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Endocrinology Vol. 142, No. 10 4389-4393
Copyright © 2001 by The Endocrine Society

ARTICLES

Generation of Human Soluble Leptin Receptor by Proteolytic Cleavage of Membrane-Anchored Receptors

M. Maamra, M. Bidlingmaier, M.-C. Postel-Vinay, Z. Wu, C. J. Strasburger and R. J. M. Ross

Division of Clinical Sciences (M.M., R.J.M.R.) Sheffield University, Sheffield S5 7AU, United Kingdom; Medizinische Klinik-Innenstadt (M.B., Z.W., C.J.S.), Muenchen 80336, Germany; and Institute National de la Santé et de la Recherche Médicale Unité 344 (M.-C.P.-V.), Endocrinologie Moleculaire, Faculte de Medecine Necker, Paris 75730 Cedex 15, France

Address all correspondence and requests for reprints to: Prof. R. J. M. Ross, Clinical Sciences, Northern General Hospital, Sheffield S5 7AU, United Kingdom. E-mail: r.j.ross{at}sheffield.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The leptin receptor (ObR) exists in multiple isoforms. In rodents, a soluble isoform is generated by alternative splicing; but in humans, there is no mRNA encoding soluble receptor (leptin binding protein). We investigated the hypothesis that human leptin binding protein can be generated by proteolytic cleavage of membrane-anchored leptin receptors (ObRb and ObRa). Leptin binding protein of similar size to that previously detected in human serum was detected by HPLC in medium of cells transfected with ObRa. ObRa exhibited higher expression at the cell surface than ObRb and generated greater levels of leptin binding protein. Ligand-mediated immunofunctional and immunofluorometric assays revealed that the leptin binding protein in medium bound both leptin and an ObR-specific antibody and that the level of leptin binding protein correlated with receptor expression at the cell surface. Phorbol 12-myristate-13-acetate and N-ethylmaleimide increased the accumulation of leptin binding protein, an indication that the production of leptin binding protein was up-regulated by PKC and sulfhydryl group activation. The protease inhibitors, TNF{alpha} protease inhibitor 1 and Immunex compound 2, could inhibit the production of leptin binding protein, indicating that the enzyme responsible for leptin binding protein cleavage belongs to the metalloprotease family. In conclusion, human leptin binding protein is generated by proteolytic cleavage of membrane-anchored leptin receptor by a metalloprotease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LEPTIN, THE PRODUCT of the Lep gene, is a hormone derived from adipocytes, and genetic deficiency results in extreme obesity. It is evident that leptin has multiple biological actions on feeding, metabolism, the neuroendocrine axis, and immune system (1). Leptin acts through a class 1 cytokine receptor (ObR), the product of a single gene, and mutations in this gene result in a similar phenotype to leptin deficiency (2). The leptin receptor is expressed in multiple isoforms. At least five splice variants of the leptin receptor have been detected in mice (3). In humans, at least four variants, which differ in the length of their cytoplasmic domains, have been reported (4). The predominant isoform is the short receptor (ObRa), which is expressed in most tissues, and at high level in the choroid plexus and brain microvessels (5, 6). In contrast, the full-length receptor (ObRb), although detectable by RT-PCR in many tissues, is only highly expressed in the hypothalamus (7), where expression exceeds that of the short form (8). ObRb is assumed to be responsible for transmitting the majority of biological signals mediating the central effects of leptin on appetite and energy expenditure, because deficient expression results in an obese diabetic phenotype (3). The function of the short form of the receptor, ObRa, has yet to be defined, although it has been suggested that it may act to transport leptin into the CSF (9).

A soluble receptor or leptin binding protein (LBP), circulating in complex with leptin, has been described in both the human and rodent. In humans, LBP can be precipitated by a leptin receptor antibody (10). In the pregnant mouse, LBP has been sequenced and confirmed as the extracellular domain of the leptin receptor (11). These results are consistent with observations made for other members of the class I cytokine family of receptors, a number of which produce soluble receptors that represent the extracellular domain of the receptor (12). The functional significance of soluble receptors is yet to be defined. They could act either as biomodulators of receptor signaling by competing for ligand binding or as transporters of cytokines in serum, reducing their degradation and clearance (13, 14). LBP is thought, in lean subjects, to play a role in restricting the availability of leptin to its hypothalamic receptor and thus inhibiting its effect on food intake and energy metabolism (10).

