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Originally published In Press as doi:10.1074/jbc.M413308200 on April 18, 2005

J. Biol. Chem., Vol. 280, Issue 24, 22632-22640, June 17, 2005
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Leptin Receptor Activation Depends on Critical Cysteine Residues in Its Fibronectin Type III Subdomains*

Lennart Zabeau, Delphine Defeau, Hannes Iserentant, Joël Vandekerckhove, Frank Peelman, and Jan Tavernier{ddagger}

From the Department of Medical Protein Research, Faculty of Medicine and Health Sciences, Flanders Interuniversity Institute for Biotechnology, VIB09, Ghent University, A. Baertsoenkaai 3, B-9000 Ghent, Belgium

Received for publication, November 26, 2004 , and in revised form, April 14, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The leptin receptor (LR) complex is composed of a single subunit belonging to the class I cytokine receptor family and exists as a preformed complex. The extracellular portion contains two cytokine receptor homology (CRH) domains, separated by an Ig-like domain and followed by two membrane-proximal fibronectin type III (FNIII) domains. The mechanisms underlying ligand-induced receptor activation are still poorly understood. LRs can exist as disulfide-linked dimers at the cell surface, even in the absence of leptin. We evaluated the role of the two unpaired cysteine residues (Cys-672 and Cys-751) in the FNIII domains in receptor clustering, leptin binding, and biological activity. Although mutation of cysteine on position 751 to serine has hardly any effect on ligand binding and receptor activation, the C672S mutant exhibits a marked reduction in STAT3-dependent signaling. The double mutant was completely devoid of biological activity, although leptin binding remained unaffected. Mutation of both residues resulted in complete loss of disulfide bridge formation of FNIII domains in solution. In contrast, no difference was observed in ligand-independent oligomerization of the membrane-bound receptor, suggesting a role for cysteines in the CRH2 domain in formation of the preformed LR complex. We propose a model wherein leptin-induced clustering of two preformed dimers forms the activated LR complex. Disulfide bridge formation involving Cys-672 and Cys-751 may be necessary for JAK activation and hence signaling.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The 16-kDa cytokine-like hormone leptin has been identified as one of the key players in the control of body weight. The product of the ob gene is produced and secreted mainly by adipocytes (1, 2), and plasma protein levels positively correlate with body fat energy stores. Leptin reduces food intake and stimulates energy expenditure by activating its receptor in specific hypothalamic nuclei (3–5). Spontaneous mutations that lead to a functional defect in either leptin (6) or its receptor (7–9) result in a complex syndrome that includes morbid obesity, hypothermia, infertility, hyperglycemia, decreased insulin sensitivity, and hyperlipidemia. Besides serving a weight-regulating function, leptin also plays a role in other processes including metabolism, reproduction, hematopoiesis, and immunity (10–13).

The leptin receptor (LR),1 the product of the db gene (14), is a member of the class I cytokine receptor family. The extracellular (EC) domain is composed of two so-called cytokine receptor homology (CRH) domains, a membrane distal CRH1 and a membrane proximal CRH2. Both domains are separated by an immunoglobulin-like (Ig) domain and are followed by two fibronectin type III (FNIII) domains proximal to the membrane. The CRH2 domain is necessary and sufficient for leptin binding (15). Despite the lack of any binding affinity for the ligand, the two FNIII and the Ig domains are needed for receptor activation (15, 16). Because of alternative splicing and ectodomain shedding, the LR can exist as six isoforms: a LR long form (LRlo or LRb), four short forms (LRa, LRc, LRd, and LRf), and a soluble isoform (LRe). LRlo contains the full cytosolic domain and is the only isoform capable of signaling. This receptor is highly expressed in hypothalamic neurons, but expression at functional levels could also be shown in several other cell types including hepatocytes, testis, immune cells etc., thereby forming the basis of the peripheral biological functions of leptin.

It has been questioned whether the LR becomes activated upon "simple" ligand-induced dimerization. Ligand-independent clustering of the receptor has been demonstrated in solution (17) and at the cellular surface (18, 19). Couturier and Jockers (18) used a bioluminescence resonance energy transfer (BRET) assay to postulate that leptin induces conformational reorganization within a preformed LR complex (18). We recently used a JAK/STAT (Janus kinase/signal transducer and activator of transcription) complementation strategy to demonstrate that the LR becomes activated upon higher order clustering, i.e. more than two receptors per activated complex (16). In this approach, two signaling-deficient receptors, one unable to activate the JAKs and the other lacking a functional STAT3 recruitment site, are only able to signal when they are co-expressed. Finally, a detailed mutagenesis study identified three receptor binding sites in the leptin molecule (20). Mutations within site II impair binding to CRH2, with only a moderate effect on signaling. Mutations within sites I and III do not affect binding to CRH2, but mainly site III plays an important role in receptor activation. The leptin/LR system therefore shows strong structural similarities with the hexameric interleukin-6 (IL-6) receptor complex.

