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First published online May 28, 2005
doi: 10.1242/10.1242/jcs.02386

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Mapping of the interface between leptin and the leptin receptor CRH2 domain

Hannes Iserentant^*, Frank Peelman^*, Delphine Defeau, Joël Vandekerckhove, Lennart Zabeau and Jan Tavernier

Flanders Interuniversity Institute for Biotechnology, Department of Medical Protein Research (VIB09), Ghent University, Faculty of Medicine and Health Sciences, Baertsoenkaai 3, 9000 Ghent, Belgium

View larger version (41K): [in a new window]   Fig. 1. (A) Diagram of the structure of the leptin receptor. (B) Alignment of the leptin receptor CRH2 sequences with CRH crystal structures. Part of the alignment used for model construction is shown. Mouse and human LR sequence numbers are indicated at the end of the line. The superposed CRH crystal structure chains are indicated by their PDB identifier and the chain number used in the superposition (1AXI: human growth hormone receptor mutant, 1HWG: human growth hormone receptor,1F6F: rat prolactin receptor, 1EER: human Epo receptor, 1I1R: human gp130, 1CD9: mouse G-CSF receptor). Binding site II residues involved in cytokine/CRH interaction in the crystal structures are coloured green. The residue with the largest contact area with its cytokine is coloured cyan blue. In all structures, this is a conserved hydrophobic residue, indicated with an asterisk under the alignment. Secondary structure elements of the mouse LR homology model are indicated: ß-strand residues are coloured yellow, and indicated by an arrow under the alignment. Cysteine residues forming a disulphide bridge in the model are connected by lines. These cysteines, and their corresponding cysteines in the CRH crystal structures are coloured black. Residues that were mutated in this work are coloured red.

View larger version (28K): [in a new window]   Fig. 2. Effect of mutations in the mouse leptin receptor on signalling. Hek293T cells were transiently co-transfected with pMET7-mLRlo_BglII plasmids encoding different leptin receptor (LR) mutants and the pXP2d2-rPAP1-luci reporter construct. The transfected cells were either stimulated with leptin for 24 hours or left unstimulated. Luciferase measurements were performed in duplicate. Results are shown as fold inductions, i.e. the luciferase signal of stimulated cells divided by the signal of unstimulated cells. Data are representative of five separate transfection experiments. As positive control, wild-type pMET7-mLRlo_BglII was used; as negative control, an empty vector was transfected. The bracket groups the six mutants with the most drastic effect on signalling: I501A, F502A, L503A, L504A, S505A and D615A.

View larger version (37K): [in a new window]   Fig. 3. Effect of mutations in the CRH2 domain on binding. (A) Leptin binding by selected mutants. Cos-1 cells were transiently transfected with pMET7-mLRCRH2-His6 plasmids encoding different polyhistidine-tagged CRH2 domain mutants. The collected supernatants were incubated in triplicate in pent-His antibody-coated plates. Binding of leptin was investigated by adding a serial dilution of a leptin-SEAP fusion protein and measuring the resulting alkaline phosphatase (AP) activity. Data were fitted to a hyperbola (corresponding to a one site binding curve) using GraphPad Prism; curves shown are results of these fits. Results shown are representative of three separate experiments. (B) Kd values of alanine mutants with effect on binding. Also included are the R2 values of the fitted hyperbola. R.E.: relative expression levels, as determined by ELISA, expressed as a percentage of wild type.

View larger version (17K): [in a new window]   Fig. 4. Effect of serine mutations in the mouse leptin receptor on signalling. The same method as in Fig. 2 was employed. Results shown are representative of three separate transfection experiments. The bracket groups the four mutants that have the most drastic effect on signalling: L504S, I501SF502S, F502SL503S and L503SL504S.

View larger version (35K): [in a new window]   Fig. 5. Effect of serine mutations in the CRH2 domain on binding. (A) Leptin binding by the different serine mutants. The same method as in Fig. 3 was applied. (B) K values of the serine mutants. Also included are the R2 values of the fitted hyperbola and the relative expression levels (R.E.), as determined by ELISA. Owing to the low expression levels, the Kd of the I501S and I501SF502S mutation was not determined. Since binding of leptin is severely reduced in the case of the double mutants, alkaline phosphatase values are far from reaching their plateau levels, resulting in an unreliable fit and very large confidence intervals.

View larger version (46K): [in a new window]   Fig. 6. Molecular models of binding site II in mouse leptin. (A) Molecular surface map of leptin, coloured according to surface hydrophobicity (blue, hydrophilic; green, hydrophobic). A hydrophobic cleft is formed by residues L13, L86, L89 and F92. (B) Residues in binding site II that affect binding to CRH2 are coloured yellow. Residues in binding site II that affect both binding to CRH2 and LR activation are coloured orange. (C) Residues that become buried in the leptin/CRH2 interface, coloured according to the area that becomes buried (cyan, <25 Å2; blue, 25-50 Å2; green, >50 Å2).

View larger version (23K): [in a new window]   Fig. 7. Competitive leptin-SEAP binding assay of leptin mutants. We tested the binding of leptin mutants using a competitive leptin-SEAP assay as described in the Materials and Methods section. Wild-type leptin and leptin mutants that bind to CRH2 lead to a decreased binding of leptin-SEAP, measured as decreased alkaline phosphatase activity. The L86 mutants show a decreased competitive binding to CRH2, whereas the L89S and F92S mutations have no effect on binding.

View larger version (12K): [in a new window]   Fig. 8. Luciferase activity induced by a leptin mutant. Hek293T cells were transiently co-transfected with pMET7-mLRlo plasmid and the pXP2d2-rPAP1-luci reporter construct. The transfected cells were either stimulated with a dilution series of leptin mutant for 24 hours or left untreated. Luciferase measurements were performed in duplicate. The L86 mutant shows a shift of its EC50 value towards higher leptin concentrations.

View larger version (69K): [in a new window]   Fig. 9. Model of the mouse leptin/CRH2 complex. The molecular surface of leptin is coloured according to the surface hydrophobicity (blue, hydrophilic; green, hydrophobic). The CRH2 model is presented as ribbons, the C atom and heavy side chain atoms of residues I501, F502, L503, L504S and D615 are displayed as white sticks. L504 of CRH2 fits into the hydrophobic cleft of leptin and interacts with L13 and L86 of leptin.

View larger version (62K): [in a new window]   Fig. 10. Molecular models of mouse LR CRH2. (A) CRH2 residues that become buried in the leptin/CRH2 interface, coloured according to the area that becomes buried (cyan, <25 Å2; blue, 25-50 Å2; green, >50 Å2). (B) CRH2 residues that lead to a drastic increase of leptin binding to CRH2 and LR activation upon mutation.

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