinity cross-linking method, Western blot analysis, and HPLC gel filtration analysis.

¹²⁵I-Leptin affinity cross-linking to the soluble human leptin receptor was carried out as follows: Two days following transfection, COS7 cells were replaced with serum-free DMEM and cultured for one additional day. The transfected COS7 cell conditioned medium was collected, centrifuged for 1 h at 10,000 x g, and the supernatant was concentrated 20-fold using Centricon-30 (Amicon). Twenty microliters of the concentrated medium was incubated for 4 h at 4 C with 100 pM of ¹²⁵I-leptin either in the presence or absence of 200 nM unlabeled mouse recombinant leptin. The cross-linker ethylene glycol bis-succinimidylsuccinate (Pierce) was then added to the mixture at a final concentration of 2 mM, and the reaction mixture was incubated at room temperature for an additional 20 min. The reaction mixture was then run onto a 4–20% SDS-PAGE under reducing conditions, and the gel was dried and exposed to x-ray film for 5 h at -70 C with an intensifying screen (Kodak).

Western blot detection of the expressed soluble human leptin receptor was carried out as follows: Transfected COS7 cell culture medium was centrifuged to get rid of cell debris. A leptin affinity column was prepared by cross-linking recombinant human leptin to Affi-gel 15 matrix (Bio-Rad, Chicago, IL) as described by manufacturer. Deactivated Affi-gel 15 with no protein cross-linked to the beads was used as a control. Twenty microliters of human leptin-coupled Affi-gel 15 were added to 1 ml of cell culture supernatant and incubated overnight at 4 C. The affinity gel was then spun down and washed three times with ice-cold Tris-buffered saline containing 0.05% Tween-20 (TBST: 20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20). SDS loading buffer (either reducing or nonreducing) was added to the affinity gel, and the samples were run on a 4–20% SDS-PAGE. The gel-separated proteins were then transferred onto an Immobilon membrane (Minipore), blocked with 1% casein in TBST, blotted first with anti-FLAG M2 antibody, second with horseradish peroxidase conjugated goat antimouse IgG (Fc specific; Sigma Chemical Co., St. Louis, MO) antibody, and detected with the enhanced chemiluminescent (ECL) system (Amersham Life Sciences, Arlington Heights, IL).

HPLC gel filtration analysis of the leptin-soluble receptor complex was carried out as follows: Five hundred microliters of serum free transfected COS7 cell conditioned medium was incubated at room temperature for 15 min with 2 µl ¹²⁵I-leptin (35,000 cpm) either in the presence or absence of 200 nM of unlabeled mouse leptin as the competitor. After incubation, 100 µl of medium was loaded on a TSK gel G3000SW column (TOSOHAAS, Montgomeryville, PN) and eluted at a flow rate 0.5 ml/min with buffer containing 0.1 M sodium phosphate, 0.1 M Na₂SO₄, 0.05% sodium azide, pH 7.2; fractions (0.2 ml) were collected and counted in a -counter.

Functional characterization of the soluble leptin receptor
The recombinant leptin receptor expressed in COS7 cells was purified as follows: Ten 150-mm dishes of COS7 cells were transfected with the soluble leptin receptor expressing plasmid. One day after transfection, the cell culture was replaced with serum-free DMEM, and the conditioned medium was collected once a day for 3 days. The collected medium was tested for ¹²⁵I-leptin binding activity as described above and the concentration of soluble leptin receptor in the conditioned medium was estimated at approximately 2 µg/ml. The medium was then loaded on a human leptin-coupled affinity column; the column was washed extensively first with PBS, then PBS containing 0.5 M NaCl, and eluted with 0.1 M glycine-HCl, pH 3.0. The concentration of the purified soluble leptin receptor was determined using a Bio-Rad protein assay kit.

