Flanders Interuniversity Institute for Biotechnology, Department of Medical Protein Research (VIB09), Ghent University, Faculty of Medicine and Health Sciences, Baertsoenkaai 3, 9000 Ghent, Belgium
Author for correspondence (e-mail: jan.tavernier{at}ugent.be)
Accepted 16 March 2005
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Summary |
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Key words: Cytokines, Leptin, Leptin receptor, Mutagenesis, Molecular modelling
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Introduction |
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The leptin receptor (LR), encoded by the db gene (Tartaglia et al., 1995), is a member of the class I cytokine receptor family. It has no intrinsic kinase activity and depends on cytoplasmic-associated Janus kinase 2 (JAK2) for signalling. The extracellular part of the receptor contains several structural domains (Fig. 1A). Amino-terminally, there is a cytokine receptor homology (CRH) module, termed CRH1, which is formed by two sub-domains that have a fibronectin type III (FNIII) fold (residues 62-178 and 235-328 in the human leptin receptor). Residues 329-427 (all numbering refers to the human leptin receptor) adopt an immunoglobulin (Ig)-like fold. The next two FNIII-like sub-domains (residues 428-535 and 536-635, respectively) form a second CRH module, called CRH2. Membrane-proximally, there are two more FNIII domains. Using several approaches, CRH2 was identified as the main high-affinity binding site for leptin on the LR (Fong et al., 1998
; Sandowski et al., 2002
; Zabeau et al., 2004
). The precise role of CRH1 remains elusive, but the Ig-like and the FN-III domains are critically involved in LR activation (Zabeau et al., 2004
; Fong et al., 1998
).
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Materials and Methods |
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The sequences of leptin receptor CRH2 domains were aligned using t_coffee. Subsequently, these two alignments were used to create two profile libraries and aligned with each other using t_coffee. This gives an optimal alignment of the LR CRH2 domain with the CRH domains of other long chain receptors.
Using this alignment, molecular models were built for the mouse LR CRH2, using the G-CSF receptor structure (1cd9) as template. According to our alignment, the two sequences are 23% identical. One model was built using `jackal' (Xiang et al., 2002), 10 models were built using `moe'.
Eleven leptin/CRH2 complex models were built using one dimer of the G-CSF/G-CSF receptor complex (1cd9) as template: the model for mouse leptin was superposed on G-CSF as described previously (Peelman et al., 2004b), and the 11 LR CRH2 models were superposed on the G-CSF receptor CRH. The complex interface was energy minimized using MULTIDOCK (Jackson et al., 1998
). The interface properties of our models were analysed using the Protein-Protein interaction server (V1.5) (http://www.biochem.ucl.ac.uk/bsm/PP/server/).
Vectors and construction of mutants
The pMET7-mLRCRH2-His6 vector allows the expression of a fusion protein consisting of the CRH2 domain of the mouse LR followed by a hexahistidine tag (Zabeau et al., 2004). pMET7-SIgK-HA-mLep allows expression of HA-tagged leptin mutants. Mutations in leptin and the CRH2 domain were generated using the Quikchange site-directed mutagenesis procedure (Stratagene). The pMET7-mLRlo vector allows expression of the mouse LR with an additional C-terminal myc tag. pMET7-mLRlo_BglII contains an extra unique BglII site at position 1903. CRH2 mutations in pMET7-mLRCRH2-His6 were transferred as BbvCI-BglII fragments to the pMET7-mLRlo_BglII vector. All constructs in this work were verified by DNA sequence analysis. The sequences of oligonucleotide primers used for mutagenesis are included as supplementary material.
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Production of leptin mutants in Cos-1 cells
HA-tagged leptin mutants were expressed in Cos-1 cells, using the pMET7-SIgK-HA-mLep plasmids and polyethyleneimine transfection, and concentrated as described previously (Peelman et al., 2004b). Expression was checked by anti-HA western blot analysis and quantified using an anti-mouse leptin ELISA kit (R&D systems) (Peelman et al., 2004b
).
STAT3 reporter assays
Hek293T cells were cultured as described for the Cos-1 cells, and transfected with pMET7-mLRlo or pMET7-mLRlo_BglII using a standard calcium phosphate transfection procedure. The day after transfection, cells were resuspended with cell dissociation buffer (Invitrogen). Some of the cells were used for a luciferase assay: these were seeded in a black 96-well plate (Nunc) and stimulated with different concentrations of mouse leptin (R&D Systems) or were left untreated. The remainder of the cells were cultured overnight in a 6-well plate, resuspended with cell dissociation buffer and used for FACS analysis (see below).