In this study we focused on the role of cysteine residues in the membrane-proximal FNIII domains (Cys-672 and Cys-751). We evaluated their role in ligand binding, receptor activation, and ligand-independent clustering at the cellular surface. We used these data to propose a model in which the LR becomes activated upon leptin-mediated clustering of two preformed receptor dimers.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Vectors—Murine leptin receptor (mLR) deletion variants mLR {Delta}CRH1 and mLR {Delta}CRH1, {Delta}Ig have been constructed using a mutagenesis strategy (16). In brief, a XhoI site was introduced immediately following the signal-peptide encoding sequence. A second XhoI site was inserted following the sequence coding for the membrane distal CRH1 domain, or for the Ig-like domain. Resulting vectors were XhoI-digested and circularized. Deletion variant mLR {Delta}CRH1, {Delta}Ig, {Delta}CRH2 was made by PCR amplification with oligonucleotides 5'-GCGCTCGAGTCAAAGTTCCTATGAGAGGGCC-3' (with XhoI site underlined) and 5'-CGCCGCAGCCGAACGACCGA-3' (50 bp downstream of the KpnI site). The resulting amplicon was XhoI-KpnI-digested and ligated into the opened pMET7 mLR {Delta}CRH1, {Delta}Ig vector. The vector pMET7 V5-mLR {Delta}EC encodes a LR variant in which the EC domain is replaced by the sequence encoding the V5 epitope. LR transmembrane and cytoplasmic domains were amplified with the forward primer 5'-GCGCTCGAGGTGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGTTCACCAAAGATGCTATCGAC-3' (with the XhoI site underlined and the V5 epitope sequence in boldface) and reverse primer 5'-CGCCGCAGCCGAACGACCGA-3' (50 bp downstream of the KpnI site). Also, after XhoI-KpnI digestion, the amplicon was inserted into the opened pMET7 mLR {Delta}CRH1, {Delta}Ig vector.

In the full-length receptor and in the deletion variants, the Myc tag was replaced by the sequence coding for the FLAG tag. Therefore, the LR sequence between nucleotides 2161 and 2684 was amplified with oligonucleotides 5'-CCCTTGTGAATTTTAACCTTACC-3' (50 bp upstream of the unique DraIII site) and 5'-CGCTCTAGATTACTTATCGTCGTCATCCTTGTAATCCACAGTTAAGTCACACATC-3' (with XbaI site underlined and FLAG epitope sequence in boldface). The amplicon was DraIII-XbaI digested and ligated in the appropriate expression vectors.

An expression vector wherein CRH2 is coupled to (i) the combined FLAG-His tag or (ii) secreted alkaline phosphatase (SEAP)-FLAG was constructed by inserting a BglII immediately following the sequence encoding the CRH2 domain in the pMET7 mLR {Delta}CRH1, {Delta}Ig vector (mutagenesis primers with BglII site underlined: 5'-GCTTGTCATGGATGTAAAGATCTCTATGAGAGGGCCTGAATTTTGG-3', and 5'-CCAAAATTCAGGCCCTCTCATAGAGATCTTTACATCCATGACAAGC-3'). The resulting vector was cut with BglII and XbaI. (i) Oligonucleotides 5'-GATCTTAGATTACAAGGATGACGACGATAAGCACCACCACCACCACCACTAAT-3' and 5'-CTAGATTAGTGGTGGTGGTGGTGGTGCTTATCGTCGTCATCCTTGTAATCTAA-3' encode both FLAG and His tags, and the ends are complementary to the BglII and XbaI sticky ends. Oligos were annealed and ligated into the opened vectors, resulting in FLAG-His-tagged protein. (ii) Alternatively, oligonucleotides 5'-GCGGCGAGATCTCTATCATCCCAGTTGAGGAGGAGAACC-3' (with BglII site underlined) and 5'-CGCCTCTAGATTACTTATCGTCGTCATCCTTGTAATCACCCGGGTGCGCGGCGTCG-3' (with XbaI site underlined and FLAG sequence in boldface) were used to amplify the sequence encoding SEAP. The amplicon was digested with the enzymes BglII and XbaI, and ligated into the opened vector.

Expression vectors pMET7 mLR FNIII-FLAG-His and pMET7 mLR FNIII-SEAP-FLAG were constructed as follows. cDNA for FNIII domains was amplified using the primers 5'-GCGCTCGAGCCGTTCCTATGAGAGGGCCTG-3' (with XhoI underlined) and 5'-CGCCGCAGATCTTCCCTGCGTCATTCTGCTGCTTGTCG-3' (with BglII underlined). The CRH2 domain in pMET7 mLR CRH2-FLAG-His and pMET7 mLR CRH2-SEAP-FLAG was replaced by a cDNA fragment encoding the FNIII domains by a XhoI-BglII digestion of the amplicon and the appropriate vectors.