The functional ability of the purified soluble leptin receptor to inhibit ¹²⁵I-leptin binding to cell membranes was evaluated. COS7 cells transfected with the full-length leptin receptor cDNA were trypsinized and seeded in 6-well tissue culture plates as described earlier. Three days after transfection, the transfected COS7 cells were incubated with 50 pM ¹²⁵I-leptin in the absence or presence of 5 nM of the purified soluble leptin receptor (sR) or 200 nM unlabeled leptin and the whole cell binding assay was carried out as described above.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Expression and characterization of the recombinant human full-length leptin receptor
The recombinant full-length human leptin receptor cDNA encoding the long cytoplasmic domain (mR) was expressed in COS7 cells and the ¹²⁵I-leptin binding characteristics in the membrane fraction, and in the medium were determined. There was a high signal to noise ratio in the cell membrane binding assay with specific (i.e. 200 nM unlabeled leptin displaceable)¹²⁵I-leptin binding representing > 90% of the total ¹²⁵I-leptin binding; no detectable membrane binding was evident in the control mock transfected COS7 cells (Ct) (Fig. 1A). The affinity of the expressed full length membrane receptor was determined in competition studies using both human and mouse leptin. Both species of leptin dose dependently inhibited binding in a monophasic manner to the membrane leptin receptor with comparable high affinities (K_i 200 pM) (Fig. 1B). No binding was present in the medium of mR transfected COS7 cells (see Fig. 3A).

View larger version (15K): [in this window] [in a new window]   Figure 1. A, 125I-Leptin binding in cell membranes of transfected COS7 cells. COS7 cells transfected with the full-length cDNA encoding the long cytoplasmic domain (mR) or the extracellular domain (sR) of the human leptin receptor or mock transfected cells (Ct) were seeded in 6-well plates. Three days after transfection, the medium was aspirated, and the cells were overlayed with 50 pM of 125I-leptin either in the presence (open column) or absence (filled column) of 200 nM of unlabeled leptin as the competitor. The mixture was incubated for 5 h at 4 C, after which the binding medium was aspirated and the cells washed three times with ice-cold PBS. The cells were solubilized and the bound 125I-leptin was counted. Data represent the mean ± SEM of three determinations within a representative experiment that was reproduced three times with comparable results. B, Competition of 125I-leptin binding to membranes of COS7 cells expressing the full-length human leptin receptor by human or mouse leptin. The mR leptin receptor cDNA transfected COS7 cells were seeded in 24-well tissue culture plates. Three days after transfection, the cells were used for cell membrane binding with 125I-leptin in the presence of different concentration of either human leptin or mouse leptin as the competitor.

View larger version (58K): [in this window] [in a new window]   Figure 3. A, Determination of the molecular mass of the 125I-leptin/soluble leptin receptor complex in cross-linking studies. Cell culture supernatants from COS7 cells transfected with the full-length cDNA encoding the long cytoplasmic domain (mR) or the extracellular domain (sR) of the human leptin receptor or mock transfected cells (Ct) were cross-linked with 125I-leptin either in the absence (lanes: 1, 2, 3) or presence (lanes: 4, 5, 6) of 200 nM of unlabeled leptin as competitor. B, Affinity precipitation and Western blot analysis of the expressed soluble leptin receptor. Medium from control COS7 cells (mock transfected, lanes 1, 2) or sR leptin cDNA transfected COS7 cells (lanes 3, 4, 5) was affinity precipitated either with control affinity gel (lanes 1, 3), or human leptin affinity gel (lanes 2, 4, 5) and run on a SDS-PAGE either under reducing conditions (lanes 1, 2, 3, 4) or under nonreducing conditions (lane 5). The proteins were then transferred to membrane and blotted with anti-FLAG M2 antibody.

View larger version (14K): [in this window] [in a new window]   Figure 2. A, Soluble leptin receptor binding. One milliliter of cell culture medium from COS7 cells transfected with the extracellular domain of the human leptin receptor (sR) or mock transfected cells (Ct) was incubated with 125I-leptin either in the absence (closed columns) or presence (open columns) of 200 nM of unlabeled mouse leptin, and the receptor-leptin complex was precipitated with anti-FLAG M2 affinity gel. The precipitated 125I-leptin was then counted. Data represent the mean ± SEM of three determinations within a representative experiment that was reproduced at least ten times with comparable results. B, Competition of 125I-leptin binding to the soluble leptin receptor by human or mouse leptin. Medium (0.2 ml) from leptin sR cDNA transfected COS7 cell supernatant was diluted to 1 ml with TBST containing 1 mM of Mg2+, and the soluble leptin receptor-leptin binding assay was performed as described. Different concentrations of either mouse or human unlabeled leptin were added to the binding mixture for competition studies.