An mRNA encoding a putative soluble ObR has been detected in mice (3), but this has not been detected in humans (15). One of the most studied soluble cytokine receptors is the GH binding protein (GHBP). In rodents, alternative mRNA splicing at exon 8 encodes the soluble receptor. In contrast, human GHBP is generated by shedding of membraneanchored isoforms. This shedding is catalyzed by the metalloprotease A disintegrin and metalloprotease (ADAM) 17 (16) and has been shown to be enhanced upon PKC activation and sulfhydryl group alkylation (17). We have investigated the possibility that human LBP could be generated by shedding of the membraneanchored receptors, ObRa and ObRb.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The long form (ObRb) and short form (ObRa) leptin receptor cDNA in PCEP4 were a kind gift from A. Welcher (Amgen, Inc., Thousand Oaks, CA).

Recombinant human leptin was obtained from R&D systems (Abingdon, UK) and radiolabeled human leptin from Biogenesis (Poole, UK). N-ethylmaleimide (NEM) and phorbol 12-myristate-13 acetate (PMA) were purchased from Sigma-Aldrich Corp. (Poole, UK). We used two hydroxyamid acid-based inhibitors, TNF{alpha} protease inhibitor 1 (TAPI-1) and modified metalloprotease inhibitor Immunex compound 2 (IC2; Immunex Corp., Seattle, WA), which are general inhibitors of mammalians shedding metalloproteases (18, 19). The small TAPI-1 inhibitor was purchased from Peptides International, Inc. (Louisville, KY). The calcium phosphate transfection kit and tissue culture solutions were purchased from Gibco Ltd. (Paisley, UK).

Cell culture
293 cells (human kidney embryonal cell line) were grown in DMEM Nut F12 medium supplemented with 10% FCS, 100 IU and 100 µg/ml penicillin/streptomycin, and 2 nM L-glutamine. Cells were plated 24 h before transfection.

Binding studies
For cell surface binding, 293 cells were transiently transfected with leptin receptor cDNA using the calcium phosphate method. The cells were incubated in serum free medium for 16 h, and the medium was then collected for soluble binding protein measurement. Cells were then washed in PBS containing 1% BSA and incubated with I125-labeled leptin (50,000 cpm/ml) for 3 h at room temperature in the presence or absence of an excess of cold leptin (3 µg/ml). Cells were subsequently washed in PBS and solubilized in 1 N NaOH for counting radiation.

HPLC assay
LBP was assayed in culture media using gel filtration. The medium from ObRa-transfected cells was concentrated 10 times by freeze drying and incubated overnight at 4 C with 105 cpm I125 leptin in PBS-0.1% BSA. A parallel incubation was carried out in the presence of an excess of cold leptin. The samples were then analyzed by gel filtration, using a liquid chromatograph (Waters Corp., Milford, MA) equipped with a sample injector (model U6K) fitted with a 250-µl loop and an analytic HPLC Protein Pak 300 SW (Waters Corp.; 0.75 x 30 cm). Elution was performed automatically using a degassed buffer (0.1 M Na2SO4, 0.1 M K2HPO4, pH 7), pumped at a rate of 0.5 ml/min. Radioactivity was recorded on line using a Berthold LB2040 {gamma}-detector (Berthold, Elancourt, France) connected to a Compaq computer.

Soluble LBP assays [ligand-mediated immunofunctional assays (LIFA) and immunofluorometric assays (IFMA)]
Medium of 293 transfected with the leptin receptors was collected after overnight incubation and concentrated 25-fold using the Centripep YM10 devices (Millipore Corp. UK Ltd., Watford, UK). Biologically active soluble leptin binding protein (LBP) was measured by LIFA and IFMA (20), and cell surface binding was performed on the same cells. In brief, LIFA involves capture of LBP from samples by an anti-LBP monoclonal antibody immobilized on microtiter plates, saturation of all leptin binding sites of the bound LBP by recombinant leptin, and detection of bound leptin by a monoclonal antileptin antibody. Both the anti-LBP and the antileptin monoclonal antibodies bind to an epitope distant from the hormone-receptor interaction site. The LIFA is expected to reflect the biologically active LBP, because only those molecules that bind leptin are translated into a signal. In the sandwich-type assay (IFMA), the protein was captured by the same anti-LBP antibody as in the LIFA; but for detection, another labeled antibody against the extracellular domain of the receptor was used. In both LIFA and IFMA, concentrated serum-free medium was used as a matrix of the assay, and recombinant human leptin receptor ectodomain was used as a standard.