Free cysteine residues in the LR variants were mutated to serines. The primers used were: 5'-CGAAAAATGACTCACTCTCGAGTGTGAGGAGGTACG-3' and 5'-CGTACCTCCTCACACTCGAGAGTGAGTCATTTTTCG-3' for C672S; and 5'-GCTTATCCCCTGAGCAGCTCGAGCGTCATCCTTTCCTGG-3' and 5'-CCAGGAAAGGATGACGCTCGAGCTGCTCAGGGGATAAGC-3' for the C751S mutation. The double mutant LR C672S,C751S was constructed by digestion of pMET7 mLR C751S with enzymes DraIII and SacI. The resulting insert of 1507 bp was ligated in the DraIII-SacI opened pMET7 mLR C672S.

Generation of the pXP2d2-rPAP1 (rat pancreatitis associated protein 1)-luciferase reporter was described previously (21). Activation of this reporter is dependent on STAT3. Over-expression of dominant-negative STAT3, but not of dominant-negative STAT1, completely blocks rPAP-luciferase reporter activation (22).

Cell Lines and Transfection Procedures—HEK293T and COS-1 cells were grown in Dulbecco's modified Eagle's medium with 4500 mg/liter glucose supplemented with 10% fetal bovine serum (all from Invitrogen) in 10% CO2 humidified atmosphere at 37 °C. For transfection experiments, 4.105 cells/10 cm2 well were freshly seeded and cultured overnight. HEK293T and COS-1 cells were transfected overnight with standard calcium phosphate precipitation or polyethyleneimine procedures, respectively. One day after transfection, cells were washed with phosphate-buffered saline-A and cultured overnight until further use (Western blot, co-precipitation, chemical cross-linking, reporter assay, or leptin-SEAP binding).

Western Blot Analysis—Expression of LR or LR (deletion) mutants was monitored using Western blot analysis. Cells expressing the receptors were lysed in 300 µl of loading buffer and sonicated. Samples were loaded on a polyacrylamide gel and blotted onto a nitrocellulose membrane. Proteins were revealed with the M2 anti-FLAG monoclonal antibody (Sigma) and sheep anti-mouse horseradish peroxidase-coupled secondary antibody (Amersham Bioscience).

For JAK2 phosphorylation HEK293T cells were transfected with the appropriate LR mutants and 0.01 µg of the JAK2 expression vector, pRK5-JAK2. After 65 h cells were starved in serum-free medium for 6 h and were left untreated or stimulated with 200 ng/ml leptin for 10 min. After gel electrophoresis and blotting, JAK2 was revealed using an anti-phospho-JAK2 or an anti-JAK2 antibody (both from Upstate Biotechnology).

Co-precipitation—HEK293T cells were transiently transfected overnight with SEAP-FLAG and FLAG-His fusion protein vectors (or empty vector as a negative control). Three days after transfection, supernatants were collected and subjected to precipitation with Talon metal affinity resin (BD Biosciences). 50 µl of bed volume resin per precipitation was washed three times with wash buffer (50 mM NaPO4, 300 mM NaCl, 0.5% Nonidet P-40, pH 7.0). Supernatant was incubated with the resin for 1 h at 4 °C. After three washes with wash buffer, the precipitated complexes were eluted with an acidic elution buffer (50 mM sodium acetate, 300 mM NaCl, pH 5.0). Co-precipitated SEAP activity was measured using the chemiluminescent CSPD substrate (PhosphaLight, Tropix) in a TopCount chemiluminescence counter (Packard).

Reporter Assays—Two days after transfection, cells expressing different combinations of LR variants were resuspended with cell dissociation agent (Invitrogen) and seeded in a 96-well plate (Costar). Cells were stimulated overnight with leptin (R&D Systems) as indicated or were left unstimulated. Lysates were prepared (lysis buffer: 25 mM Tris, pH 7.8, 2 mM EDTA, 2 mM dithiothreitol, 10% glycerol, 1% Triton X-100), and 35 µl of luciferase substrate buffer (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 µM coenzyme A, 470 µM luciferin, 530 µM ATP, final pH 7.8) was added per 50 µl of lysate. Light emission was measured for 5 s in a TopCount chemiluminescence counter (Packard).