Molecular mass determination.
The molecular mass of the ¹²⁵I-leptin/soluble leptin receptor complex was determined in supernatants of the soluble leptin receptor cDNA transfected COS7 cells using a radioligand/receptor affinity cross-linking method. Two specific bands that migrate at molecular masses of approximately 130–150 kDa (major band) and approximately 300 kDa (minor band) represent the cross-linked complex of ¹²⁵I-leptin and the soluble leptin receptor (lane 2 in Fig. 3A). The leptin binding specificity of these bands was demonstrated by their absence in the supernatant of mock transfected cells (lane 1 in Fig. 3A) and the fact that they were competed by 200 nM of unlabeled mouse leptin (lane 5 in Fig. 3A). A band with molecular mass of approximately 33 kDa appeared in every lane, presumably representing the dimer of ¹²⁵I-leptin.

The recombinant soluble leptin receptor coding sequence predicts a 92-kDa protein, but the soluble receptor/¹²⁵I-leptin complex has a major band with a molecular mass of approximately 130–150 kDa and a minor band with a molecular mass of approximately 300 kDa (Fig. 3A). The higher than predicted molecular mass of the complexes may result from either glycosylation of the soluble leptin receptor or the requirement for more than one subunit to participate in the receptor/ligand binding complex. Characterization of the molecular mass of the soluble leptin receptor in the absence of ligand (i.e., leptin) addresses the former hypothesis. Because a FLAG sequence was tagged to the C-terminus of the soluble leptin receptor, Western blot analysis using anti-FLAG antibody was used to detect the molecular mass of the soluble leptin receptor itself. The soluble leptin receptor was first affinity precipitated using a human leptin affinity gel, run on SDS-PAGE, transferred onto membrane, and blotted with anti-FLAG antibody. Our results indicated that the soluble leptin receptor migrates with a molecular mass of approximately 130 kDa both under reducing (lane 4 in Fig. 3B) and nonreducing (lane 5 in Fig. 3B) conditions; this band corresponds to the major band identified in the cross-linking studies. Further evidence that the soluble leptin receptor has a molecular mass of approximately 130 kDa was obtained with the affinity purified receptor using a human leptin-coupled affinity column. The purified protein was run on SDS-PAGE and Coomassie staining of the gel indicated that a single protein band with a molecular mass of approximately 130 kDa was purified (data not shown).

To further characterize the binding requirements and stoichiometry between leptin and the leptin soluble receptor, the medium of the COS7 cells transfected with the soluble receptor was incubated with ¹²⁵I-leptin, and the mixture was run through a gel filtration HPLC column to analyze the molecular mass of the binding complex under native conditions. Our results indicated that a single peak representing the soluble leptin receptor/¹²⁵I-leptin complex runs at a molecular mass of approximately 340 kDa (corresponding to the minor band in the cross-linking studies), whereas the free ¹²⁵I-leptin migrates close to a molecular mass of approximately 10–16 kDa (Fig. 4). The specificity of the 340-kDa peak was demonstrated by its absence in the presence of 200 nM unlabeled leptin (Fig. 4). In addition, the 340-kDa peak was absent when the medium from mock transfected COS7 cells was used in a parallel experiment (data not shown). These data suggest that the soluble leptin receptor binds leptin as a dimer.

View larger version (16K): [in this window] [in a new window]   Figure 4. Gel filtration analysis of the molecular weight of the leptin/soluble leptin receptor complex. Conditioned medium from COS7 cells transfected with the extracellular domain of the human leptin receptor (sR) was incubated with 125I-leptin at room temperature for 15 min either in the presence or absence of 200 nM of unlabeled mouse leptin as the competitor. After incubation, the mixture was loaded on a HPLC gel filtration column and eluted with buffer containing 0.1 M sodium phosphate, 0.1 M Na2SO4 and 0.05% sodium azide, pH 7.2. The sample was collected in fractions of 0.2 ml and counted on a -counter. The soluble leptin receptor/125I-leptin complex runs at a molecular mass of approximately 340 kDa, whereas unbound 125I-leptin migrates at a molecular mass of approximately 10–16 kDa.