Effect of NEM and PMA on LBP production
ObR-transfected cells were incubated at 37 C in serum-free medium supplemented with 5 nM NEM, 1 µg/ml PMA, or vehicle dimethylsulfoxide (DMSO) for 90 min. The media were concentrated and analyzed by LIFA.

For the metalloprotease experiments, cells were preincubated for 15 min with 30 µM metalloprotease inhibitor TAPI1 or 45 µM of IC2, or vehicle, before treatment with DMSO, PMA, or NEM. The media was then concentrated and analyzed by LIFA.

Statistics
For analysis of binding data and LIFA, ANOVA with post hoc Bonferroni analysis was used, and the level of significance was accepted as P < 0.05. LIFA and IFMA results were compared by regression analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Binding studies
We compared binding of iodinated leptin at the cell surface of cells transfected with ObRa, ObRb, or control salmon sperm DNA. Transient transfection studies were performed in 293 cells. Transfection of the leptin receptor lead to significant binding activity of leptin to the cells and cells transfected with ObRa showed significantly greater binding of iodinated leptin compared with cells expressing ObRb, P < 0.01 (Fig. 1AGo). In simultaneous experiments studying signaling, ObRa and ObRb were cotransfected with a ß-galactosidase expression vector. In these experiments, transfection efficiency for ObRa and ObRb were equivalent (data not shown).



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  		Figure 1. Percentage specific binding of iodinated leptin to 293 cells in monolayer transiently transfected with leptin receptor and LBP concentration in the medium. A, 293 cells transiently transfected with ObRa, ObRb, or control salmon sperm were incubated overnight in starvation medium, which was then removed before addition of 125I-leptin plus or minus an excess of cold leptin. The results of cell monolayer binding are expressed as percentage specific binding. B, The media from the same cells (overnight incubation) were concentrated, and the LBP concentration (ng/ml) was measured by LIFA. *, P < 0.01, ObRa vs. ObRb.

 
LBP
We investigated whether a LBP could be generated from membrane-anchored receptor. Medium from 293 cells transiently transfected with ObRa, ObRb, or control salmon sperm was concentrated. LBP was measured by HPLC, LIFA, and IFMA. HPLC on concentrated medium from cells transfected with ObRa revealed 9.0% specific binding of iodinated leptin with an elution time similar to the leptin binding activity we have previously reported in normal human serum (21) (Fig. 2Go). The results for the LIFA assays are given in Fig. 1BGo. LBP was detectable in concentrated medium from cells transfected with ObRb and ObRa. The level of LBP correlated with specific binding of leptin on the cell surface (r = 0.80, P < 0.05), and the LIFA and IFMA levels correlated (r = 0.96, P < 0.001), confirming the molecular identity between the leptin binding activity detected in the medium and the soluble leptin receptor (Fig. 3Go).



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  		Figure 2. Elution profile of 125I leptin incubated with concentrated culture medium of 293 cells transfected with ObRa. Medium from ObRa-transfected cells was concentrated, and incubation was performed with 125I leptin plus (thin line) or minus (thick line) an excess cold leptin, and the samples were analyzed by HPLC gel filtration column. The chromatograph shows a specific peak for LBP with 9% binding of tracer.

 


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  		Figure 3. Comparison of LBP concentration by LIFA and IFMA. To determine whether all the extracellular domain of the leptin receptor present in the medium is able to bind leptin, several samples were analyzed by IFMA (uses antibody specific to the receptor) and in parallel by LIFA (uses ligand, leptin). IFMA concentrations of LBP were plotted against the corresponding LIFA measurement, and this confirmed a close correlation between the two assays (r = 0.96, P < 0.001).

 
Effect of PMA and NEM on LBP production
We investigated whether PMA and NEM could regulate LBP production. For these experiments, medium from 293 cells transfected with the ObRa leptin receptor was collected after 90 min incubation with PMA, NEM, or vehicle (DMSO). The phorbol ester PMA was used to activate PKC, and NEM was used for alkylation of sulfhydryl groups. Both PMA and NEM promoted the appearance of soluble receptor in the medium of cells transfected with membrane-anchored receptors (Fig. 4Go). In DMSO-only treated cells, the levels of LBP detected was very low because of the short incubation period. PMA treatment significantly increased the levels of LBP in ObRa-transfected cells by about 2-fold (2.16 ± 0.38, mean ± SEM fold induction) and NEM by about 30-fold (31.36 ± 4.12, mean ± SEM fold induction).