Leptin-SEAP Binding—Cell surface expression of wild type LR or LR mutants was measured using a binding assay with a mouse leptin-SEAP chimeric protein. Two days after transfection, cells were washed (wash buffer: Dulbecco's modified Eagle's medium, 0.1% NaN3, 20 mM Hepes, pH 7.0, 0.01% Tween 20) and incubated for 90 min at room temperature with a 1/50 dilution of a COS-1 conditioned medium containing the leptin-SEAP chimera (final concentration, ± 10 ng/ml). After 3 washing steps, cells were lysed (lysis buffer: 1% Triton X-100, 10 mM Tris-HCl, pH 7.4). Endogenous phosphatases in the lysates were inactivated (65 °C, 30 min), and secreted alkaline phosphatase activity was measured as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Leptin Receptors Exist as Preformed Disulfide-linked Oligomers on the Cell Surface—A series of mLR deletion variants lacking EC subdomains was generated and are shown in Fig. 1A. All variants were expressed in COS-1 cells and analyzed under reducing ({beta}-mercaptoethanol) or nonreducing conditions. As shown in Fig. 1B, all deletion variants, except the one lacking the complete EC domain (mLR {Delta}EC), formed ligand-independent oligomers on the cellular surface. All receptors (full-length, ~ 160 kDa; mLR {Delta}CRH1, ~120 kDa; mLR {Delta}CRH1, {Delta}Ig, ~105 kDa; mLR {Delta}CRH1, {Delta}Ig, {Delta}CRH2, ~80 kDa; and mLR {Delta}EC, ~45 kDa) appeared as monomers under reducing conditions. This suggests that disulfide bridges, possibly between the FNIII domains, are involved in LR dimerization. The two mLR FNIII subdomains each contain a single free cysteine residue at positions 672 and 751 (23). Cross-species alignment revealed complete conservation of both residues during evolution, suggesting their functional importance.



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  		FIG. 1.
Expression and ligand-independent clustering of LR deletion variants. A, schematic representation of the wild type mLR and deletion variants thereof. The domain structure of the EC part is outlined, as well as determinants for signaling in the cytoplasmic tail. Aa, amino acid; Y, tyrosine residue. See the Introduction for details. B, COS-1 cells were transfected with plasmids encoding different LR deletion variants: full-length (lane 1); mLR {Delta}CRH1 (lane 2); mLR {Delta}CRH1, {Delta}Ig (lane 3); mLR {Delta}CRH1, {Delta}Ig, {Delta}CRH2 (lane 4); and mLR {Delta}EC (lane 5). Cells were lysed in either a reducing, {beta}-mercaptoethanol-containing loading buffer (R) or in a nonreducing loading buffer (NR). Protein complexes were separated with SDS-PAGE, blotted onto a nitrocellulose membrane, and revealed with an anti-FLAG antibody.

 
Spontaneous Aggregation of FNIII Domains in Solution and the Role of Disulfide Linkage—We next analyzed the ligand-independent clustering of the FNIII domains in more detail. Constructs were generated to produce secreted forms of the FNIII domains either as a FLAG-His- or a SEAP-FLAG-tagged protein. When supernatants from FNIII-FLAG-His-expressing COS-1 cells were analyzed under nonreducing conditions, dimers, trimers, and even higher order clusters were observed (Fig. 2A). This clustering is independent of protein concentration since transfection with smaller amounts of expression vector gave the same clustering pattern (data not shown).

To confirm this FNIII clustering, we set up a His/SEAP co-precipitation experiment. In this assay, FNIII-SEAP-FLAG proteins were co-expressed with FNIII-FLAG-His or with empty vector as a negative control. Supernatants were analyzed in a precipitation experiment with a Co2+ affinity resin. Co-precipitated SEAP activity reflected interaction between the proteins. Fig. 2B clearly confirms homotypic interaction of the FNIII domain.

Both FNIII cysteine residues were mutated to serines (C672S and C751S) in the soluble FNIII-FLAG-His construct. As shown in Fig. 2C, both single mutations only slightly altered disulfide complex formation. In contrast, the double mutant (C672S,C751S) appeared in a monomeric form under nonreducing conditions, indicating that both cysteines are involved in the observed disulfide bonding.

Conserved Cysteines in the FNIII Domain Are Essential for LR Activation—To examine the role of the FNIII Cys-672 and Cys-751 residues in activation of the LR, we analyzed the effect of Cys to Ser mutations in the membrane-bound full-length LR long isoform. Single and double mutants were tested for their signaling capacity using a STAT3-dependent reporter assay in HEK293T cells. Results are shown in Fig. 3A. Mutation C672S showed clear reduction on the activation of the rPAP1-luciferase reporter, whereas mLR C751S had signaling capacities comparable with the wild type receptor. Combined mutation resulted in a receptor almost completely devoid of biological activity. As shown in Fig. 3B, cell surface expression and leptin binding of all mutant receptors was comparable with the wild type LR.

We next evaluated the effect of these mutations on specific steps in signaling, i.e. JAK2 and STAT3 activation. Data shown in Fig. 3C clearly illustrate that whereas mutation of Cys-751 had no effect, only a very weak JAK2 phosphorylation could be shown for the C672S mutant. Activation of the kinase is completely blocked by the combined mutation, thereby illustrating the role of these cysteines in activation of the kinase.