View larger version (20K): [in this window] [in a new window]   Figure 5. Inhibition of cell membrane 125I-leptin binding by the soluble leptin receptor. COS7 cells transfected with the full length leptin receptor cDNA were seeded in 6-well tissue culture plates and were used for cell membrane binding with 125I-leptin in the absence or presence of approximately 5 nM of the purified soluble leptin receptor (sR) or 200 nM of unlabeled leptin. The results show the mean ± SEM of triplicate determinations.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Expression of the full-length human leptin receptor cDNA encoding the long cytoplasmic domain of leptin receptor in COS7 cells resulted in high affinity membrane binding of mouse and human leptin (K_i 200 pM) in the range reported in a previous study (4). Furthermore, the high affinity of leptin for the full length receptor is in keeping with the concentrations of ligand necessary for signal transduction (9, 10). JAK and STAT interaction motifs are predicted in the long cytoplasmic domain of the full length leptin receptor and recent studies have demonstrated that this form of the receptor activates STAT-3, STAT-5 and STAT-6 (9, 10).

Expression of the cDNA encoding the recombinant human soluble leptin receptor in COS7 cells demonstrated, for the first time, high affinity binding of leptin to this form of the receptor, which is present in the medium. The affinity and pharmacological characteristics of leptin for the putative soluble receptor were similar to the membrane binding characteristics of the full-length receptor. In addition to binding in the conditioned medium, COS7 cells transfected with the soluble receptor also displayed ¹²⁵I-leptin binding in the cell membrane, albeit at a significantly lower level than that seen with the full length receptor. These data suggest that a portion of secreted soluble leptin receptor was associated with cell membrane protein(s). Since the extracellular domain of leptin receptor consists of 15 pairs of cysteine residues, the possibility exists that the complexes may result from disulfide linkages. The SDS-PAGE profiles were comparable under reducing and nonreducing conditions, suggesting that the soluble receptor is not covalently linked to other proteins by disulfide linkages. Furthermore, these data suggest that only intramolecular disulfide bond formation probably exists in the functional leptin receptor. Because the leptin receptor is homologous to gp130, a common subunit of the LIF, IL-6, and CNTF receptors (8), it is not surprising that it may associate with other subunit(s) to form functional complexes necessary for signal transduction. The relevance of the membrane complex formation to enhance the local decoy function of the naturally occurring soluble leptin receptor (6) remains to be demonstrated.

The leptin receptor is homologous to the cytokine receptor superfamily where both transmembrane and soluble receptors exist (5, 6, 12). The soluble forms of cytokine receptors such as the type II IL-1 receptor as well as the IL-6 receptor have been postulated to be primarily derived by posttranslational processing and cleavage of the extracellular portion of the receptor (13, 14). Recent data also suggest the presence of an alternatively processed type II IL-1 receptor mRNA encoding a soluble receptor (15). To determine if posttranslational processing of the full-length leptin receptor may contribute to the soluble receptor pool, we measured ¹²⁵I-leptin binding in the conditioned medium of COS7 cells expressing the full length receptor. No detectable leptin binding activity was found in the supernatant indicating the absence of soluble receptor protein. These data suggest that the soluble leptin receptor is exclusively derived from the splice variant expressing the extracellular portion of the protein.

The coding sequence of the recombinant human soluble leptin receptor predicts a protein with a molecular mass of 92 kDa. Affinity cross-linking assays followed by SDS-PAGE demonstrated that the soluble receptor/¹²⁵I-leptin complex has a major band with a molecular mass of approximately 130–150 kDa and a minor band with a molecular mass of approximately 300 kDa. The higher than predicted molecular mass of the complexes may result from either glycosylation of the soluble leptin receptor or the requirement for more than one subunit to participate in the receptor/ligand binding complex. Three independent lines of evidence suggest that the 130- to 150-kDa band represents the leptin receptor rather than complexes with other proteins. First, the 130 kDa band is present when electrophoretically fractionated under both reducing and nonreducing conditions, suggesting the absence of disulfide complexes with other proteins. Second, Western blot analysis of an affinity purified FLAG-tagged version of the soluble receptor identified a single band with a molecular mass of approximately 130 kDa. Finally, when the affinity purified soluble receptor protein was run on SDS-PAGE, Coomassie Brilliant Blue staining of the gel indicated the presence of a single band of protein with a molecular mass of approximately 130 kDa. Overall, these data suggest that the molecular mass of the soluble leptin receptor is approximately 130 kDa and that the higher than predicted molecular weight is most likely a consequence of glycosylation of the protein. The extracellular portion of the leptin receptor protein predicts 20 potential N-linked glycosylation sites (4).