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  		Figure 4. Effect of NEM and PMA on LBP production. A total of 293 cells transfected with leptin receptor (ObRa) were incubated for 90 min (not overnight as in Fig. 1BGo) in serum-free medium supplemented with DMSO (vehicle), 1 µg/ml PMA, or 5 nM NEM. The media were concentrated, and LBP was measured by LIFA and IFMA.

 
Effect of metalloprotease inhibitors on basal and enhanced LBP production
We investigated whether two metalloproteases inhibitors, IC2 and the small TAPI1 inhibitor could inhibit basal, PMA-, or NEM-induced LBP production (Fig. 5Go). For these experiments, cells transfected with ObRa were preincubated for 15 min with the metalloprotease inhibitors or vehicle (DMSO), before the incubation with PMA, NEM, or vehicle. The LBP levels in the conditioned media were assayed by LIFA. The basal levels of LBP were low because of the short period of incubation. Preincubation with the metalloprotease inhibitors IC2 or TAPI1 inhibited LBP production in the conditioned medium induced by PMA and NEM.



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  		Figure 5. Effect of a metalloprotease inhibitor on PMA- and NEM-induced LBP production. 293 cells transfected with ObRa were treated in serum-free medium for 15 min with metalloprotease inhibitors (IC2 or TAPI1) before adding PMA (1 µg/ml) or NEM (5 mM) or vehicle (DMSO) for 90 min. The medium was then collected and concentrated, and LBP was measured by LIFA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated that generation of the human LBP may occur by proteolytic cleavage of membrane-bound leptin receptor. Expression of the membrane-anchored receptor isoforms in 293 cells resulted in the appearance in the medium of LBP, as assessed by HPLC. Thus, cells could release, in the medium, a molecule bioactive for the binding of leptin. The detected LBP was similar to that we have previously reported for leptin binding activity in human serum (21). The nature of the ligand binding activity was further characterized by 2 separate immunoassays, which gave similar results. The IFMA (antibody recognition of LBP) and the LIFA (measurement of leptin binding) assays confirmed that the leptin binding activity that we detected in the cells corresponds to the extracellular domain of the leptin receptor. Experiments using inhibitors demonstrated that cleavage of LBP was through a metalloprotease.

The leptin receptor is homologous to the cytokine receptor family. A feature of cytokine receptors, including GH and IL-6 receptors, is the production of soluble receptor, which circulates as binding protein. Two distinct mechanisms for soluble receptor production, not mutually exclusive, have been described. The soluble IL-6 and IL-1 receptors may be generated both by proteolytic cleavage and alternative splicing (22, 23, 24). Similarly, the soluble GH receptor (GHR) is generated by alternative splicing in rodents (25), proteolysis in humans (17), and both mechanisms in the monkey (26). In rodent tissues, a splice variant encoding LBP has been characterized (3), but this does not occur in the human, where the gene lacks a polyadenylation signal in the potential 3' terminal exon (15). Our findings do not exclude the possibility that an uncharacterized alternative spliced mRNA could contribute to the pool of human LBP in vivo, but our results do demonstrate that human LBP may be generated by shedding of both the long and short forms of the leptin receptor, and that LBP levels are related to receptor expression. Changes in levels of LBP could reflect changes in expression of ObR receptor, as seen with the GHR (27). Previously, Liu et al. (28) reported that no soluble receptor could be detected by radioligand binding and immunoprecipitation in the medium of COS7 cells transfected with ObRb. However, in our system, we could detect LBP in the medium 293 cells transfected with ObRb by LIFA and IFMA. The difference is likely to be attributable to the higher sensitivity of the IFMA and LIFA. ObRa consistently shows a higher level of expression at the cell surface, as previously reported. This is not related to a difference in affinity for leptin (29). We now show that ObRa also generates more LBP. The level of LBP detected in the medium from cells expressing the ObR was closely related to the level of expression of the receptor at the cell surface. This suggests that the availability of membrane receptor may, in part, determine the production of LBP. Because ObRa is expressed at a higher level than ObRb and is the predominant receptor isoform in peripheral tissues, it is likely that ObRa makes a significant contribution to circulating LBP. The soluble receptor has been shown, in vitro, to be able to compete for binding of leptin to ObRb (28). Thus, the differential expression of the leptin receptor isoforms ObRa and ObRb may determine the level of circulating LBP and the signaling profile activated in individual cells.