The FNIII Domains Induce Ligand-independent Signaling—We observed spontaneous, ligand-independent STAT3-dependent signaling in cells expressing a mLR variant with only the FNIII domains (mLR {Delta}CRH1, {Delta}Ig, {Delta}CRH2) when compared with cells expressing a LR variant in which the complete EC domain is replaced by the V5 tag (Fig. 4A). To test whether homotypic FNIII-FNIII interactions were responsible for this ligand-independent signaling, we analyzed the effect of co-expression of signaling-deficient mLR-F3 deletion variants. mLR {Delta}CRH1, {Delta}Ig, {Delta}CRH2, and an increasing amount of vector as indicated encoding the F3 mutants mLR-F3 {Delta}CRH1, {Delta}Ig, {Delta}CRH2, and mLR-F3 {Delta}EC were transiently co-transfected in HEK293T cells (Fig. 4B). Clearly, leptin-independent activation of the mLR {Delta}CRH1, {Delta}Ig, {Delta}CRH2 variant was reduced only when the F3 variant had an EC part containing the FNIII domains (Fig. 4B). The F3 variant lacking the EC domain is not able to reduce background signaling. It is notable that differences in ligand-independent activity could not be explained simply by differences in the expression levels of the different LR deletion variants as measured by Western blot analysis (see also Fig. 1) or fluorescence-activated cell sorter analysis (data not shown). Together, these data lend further support to the role of (spatially correct) FNIII domain clustering for JAK activation and subsequent STAT3-dependent signaling. These data also rule out an important role of the transmembrane domain in this process.

Inhibition of LR Signaling by Homotypic FNIII-FNIII Interaction—Previous experiments showed that the FNIII domains and cysteine residues therein play a crucial role in LR activation. We next questioned whether preventing homotypic FNIII-FNIII interaction could inhibit leptin receptor signaling. In a first approach, we evaluated the effect of two STAT3 signaling-deficient F3 mutant receptors on wild type LR signaling: mLR-F3 {Delta}CRH1, {Delta}Ig, {Delta}CRH2 (with only the FNIII domains), and mLR-F3 {Delta}EC (wherein the complete EC domain was replaced by the V5 epitope). A vector encoding the wild type mLR was co-transfected with increasing amounts of vector encoding the mLR-F3 variants, and STAT3-dependent reporter activity was measured (Fig. 5). Results clearly showed that only the LR-F3 variant with the FNIII domains could inhibit LR signaling, in strong contrast to the mLR-F3 {Delta}EC variant. Because the FNIII domains cannot bind leptin themselves, these data implied that the mLR-F3 {Delta}CRH1, {Delta}Ig, {Delta}CRH2 mutant is recruited in the complex only via FNIII-dependent receptor-receptor interactions.



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  		FIG. 2.
FNIII clustering in solution. A, the plasmid encoding FNIII-FLAG-His was transfected in COS-1 cells. Supernatants was subjected to Western blot analysis under reducing (R) or nonreducing (NR) conditions with an anti-FLAG antibody. B, HEK293T cells were co-transfected with cDNA encoding FNIII-SEAP-FLAG in combination with FNIII-FLAG-His or with empty vector as indicated. Three days after transfection, supernatants were collected and subjected to precipitation with the Co2+ metal affinity resin. After three successive washes and subsequent elution, co-precipitated alkaline phosphatase activity was measured. Error bars represent mean ± S.D. of triplicate measurements. CPS, counts/s. C, FNIII-FLAG-His and mutants thereof, as indicated, were transiently expressed and analyzed as described in A.

 
Role of FNIII Cysteines in Disulfide-dependent LR Oligomerization at the Cell Surface—As with the soluble FNIII domains, combined mutation of residues Cys-672 and Cys-751 in LR {Delta}CRH1, {Delta}Ig, {Delta}CRH2 completely abolished disulfide linkage on the cell surface (Fig. 6A). The single C751S mutant receptor still showed dimerization, although to a lesser extent when compared with mLR wild type and C672S. In strong contrast to this truncated receptor, mutation of Cys-672 and Cys-751 appeared to have no effect at all on disulfide bridging of the full-length receptor (Fig. 6B) or on the deletion variant LR {Delta}CRH1, {Delta}Ig (Fig. 6C). This suggests that the free FNIII cysteine residues are not critical for the ligand-independent disulfide linkage of the wild type receptor. This covalent clustering seems to be mediated by residues in CRH2.

Ligand-binding CRH2 Domain Clusters in Solution—Because the FNIII domains appear not to be involved in leptin-independent clustering of the full-length receptor, we next focused on the ligand-binding CRH2 domain. Like FNIII, this domain was expressed either as a FLAG-His-tagged or SEAP-FLAG fusion protein. Western blot analysis showed that CRH2-FLAG-His was, at least in part, expressed as an oligomeric complex, which, similar to the FNIII domains, was sensitive to reduction (Fig. 7A). This homotypic CRH2-CRH2 interaction was confirmed using a co-precipitation experiment as described for the FNIII domains (Fig. 7B). This assay also allowed us to test whether the CRH2-CRH2 clustering occurs during biosynthesis. Fig. 7C clearly shows that co-precipitation, and thus clustering, was strictly dependent on co-expression of both of the tagged CRH2 proteins in the same cell.