Receptors with homology to gp130 normally require more than one subunit to bind ligand with high affinity. To determine if the 300-kDa band in the cross-linking studies represented multiple subunits of the receptor and ligand, we further characterized the binding requirements and stoichiometry between leptin and the leptin soluble receptor. The medium containing the soluble receptor was incubated with ¹²⁵I-leptin, and the mixture was run through a gel filtration HPLC column to analyze the molecular mass of the complex under native conditions. Our results indicated that a single specific peak representing the soluble leptin receptor/¹²⁵I-leptin complex runs at a molecular mass of approximately 340 kDa. These data suggest that the soluble leptin receptor binds leptin as a dimer and further substantiates the multiple subunit requirement for high affinity binding for this member of the gp130 family of receptors.

The functional relevance of the naturally expressed soluble receptor in modulating the actions of endogenous leptin is at present unclear. It could serve as a decoy receptor to limit the actions of leptin. If the soluble receptor is present in blood, then measurement of immunoreactive levels of leptin may be misleading because they may not accurately reflect the bioactive levels. Immunoreactive levels of leptin are normal or elevated in obese individuals (16, 17, 18), suggesting that obesity is not a simple consequence of leptin deficiency as is seen in the genetically deficient ob/ob mouse (3) but rather a result of decreased biological activity of leptin. The reduced biological activity could result from a point mutation in the leptin receptor similar to that seen in the db/db mouse resulting in reduced expression of the long signaling isoform of the receptor (5, 6, 10, 11); however, the absence of the mutation in humans (16) argues against this possibility. Alternatively, elevated expression and secretion of the soluble leptin receptor under conditions of obesity may neutralize and reduce the biological effects of leptin to account for some aspects of obesity. The soluble leptin receptor may also subserve other functions more analogous to those seen for other members of the gp130 family such as the IL-6 receptor (14). Evidence for dimerization of the leptin/leptin receptor complex seen in the present study raises the possibility that leptin bound to the soluble receptor may associate with either the full-length leptin receptor or some other protein that may transduce a signal. Additional studies are necessary to fully elucidate the role of the soluble leptin receptor.

We have also demonstrated that the soluble leptin receptor binds leptin with high affinity and can inhibit binding to the membrane bound leptin receptor. Expression and purification of soluble leptin receptor provides a valuable tool to study the role of leptin under physiological and pathological conditions. The ability to functionally inactivate leptin has advantages over immunoneutralization with an antibody that may be directed at a portion of leptin that is not required for receptor binding. The functional inactivation of leptin using the soluble receptor can be controlled, resulting in a temporary knockdown that may have certain advantages over genetically deficient (e.g. ob and db mice) or knockout animals where compensatory mechanisms may be present.

In summary, we have expressed the soluble leptin receptor splice variant in COS7 cells and demonstrated high affinity binding of ¹²⁵I-leptin in the cell culture supernatant with pharmacological characteristics similar to the membrane binding of the full-length transmembrane receptor. Affinity cross-linking followed by SDS-PAGE, Western blot analysis, and affinity purification studies demonstrate that the soluble leptin receptor has a molecular mass (130 kDa) higher than that predicted from the amino acid sequence, suggesting that it is glycosylated. In addition, we provided evidence that the soluble leptin receptor binds leptin as a dimer and may have utility as a functional tool for determining the role of endogenous leptin. The functional relevance of the naturally expressed soluble receptor as well as its regulation and physiological role in limiting the actions of leptin await future studies.

	Acknowledgments

 
We thank Dr. Dimitri Grigoriadis for his help in analyzing the radioligand binding data.

Received February 10, 1997.

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