Luciferase assays were performed as described previously (Eyckerman et al., 2000). In brief, Hek293T cells were co-transfected with the pXP2d2-rPAP1 plasmid and the pMET7-mLRlo plasmid containing the LR mutations. The pXP2d2-rPAP1 plasmid contains the luciferase gene under control of the STAT3-inducible rat pancreatitis-associated protein 1 promoter. Leptin-induced luciferase activity was measured by chemiluminescence. Cells were lysed in 50 µl of lysis buffer (25 mM Tris, pH 7.8, 2 mM EDTA, 2 mM dithiothreitol, 10% glycerol, 1% Triton X-100) for 10 minutes, then 35 µl of luciferase substrate buffer were added (20 mM Tricine, 1.07 mM (MgCO3)4Mg(OH)2.5H2O, 2.67 mM MgSO4.7H2O, 0.1 mM EDTA; 33.3 mM dithiothreitol; 270 mM coenzyme A; 470 mM luciferin; 530 mM ATP; final pH 7.8) Luciferase activity was measured in a Topcount Chemiluminescence counter (Packard). Data were fitted to a hyperbola, or, if more appropriate, a first order polynomial, using GraphPad Prism 2.0 software, to determine the median effective concentration (EC50) values.
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Solid plate leptin-SEAP binding assay
F96 Maxisorp white microwell plates (Nunc) were coated overnight at 4°C with 0.25 µg/ml penta-His antibody (Qiagen). After washing with PBS-T (phosphate buffered saline containing 0.1% Tween 20), plates were blocked at room temperature for 2 hours with PBS containing 0.1% casein. Plates were washed with PBS-T and supernatants containing the CRH2 domain of the LR (or mutants thereof) were added and incubated overnight at 4°C, after which the plates were washed again with PBS-T. An appropriate dilution of a leptin-SEAP (secreted alkaline phosphatase) fusion construct (see Tartaglia et al., 1995) was added, and after 2 hours incubation at room temperature and a final washing step, plates were measured in a Topcount chemiluminescence counter (Packard) using a SEAP assay (Phospha Light gene assay system, Tropix). This was tested for different concentrations of leptin-SEAP and data were fitted to a hyperbola using GraphPad Prism 2.0 and the Kd values were calculated.
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ELISA
The CRH2 mutant domains were quantified using ELISA. Clear Maxisorp 96-well plates (Nunc) were directly coated overnight with supernatants containing the His-tagged CRH2 domain, washed with PBS-T and subsequently blocked with 1% bovine serum albumin (fraction V, Sigma-Aldrich) plus 5% sucrose in PBS. After washing with PBS-T, plates were incubated with 0.2 µg/ml of penta-His antibody (Qiagen) in 1% bovine serum albumin in PBS and incubated for another 2 hours. As secondary antibody, anti-mouse IgG conjugated to HRP (Amersham Bioscience, NA931V) was used (1:2000, 1 hour). 100 µl TMB 2-component peroxidase substrate solution (KPL) was added, and after sufficient colouring, the reaction was stopped by addition of 1 M H3PO4. Optical densities were measured in a plate reader spectrophotometer (Multiskan EX, Thermo Labsystems, Waltham, MA) at a wavelength of 450 nm. A dilution series of the wild type was used as a standard and could be fitted to a four-parameter logistic curve using GraphPad Prism (R2=0.99). The equation of this fit was used to calculate the relative amount of CRH2.
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Results |
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Alanine scanning of the LR CRH2: effect on signal transduction
We mutated each of the 16 selected residues separately to an alanine residue, both in the full size mouse LR and in a construct containing the CRH2 domain fused to a polyhistidine tag. We first checked the effect of these mutations on leptin signalling using the rat-PAP1-luciferase reporter assay. The effect of the mutations on LR signalling is shown in Fig. 2.
While most of the mutations seem not to have a drastic effect on signalling, six mutants show clear and reproducible lower signalling capacity, namely I501A, F502A, L503A, L504A, S505A and D615A (Fig. 2). This lower signalling capability is not due to expression levels, since all these mutants are expressed at the plasma membrane to levels equal to or higher than that of the wild type when tested by FACS analysis (see supplementary material). These six mutants appear to be less sensitive to leptin, and the generated signal is lower than that of the other mutants. The I501A, F502A, L503A, L504A and S505A mutations are conspicuous as having the most drastic effect, and have increased EC50 values (33.5-55.0 nM), when compared to the wild-type receptor (6.44 nM). The effect of the D615A mutation (EC50=24.1 nM) is less severe than the other five. It has to be stressed, though, that even this effect is clear and reproducible.