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  		FIG. 3.
Role of Cys-672 and Cys-751 in receptor activation, expression, and ligand binding. A, pMET7 expression plasmids with mLR, mLR C672S, mLR C751S, or mLR C672S,C751S were transfected in HEK293T cells. The pXP2d2-rPAP1-luciferase reporter construct was co-transfected to follow STAT3 activation. Transfected cells were stimulated overnight with a serial dilution of leptin as indicated. Luciferase reporter activity is plotted as a function of the leptin concentration. CPS, counts/s. B, effect of cysteine mutations on leptin binding. Transfections were as described in A. Cells were incubated with leptin-SEAP, with (open bars) or without (filled bars) excess unlabeled leptin for 2 h. After four successive washing steps, bound alkaline phosphatase activity was measured. Error bars represent mean values of triplicate measurements. C, JAK2 phosphorylation. Similar transfections were performed to check for phosphorylation of JAK2. Serum-starved (6 h) cells were stimulated with 200 ng/ml leptin for 10 min or were left unstimulated. Lysates were blotted onto a nitrocellulose membrane and analyzed using phosphospecific JAK2 antibody. Total amounts of JAK2 are also shown.

 



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  		FIG. 4.
Ligand-independent signaling by mLR deletion variants. A, LR deletion variants mLR {Delta}CRH1, {Delta}Ig, {Delta}CRH2, and mLR {Delta}EC were transiently transfected together with the pXP2d2-rPAP1-luciferase reporter construct in HEK293T cells. Four days later, luciferase activity was measured in triplicates. Error bars represent mean luciferase values. B, 1 µg of plasmid encoding the deletion variants mLR {Delta}CRH1, {Delta}Ig, and {Delta}CRH2 was transfected alone or with increasing amounts, as indicated, of cDNA encoding the F3 variants mLR-F3 {Delta}CRH1, {Delta}Ig, {Delta}CRH2, and mLR-F3 {Delta}EC. Differences in quantities of transfected DNA were adjusted with empty pMET7 vector. Also, STAT3 activation was measured using the pXP2d2-rPAP1-luciferase reporter.

 
The CRH2 Domain Prevents Spontaneous FNIII-FNIII Clustering—Because both FNIII and CRH2 domains show homotypic clustering, we next questioned whether the CRH2 domain influences the spontaneous receptor activation elicited by FNIII domain interactions. Different LR deletion variants were transiently transfected, and leptin-independent signaling was measured by co-transfection of the rPAP1-luciferase reporter (Fig. 8). Results clearly indicate that the presence of CRH2 (alone or in combination with Ig and CRH1) reduces FNIII-mediated signaling to background level. These data strongly suggest that the CRH2 domains keep the FNIII domains spatially apart (or in a nonproductive orientation) so that no spontaneous JAK activation and hence no signaling can occur.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The structure of the EC domain of the class I cytokine receptor superfamily is quite diverse. In their simplest form, these receptors come with only one CRH domain, which is sufficient for ligand binding and receptor activation. Other receptors, both homomeric and heteromeric, can have two CRH modules and/or additional subdomains with an Ig-like or FNIII-fold. Signaling via all of these receptors is critically dependent on the activation of the cytoplasmically associated JAKs. To achieve activation, the {alpha}-helical bundle cytokine ligands must organize their receptor complexes in such a way that the kinases are correctly juxtaposed allowing activation by cross-phosphorylation.



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  		FIG. 5.
Inhibition of LR signaling by truncated mutants. 0.1 µg of plasmid encoding the mLR {Delta}CRH1 was co-transfected with different amounts of mLR-F3 {Delta}CRH1, {Delta}Ig, {Delta}CRH2 (A) and V5-mLR-F3 {Delta}EC (B) as indicated. Differences in quantities of transfected DNA were adjusted with empty pMET7 vector. The rPAP1-luciferase reporter construct was also transfected to measure STAT3 activation. Cells were stimulated overnight with a serial dilution of leptin as indicated. Luciferase measurements were performed in triplicate and plotted as a function of the leptin concentrations.

 
Over the past two decades, the binding of cytokines to their receptors has been the subject of many studies. For most cytokines, receptor binding sites have well been mapped with mutagenesis experiments. Furthermore, co-crystallization data on several {alpha}-helical bundle cytokine-receptor complexes has been obtained. Such studies describe in great detail the assembly of ligands with their soluble EC receptor domains but cannot always account for the dynamic processes underlying ligand-dependent reorganization and activation of the membrane-anchored receptor complexes. Such insights will require functional studies that dissect ligand-induced effects on membrane-bound receptor complexes.