LBP has been measured in physiological and clinical situations. Circulating levels of LBP are low at birth, high in the prepubertal years, then fall (through puberty) to remain stable during adult life (21). LBP levels are lower in obese patients, compared with lean (30), whereas leptin levels are higher in the obese, suggesting that obese individuals are resistant to free leptin. In adults, transport of free leptin into the CSF seems to be saturated at low concentrations of serum free leptin, whereas bound leptin increases in parallel to serum concentrations over the whole physiological ranges (31), and the ratio of leptin in cerebrospinal fluid to serum fluid is decreased in obesity (32). Bound leptin levels are higher in pregnant women during the third semester of pregnancy, whereas free leptin levels are similar (33). It has been suggested that bound leptin may regulate maternal metabolism in the pregnant woman. Thus, LBP may act to carry leptin as well as modulate metabolism.

Several cell surface receptors have been shown to undergo proteolytic cleavage (12). The protease involved in the cleavage of TNF has been identified and belongs to the ADAM (a disintegrin and metalloprotease) family (34). For most cytokine receptors, the enzyme responsible for proteolysis has not yet been identified, but metalloprotease inhibitors have been shown to inhibit shedding of many receptors. The mechanism for proteolytic cleavage of GHR, another cytokine receptor, have now been well characterized. GHR shedding can be promoted by PKC activation and sulfhydryl alkylation and be inhibited by the metalloprotease inhibitor IC3 (17). Shedding of TNF and IL6 soluble receptors have also been shown to be promoted by PMA. In our experiments, PMA induced a 2-fold increase in LBP, similar to that reported for GHBP (35), suggesting that PKC could activate shedding. The sulhydryl alkylating agent NEM led to an even-higher increase in LBP, again similar to that observed with GHBP (17). The metalloprotease ADAM17 is an enzyme required for PMA-induced GHBP shedding (16). So, our findings raised the possibility that human LBP could, in vivo, be generated by shedding that involves a metalloprotease of the same family. We confirmed this by demonstrating that LBP production could be inhibited by the use of two metalloprotease inhibitors, IC2 and TAPI1.

Cleavage of cytokine receptors by shedding is largely accepted as a mechanism for the production of soluble receptor; however, the precise location of the event is not yet established. It is not clear whether proteolysis occurs intracellularly, at the cell surface or after internalization. There is good evidence for the TNF receptor, that shedding occurs intracellularly (36). For the GHR, it seems that proteolysis occurs before internalization, as a truncated receptor that fails to internalize generates high levels of GHBP (37). NEM is an internalization inhibitor (38), and incubation of leptin receptor-expressing cells with NEM resulted in high levels of LBP, suggesting that shedding of the leptin receptor occurs at the membrane. We do not exclude a specific effect of NEM on the leptin receptor shedding, because at least one free cysteine in the extracellular domain of ObR can be alkylated by NEM (39).

In summary, we have shown that cell surface human leptin receptor can be shed by a metalloprotease to produce LBP and that ObRa generates a larger proportion of LBP than ObRb. This shedding most likely occurs at the cell surface and is activated by PKC. A possible role for the short receptor isoform, ObRa, could be to provide soluble receptor to act as carrier of leptin from its peripheral site of production to its central site of neuroactions.


   Footnotes
 
We are grateful for support from Serono Laboratories, Inc. M. Maamra is supported by a Ph.D. studentship from the Society for Endocrinology.

Abbreviations: ADAM, A disintegrin and metalloprotease; DMSO, dimethylsulfoxide; GHBP, GH binding protein; GHR, GH receptor; IC2, Immunex compound 2; IFMA, immunofluorometric assays; LBP, leptin binding protein; LIFA, ligand-mediated immunofunctional assays; NEM, N-ethylmaleimide; PMA, phorbol 12-myristate-13-acetate; TAPI-1, TNF{alpha} protease inhibitor 1.

Received April 13, 2001.

Accepted for publication June 27, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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