Alanine scanning of the LR CRH2 domain: effect on leptin binding
All 16 mutations were also introduced in a plasmid containing the CRH2 domain fused to a poly-histidine tag. Supernatants from Cos-1 cells transfected with these plasmids were incubated in anti-His5 antibody-coated plates, and a leptin-SEAP binding assay was performed on these plates. To this end, a fusion protein of mouse leptin coupled to secreted alkaline phosphatase (leptin-SEAP) was added and incubated. The resulting phosphatase activity is a measure for binding of leptin to the CRH2 domain. Most mutations showed no significant effect on Kd or maximal phosphatase activity, except those that also affect signalling, strongly indicating that the reduced signalling is at least in part due to reduced binding (Fig. 3).
Identification of a hydrophobic loop in CRH2 involved in leptin interaction
It is striking that five of the mutants that affect leptin binding to CRH2 and LR signalling are successive residues of the ß5-ß6 loop, which is predicted to be a central part of the leptin-CRH2 interface. The first four residues of this loop (IFLL) are very hydrophobic. Therefore, we postulated that the binding of leptin to its receptor is based on hydrophobic interactions. To check this hypothesis and to refine our data, we mutated the residues 501-504 to more hydrophilic serine residues, both alone and in combination. The resulting mutants, I501S, F502S, L503S, L504S, I501S/F502S, F502S/L503S and L503S/L504S were all tested for their effect on LR signalling using the rat-PAP1-luciferase reporter assay; results are shown in Fig. 4. All LR mutants are expressed at the plasma membrane at levels at least equal to that of the wild type, when tested by FACS analysis. The F502S and L503S mutants do not differ much in signalling capacity from their alanine-mutated counterparts, in contrast to the I501S and L504S mutations, which show a more severe effect. However, in the case of I501S, background signalling is significantly increased, so the fold induction (ratio of stimulated versus non-stimulated cells) is lower, but the signalling capacity is not as severely affected. A LR carrying the L504S mutation is almost completely deficient in signalling: even with 300 ng/ml leptin, only very weak signalling is observed. All double mutants show no apparent reporter induction upon stimulation with leptin.
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Discussion |
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We developed molecular models to predict the interactions between leptin and CRH2, and tested the proposed models by mutagenesis studies. We were able to provide evidence for the important role of six residues located in the CRH2 domain in the leptin/LR interaction, namely I501, F502, L503, L504, S505, and to a lesser extent, D615. Mutations of these residues in CRH2 lead to decreased leptin binding and decreased signalling.
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The leptin molecule shows a hydrophobic cleft between helix A and C, consisting of residues L13, L86, L89 and F92. In our models, hydrophobic interactions of L13 and L86 in leptin with L504 in CRH2 form the centre of the leptin/CRH2 interface (Fig. 9). Mutations of L86 in leptin have a major impact on the binding to CRH2 and on receptor activation. Hydrophobic cleft residues L89 and F92 are not part of the predicted model interface (Fig. 6C), and mutation of these residues does not affect CRH2 binding or LR signalling, arguing against a major role of these residues in interactions with CRH2. Fig. 10A shows the model structure of CRH2, with indication of the residues that become buried in the leptin/CRH2 interface. I501, F502, L503, L504, S505 are all part of the interface (Fig. 10B) and become buried upon leptin/CRH2 binding. These five residues account for 40.5% of the solvent accessible surface area (ASA) of CRH2 that becomes buried upon interaction with leptin. L504 alone accounts for 18.5% of the total buried ASA. Desolvation of the hydrophobic residues in the ß5-ß6 loop might play an important role in the high affinity binding of leptin.
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Five possible salt bridges are formed in our model complexes, as shown in Table 1. The K15S, D9ST12Q and N82SD85S mutations in leptin decrease the affinity for CRH2 (Peelman et al., 2004b). The K15S, D9S and D85S mutations break possible salt bridges with E563, R613 and R466, respectively, in CRH2. However, mutation of E563 or R613 in CRH2 to alanine does not have a drastic effect on the affinity for leptin. The D615A mutation in CRH2 breaks a predicted salt bridge with K5 in leptin and has a pronounced negative effect on leptin binding and receptor signalling. Structural and functional analysis of binding site I in growth hormone/receptor complex by mutagenesis revealed that in this system, charged headgroups contribute to specificity, rather than affinity (Clackson et al., 1998
). In cases where charged side chains do contribute to the affinity, this contribution is largely due to Van der Waals interactions of the aliphatic part of the side chain (Clackson et al., 1998
). This might explain why some mutations of the charged side chains predicted to form salt bridges have only minor effects on affinity.