An important aspect in cytokine receptor activation is that many (if not all) cytokine receptors appear to exist as inactive, preassembled complexes at the cellular surface. This has been demonstrated for the receptors for erythropoietin (24–26) growth hormone (27), interferon-{gamma} (28), the interleukin-6 receptor {alpha}-chain (29), and the {beta}c signaling component in the receptors for IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor (30). This implies that ligand binding induces a spatial reorganization within the receptor complex, thereby triggering intracellular signaling. According to this model, receptor clustering correctly positions the receptor-associated JAK tyrosine kinases, so that JAK cross-phosphorylation can occur followed rapidly by activation of the intracellular signaling machinery. Insights into the reorganization of the cytokine receptors underlying their activation at the molecular level is, however, still very limited.

We used the single subunit LR complex as a model to study cytokine receptor activation. Ligand-independent formation of the LR complex has been demonstrated in solution (17) and at the cellular surface (19). More recently, Couturier and Jockers (18) used a BRET approach to show that about 60% of the cell surface receptors exist as oligomers and that a conformational change is the basis for activation of the LR. Here, we propose a novel model for LR activation based on several observations from functional studies using LR mutants.

First, the LR can exist as a disulfide-linked complex on the cell surface. The observation that only a part of the expressed receptors are found as oligomeric complexes is in line with the previous BRET study (18). Belouzard et al. (37) have demonstrated that only a small fraction of the LRs is expressed on the membrane, with the majority of the receptors localizing to the trans-Golgi network and in endosomes. This ligand-independent clustering most likely involves cysteine residues in the CRH2 and not in the FNIII domains, since the double mutant receptor LR {Delta}CRH1, {Delta}Ig C672S,C751S exists as dimers under nonreducing conditions (Fig. 6C). Disulfide-dependent dimerization in the absence of leptin has also been demonstrated for bacterially expressed CRH2 protein (31). Our co-precipitation data furthermore indicate that this CRH2-dimerization occurs intracellularly (Fig. 7).



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  		FIG. 6.
Effect of mutation of Cys-672 and Cys-751 on disulfide linkage on the cellular membrane. A, plasmids encoding mLR {Delta}CRH1, {Delta}Ig, {Delta}CRH2 (lane 1), mLR {Delta}CRH1, {Delta}Ig, {Delta}CRH2 C672S (lane 2), mLR {Delta}CRH1, {Delta}Ig, {Delta}CRH2 C751S (lane 3), and mLR {Delta}CRH1, {Delta}Ig, {Delta}CRH2 C672S,C751S (lane 4) were transiently transfected in COS-1 cells. Transfected cells were lysed in a reducing (R) or nonreducing (NR) loading buffer. Lysates were loaded on a SDS-polyacrylamide gel, blotted to a nitrocellulose membrane, and subjected to Western blot with an anti-FLAG antibody. Monomers and dimers are indicated with arrows. B, cysteine mutants of the following full-length receptors were analyzed as described in A: lane 1, mLR; lane 2, mLR C672S; lane 3, mLR C751S; lane 4, mLR C672S,C751S. C, cysteine mutants of mLR {Delta}CRH1, {Delta}Ig were analyzed as described in A: lane 1, mLR {Delta}CRH1, {Delta}Ig; lane 2, mLR {Delta}CRH1, {Delta}Ig C672S; lane 3, mLR {Delta}CRH1, {Delta}Ig C751S; lane 4, mLR {Delta}CRH1, {Delta}Ig C672S,C751S.

 
Second, a crucial role for the FNIII domains in LR activation has been suggested by Fong et al. (15), who showed that a LR mutant, min-BD (with an EC domain containing only CRH2 and Ig), is unable to signal. We show here that Cys-672, and to a lesser extent Cys-751, plays an important role in the activation of the JAKs and thus in leptin signaling (Fig. 3). Mutations of these residues do not alter expression of the secreted or membrane-bound receptor and also do not affect ligand binding. These data argue against a strictly structural role for these residues.

Third, a LR with an EC domain consisting of only the FNIII domains is constitutively active (Fig. 4). This implies that FNIII-FNIII interactions can bring the cytoplasmic receptor tails in close proximity and correct orientation so that JAK activation becomes possible. This might simulate the situation that occurs upon leptin stimulation. The presence of the CRH2 domain apparently prevents this constitutive, FNIII-mediated signaling (Fig. 8), suggesting that CRH2 dimers keep the FNIII domains spatially apart.