We compared our model complex with the reported CRH/long chain 4-helix bundle cytokine complexes, by superposing the CRH domains. L504 in the LR CRH2 superposes with a hydrophobic residue, involved in similar hydrophobic interactions in all the examined CRH2/cytokine complexes (asterisk in Fig. 1). The importance of this conserved hydrophobic residue was first revealed in the crystal structure of the growth hormone/growth hormone receptor complex (de Vos et al., 1992). The corresponding residue, W104, in the growth hormone receptor makes hydrophobic contacts with binding site II in growth hormone, and buries the most surface area (210 Å2) upon complex formation (de Vos et al., 1992
). A survey of different related 4-helix bundle cytokine/receptor binding site II crystal structures shows that the corresponding hydrophobic residue contributes the largest ASA in all these crystal structures. In each crystal structure, the residue fits in a hydrophobic pocket between helices A and C. As argued by Bravo and Heath, this also holds true for cytokines of the gp130 family, and seems to be a conserved feature of site II CRH/cytokine interactions (Bravo and Heath, 2000
). Our model, supported by our mutagenesis data, thus supports the view that leptin binding to CRH2 is very similar to the binding of other long chain 4-helix bundle cytokines via binding site II, with an important role of the conserved hydrophobic residue L504 in the ß5-ß6 loop that fits into a hydrophobic hole between helix A and helix C.
To our knowledge, two studies present a homology model of the leptin/LR interaction. Sandowski et al. (Sandowski et al., 2002) constructed a model of the 1:1 human LR CRH2/human leptin complex, based upon the structures of gp130 and human growth hormone receptor. They suggested that Y441 and F500 would have impact on leptin binding. The corresponding mouse LR residues Y439 and F498 are not involved in leptin/CRH2 interactions in our models and were not included in our mutation analysis.
A model described in more detail is provided by Hiroike and co-workers (Hiroike et al., 2000). They produced a model structure of a 2:2 human leptin/human LR CRH2 complex, based upon the G-CSF/G-CSF receptor complex, and distinguished a major and minor interface for leptin/LR interaction. The minor interface in this model was based on the minor interface seen in the G-CSF/G-CSF receptor crystal structure (Aritomi et al., 1999
). This type of interface is not seen in the crystal structures of gp130 cytokine/receptor complexes, and does not fit with structure-function analysis studies of the G-CSF/G-CSF receptor studies using mutagenesis and antibody binding (Layton et al., 2001
). It was therefore proposed to be a crystallization artefact (Layton et al., 2001
). The residues involved in the minor interface were not part of our mutation analysis, but the interaction of the major interface is in very good agreement with our model. Of the 14 residues proposed to be involved in the major interface, we analysed eight. Of these eight, E565, R615 and D617, corresponding to E563, R613 and D615 in mouse LR are involved in electrostatic interactions, according to both their and our model. Hydrophobic interactions are proposed for residues L505 and L506, corresponding to L503 and L504 in our model. Hydrogen bonds between leptin and the LR are suggested for residues F504, L505, N566 and N567, corresponding to residues F502, L503, N564, N565. Hydrogen bonding was predicted for N564 and N565 in our model, but mutation of these last two residues gave no indication for a role in leptin binding. Interestingly, the model of Hiroike et al. suggests a contribution of Y441 in leptin binding, but not of F500, as proposed by Sandowski et al. However, we also found effects of I501 and S505 (I503 and S507 in the human leptin receptor) mutants that are not predicted by this model.
Based upon structural superposition of leptin on the G-CSF/G-CSF receptor structure, Gonzalez and Leavis proposed interactions between the leptin receptor and leptin helices A and C (Gonzalez and Leavis, 2003). In this study, a synthetic peptide (LPA-2) comprising helix C (residues 70-95) was found to bind to the LR with high affinity, and to be a leptin antagonist. This agrees with our model, in which some major contributions in leptin/LR interaction happen through residues in helix C, including the L86 interaction with the receptor residue L504, and two predicted salt bridges (Table 1).
In summary, we produced a molecular model for the leptin/CRH2 interaction, and identified several residues that critically contribute to this interaction. The precise identification of these crucial residues in leptin binding may be beneficial in the rational design of small molecules, peptides or leptin mutants with higher affinity for the LR. These compounds could find an application as leptin receptor antagonists (Peelman et al., 2005).
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Acknowledgments |
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Footnotes |
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* These authors contributed equally to this work
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References |
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