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  		FIG. 7.
Clustering of CRH2 in solution. A, plasmid pMET7 CRH2-FLAG-His was transiently transfected in COS-1 cells. Supernatant was subjected to Western blot analysis with an anti-FLAG antibody under reducing (R) and nonreducing (NR) conditions. B, proteins CRH2-FLAG-His and CRH2-SEAP-FLAG were tested in the co-precipitation assay as described for Fig. 2. Error bars represent mean values ± S.D. of triplicate measurements. C, the effect of co-expression was tested in the same experiment. HEK293T cells were transfected individually with plasmids encoding CRH2-FLAG-His, CRH2-SEAP-FLAG, or empty vector. The next day, cells were resuspended and equal amounts of cells expressing CRH2-FLAG-His and CRH2-SEAP-FLAG (filled columns) or cells transfected with CRH2-SEAP-FLAG and empty vector (open columns) were mixed. As a positive control, cells were transfected with a combination of FLAG-His and SEAP-FLAG fusion or SEAP-FLAG and empty vector (see panel B). These transfected cells were also resuspended but not mixed. Two days later, the formation of oligomers was determined by subjecting supernatants of cell mixtures to co-precipitation with the Co2+ metal affinity resin. Co-precipitated alkaline phosphatase activity was measured in triplicate.

 
We used these data to propose a model for LR activation (Fig. 9). Herein, the receptor exists as preformed dimmers, based mainly on the homotypic, possibly S-S linked, interactions between the CRH2 domains of two LRs. In this configuration, FNIII domains, and therefore also the cytoplasmic receptor tails, are held apart spatially, so that there is no constitutive JAK activation. Based on the previous observation that the receptors become activated upon higher order (i.e. more than two) clustering (16), it is tempting to believe that two leptin molecules cluster two such LR dimers. Leptin site II interacts with the LR CRH2, whereas residues of site III interact with the LR Ig-like domain (20). FNIII domains of two different preformed dimers are brought in close proximity, whereby subsequent S-S bridge formation may fix the complex in the correct orientation.

In addition to its weight-regulating activities, leptin appears to be involved in the onset and progression of several autoimmune diseases, including multiple sclerosis, Crohn's disease, and rheumatoid arthritis. The fact that additional LR FNIII receptor-receptor interactions are needed for the formation of an activated receptor complex opens the possibility of blocking leptin signaling in these diseases with high affinity molecules directed against the receptor, without interfering with ligand binding. Possible candidate antagonists are purified receptor subdomains and antibodies directed against the FNIII domains.



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  		FIG. 8.
Ligand-independent signaling by mLR deletion variants. Several mLR deletion variants, as indicated, were expressed in HEK293T cells along with the rPAP1-luciferase reporter. Four days later, luciferase activity was measured. Error bars represent mean luciferase values ± S.D. of triplicate measurements.

 



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  		FIG. 9.
Model for LR activation. For reasons of clarity, the membrane distal CRH1 domain was not included in the model. Arrows indicate covalent interactions between the FNIII domains.

 
Our findings may not be restricted to the LR system, because certain data suggest that FNIII domains play a role in the activation of other class I cytokine receptors. A well studied example is the gp130 chain in the IL-6 receptor complex. (i) Pflanz et al. (34) showed that the bacterially expressed membrane distal gp130 FNIII domain (D4) oligomerizes in solution. (ii) In isothermal titration calorimetry experiments, formation of the hexameric IL-6·IL-6R{alpha}·gp130 complex with the gp130 FNIII domains resulted in the generation of a lower overall level of free energy when compared with complex formation in the absence of these domains (35). (iii) A neutralizing antibody directed against the gp130 FNIII D4 domain is able to block IL-6 signaling, further supporting its role in receptor activation (36).

It is noteworthy that ligand-induced inter-subunit disulfide linkage has been described for the IL-3 receptor (32, 33). In this case, cysteine residues in the N-terminal domain of the {beta}c chain covalently bind to cysteines in the ligand-specific {alpha}-receptors upon stimulation. Disulfide linkage appears to be essential for phosphorylation of the {beta}c receptor.

In summary, we have demonstrated the homotypic interaction of the LR FNIII domains in solution and at the cellular membrane. Two conserved free FNIII cysteine residues, on positions 672 and 751, appear to be critical for ligand-induced JAK activation and hence for biological activity. Neither of these residues has a structural or ligand binding role or is involved in ligand-independent covalent clustering. This oligomerization appears to be based on CRH2-CRH2 interactions. These may keep the FNIII domains spatially apart and prevent spontaneous signaling. Ligand-induced disulfide bridge formation involving these residues may be a mechanism to irreversibly lock the receptor complex in its active configuration.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 32-9-264-93-02; Fax: 32-9-264-94-92; E-mail: Jan.Tavernier{at}UGent.be.

1 The abbreviations used are: LR, leptin receptor; mLR, murine leptin receptor; CRH, cytokine receptor homology; EC, extracellular; FNIII, fibronectin type III; Ig, immunoglobulin-like; IL, interleukin; JAK, Janus kinase; rPAP, rat pancreatitis-associated protein; SEAP, secreted alkaline phosphatase; STAT, signal transducer and activator of transcription; BRET, bioluminescence resonance energy transfer; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; HEK, human embryonic kidney. Back



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TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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